Publications

Banday AR, Azim S, Tabish M. Alternative promoter usage and differential expression of

multiple transcripts of mouse Prkar1a gene. Mol Cell Biochem. 2011 Jun 3. [Epub ahead

of print]

Banday AR, Azim S, Tabish M. Differentially expressed three non-coding alternate exons

at 5' UTR of regulatory type I beta subunit gene of mouse. Mol Biol Rep. 2011 Jun 26.

[Epub ahead of print]

Azim S, Banday AR, Tabish M. Alternatively Spliced Three Novel Transcripts of gria1 in

the Cerebellum and Cortex of Mouse Brain. Neurochem Res. 2011 Sep 17. [Epub ahead of

print]

Banday AR, Azim S, Tabish M. Two Novel N-terminal coding exons of Prkar1b gene of

mouse- Identified using novel in silico pipeline confirmed by molecular biology

techniques. Communicated

Banday AR, Azim S, Tabish M. Identification and expression analysis of three novel

splice variants of PKA catalytic beta subunit gene in mouse: combinatorial in silico and

molecular biology approaches. Communicated

Banday AR, Azim S, Tabish M. Prediction and identification of new splice variant of

Prkaca gene in mouse. Communicated

Banday AR, Azim S, Ola M, Tabish M. PKA at interfaces of different disease status: an

overview. Communicated

Abstracts in international conferences

Banday AR, Azim S, Tabish M. Novel transcript variants arise from alternate promoter

usage and alternative splicing in 5' regions of cAMP-dependent Protein kinase A subunit

genes in mouse FASEB J March 17, 2011 25:900.3. Presented paper in Experimental

Biology-2011, April 9-13, Walter E. Washington Convention Center, Washington, DC.

Banday AR, Azim S, Tabish M “Computational and molecular analysis of gene encoding

PKA Cα-subunit in mouse” Abstract No. 105 (Page 23). The Eight Asia-Pacific

Bioinformatics Conference, January 18-21, 2010. J.N. Tata Auditorium, Indian Institute of

Science, Bangalore

Azim S, Banday AR, Tabish M. Alternative splicing of chrng gene generates Novel

Isoforms with different N-terminals FASEB J March 17, 2011 25:900.2

Alternative promoter usage and differential expression of multipletranscripts of mouse Prkar1a gene

Abdul Rouf Banday • Shafquat Azim •

Mohammad Tabish

Received: 14 February 2011 / Accepted: 19 May 2011

� Springer Science+Business Media, LLC. 2011

Abstract Prkar1a gene encodes regulatory type 1 alpha

subunit (RIa) of cAMP-dependent protein kinase (PKA)

in mouse. The role of this gene has been implicated in

Carney complex and many cancer types that suggest its

involvement in physiological processes like cell cycle

regulation, growth and/or proliferation. We have identi-

fied and sequenced partial cDNA clones encoding four

alternatively spliced transcripts of mouse Prkar1a gene.

These transcripts have alternate 50 UTR structure which

results from splicing of three exons (designated as E1a,

E1b, and E1c) to canonical exon 2. The designated

transcripts T1, T2, T3, and T4 contain 50 UTR exons as

E1c, E1a ? E1b, E1a, and E1b, respectively. The tran-

script T1 corresponded to earlier reported transcript in

GenBank. In silico study of genomic DNA sequence

revealed three distinct promoter regions namely, P1, P2,

and P3 upstream of the exons E1a, E1b, and E1c,

respectively. P1 is non-CpG-related promoter but P2 and

P3 are CpG-related promoters; however, all three are

TATA less. RT-PCR analysis demonstrated the expres-

sion of all four transcripts in late postnatal stages; how-

ever, these were differentially regulated in early postnatal

stages of 0.5 day, 3 day, and 15 day mice in different

tissue types. Variations in expression of Prkar1a gene

transcripts suggest their regulation from multiple pro-

moters that respond to a variety of signals arising in or

out of the cell in tissue and developmental stage-specific

manner.

Keywords Regulatory type 1 alpha subunit � Alternate

promoter � Differential expression � Transcription factors �Alternative splicing

Abbreviations

PKA Cyclic AMP-dependent protein kinase

R1a Regulatory type 1 alpha subunit

PN Postnatal

T Transcript

P Promoter

RT-PCR Reverse transcriptase polymerase chain

reaction

RACE Rapid amplification of cDNA ends

Introduction

Cyclic AMP-dependent protein kinase (PKA) is the

enzyme that phosphorylates the downstream molecules on

activation by secondary messenger called cAMP. This

phosphorylation plays a key regulatory role in different

cellular events like transcription, metabolism, cell cycle

progression, and apoptosis. The PKA holoenzymes contain

two catalytic (C) subunits bound to homo or heterodimers

of either regulatory type I (RI) or regulatory type II (RII)

subunit. There are four different R subunits (Ia,Ib and

IIa,IIb) and three different C subunits (Ca,Cb, and Cc),

each encoded by a separate gene. These holoenzymes and

individual R subunits show different biochemical proper-

ties and also play specific physiological functions.

Among all the subunits of PKA, RIa is studied with

immense importance in cancer cases. Increase in the

expression of RIa has been found to be associated with

A. R. Banday � S. Azim � M. Tabish (&)

Department of Biochemistry, Faculty of Life Sciences,

A.M. University, Aligarh, Uttar Pradesh 202002, India

e-mail: tabish.bcmlab@gmail.com

123

Mol Cell Biochem

DOI 10.1007/s11010-011-0897-z

neoplastic cell growth in case of cancers of breast [1],

ovary [2], lung [3], and colon [4]. Downregulation of RIaexpression with sequence-specific oligonucleotide results

in inhibition of growth and modulation of cAMP signaling

in cancer cells like LS-174T, HCT-15, and Colo-205 colon

carcinoma cells; A-549 lung carcinoma cells; LNCaP

prostate adenocarcinoma cells; Molt-4 leukemia cells; and

Jurkat T lymphoma cells. Also inhibition of RIa expression

has shown compensatory changes in expression of the

isoforms of R and C subunits and cAMP signaling in a cell

type-specific manner [5]. Recently, in case of cholangio-

carcinoma (CCA), abrogation of PRKAR1A gene (gene

symbol for human) expression showed significant CCA

cell growth inhibition, oncogenic signaling, and coupled

apoptosis induction, suggesting potential of this gene as a

drug target for CCA therapy [6]. In Carney complex,

mutations leading to the loss of function of one RIa allele

give a phenotype displaying variable defects in cellular

growth control [7]. In common variable immunodeficiency

(CVI) patients, hyperactivation of PKA type I contribute to

T cell dysfunction [8]. One of the studies has demonstrated

that isoform-specific antagonists of PKA regulatory sub-

units may be targeted for the novel drug discovery to be

used in many cancer types [9].

Therefore, the study of isoforms of PKA regulatory

subunits would be significant for designing of isoform-

specific drugs. Alternative splicing mechanism is one of the

main sources for generation of isoforms. For RIa subunit

gene, 11 splice variants in Drosophila [10, 11] and three in

human [12] have been studied as per records available in

GenBank. To analyze the isoforms resulting from alterna-

tive splicing, we have studied the mouse PKA RIa subunit

gene as mouse is the best model for correlating gene

structure and function in human. In GenBank single tran-

script was reported for mouse Prkar1a gene with accession

number NM_021880 [13]. However, partial sequences of 50

UTR exons have been previously described [14] which

were shown to be different by our methodology using 50

RACE kit and further supported by EST data. In this arti-

cle, we have demonstrated multiple transcripts that arise

due to alternative splicing and alternative promoter usage

in 50 UTR of Prkar1a gene. We detected three novel tran-

script variants of Prkar1a gene of mouse, which differ from

the previously reported transcript in first exon and studied

their expression profile in brain, heart, skeletal muscle, and

liver across different postnatal development stages. Three

promoter regions were also characterized. The results

provide evidence for a complex regulation of Prkar1a

expression from three promoters which showed consider-

able differences for transcription factor-binding sequence

motifs. We have also found that the transcripts of RIasubunit gene of human and mouse share less homology for

50 UTR exons, could be considered as different, but have

the same coding exons.

Materials and methods

Materials

Mice (C57BL/6J) were purchased from NII (National

Institute of Immunology), New Delhi, India and bred in-

house. All the animals were housed according to the

Institutional Animal Care and Use Committee and Guide-

lines of the Committee on Care and Use of Laboratory

Animal Resources, National Research Council (Depart-

ment of Health, Education and Welfare; National Institutes

of Health). The total RNA extraction kit was purchased

from iNtRON Biotechnology, Inc. (Gyeonggi-do, Korea).

M-MuLV-Reverse Transcriptase, PCR nucleotide mix,

1 kb DNA ladders were purchased from Fermentas Life

Sciences, USA. The TOPO-TA cloning kit II was obtained

from Invitrogen Corp. (Carlsbad, CA). The Plasmid DNA

miniprep kit and Qiaquick PCR gel purification kits were

purchased from Qiagen, Inc. (Santa Clarita, CA). The 50

RACE kit was purchased from Clontech (Palo Alto, CA).

Primers were custom synthesized from MWG Biotech, Pvt.

Ltd., India. All the other chemicals used in the experiments

were of molecular biology grade.

Preparation of RNA from different tissues of mouse

Total cellular RNA was isolated from mouse tissues like

brain, liver, skeletal muscle, and heart using RNA extrac-

tion kit according to manufacturer’s instructions. Finally,

the RNA was re-dissolved in diethyl pyrocarbonate-treated

water and quantitated spectrophotometrically. Integrity of

RNA was confirmed by ethidiumbromide staining after

denaturing agarose gel electrophoresis. RNA prepared was

either used immediately or stored at -80�C.

Primers

The following oligonucleotide primers were used:

PF1: 50-GCGACTCGGTCTTTAATCTTCCACTCTA

(from exon E1a)

PF2: 50-AGTCGCCCACCTGTCACCGAATCGT (from

exon E1b)

PF3: 50-CTATCGCAGAGTGGTAGTGAGGCT (from

exon E1c)

PR1: 50-CTCCTTGATCAATCACATAGAAGTTATCC

(from exon E6)

PR2: 50-AATGTCACTTCTCTCGTTATCATCAAGGT

(from exon E4/E5 junction)

Mol Cell Biochem

123

PR3: 50-AACAGCACATTCTTTTCGATGGCCTTGGC

(from exon E4)

50-Rapid amplification of cDNA ends (RACE)

50-RACE reactions were performed with 3 lg of total RNA

using a 50 RACE kit. The total RNA was annealed with

gene-specific reverse primer 50-AACAGCACATTCTTTT

CGATGGCCTTGGC-30 (PR3) according to the instruc-

tions provided with the kit. 50-RACE followed by PCR was

performed with cycling conditions as follows: after an

initial denaturation for 3 min at 94�C, 35 cycles at 94�C for

30 s, 62�C for 30 s, and 72�C for 30 s were performed,

followed by a final extension at 72�C for 7 min in an

Amplicator Block (Taurus Scientific, USA). The RACE

product was visualized on 1.5% agarose gels. Several

bands were excised from the gel, purified, and subcloned

into the TOPO-TA vector (a plasmid vector). E. coli

JM109-competent cells were transformed. Transformed

colonies were grown overnight at 37�C. Plasmid DNA was

purified using a plasmid purification kit. Plasmids con-

taining the insert were sequenced using an automatic

sequencer using either M13 forward or reverse primers.

Reverse transcriptase (RT)-PCR

To confirm the RACE data, we performed RT-PCR anal-

ysis in touchdown mode. Tissue-specific total RNA (2 lg)

was primed with gene-specific primer PR1 for the synthesis

of cDNA using M-MuLV-Reverse Transcriptase at 42�C

for 1 h in a total volume of 20 ll. Subsequently, 2 ll of the

single-stranded cDNA thus synthesized was amplified by

touchdown PCR with an appropriate set of primers using

PCR master mix in a total volume 50 ll. PCR was per-

formed for 35 cycles as denaturation at 94�C for 4 min, 1

cycle; 93�C, 30 s; annealing 65�C, 30 s with a decrease of

0.3�C per cycle, extension 72�C for 45 s, with final

extension at 72�C for 8 min. The PCR products obtained

were subjected to electrophoresis on a 1.5% (w/v) agarose

gel, stained with ethidium bromide, and photographed on a

UV transilluminator.

Semi-nested RT-PCR

To further validate the genuine PCR product, semi-nested

PCR was performed. After the RT reaction, as described

above, PCR amplification was carried out for 30 cycles (as

above) using gene-specific internal reverse primer PR2 and

first-exon-specific primers as down and upstream primers,

respectively. The resulting RT-PCR product (1 ll) was

used as a template for further amplification by PCR using

the same upstream primer but downstream primer from the

junction of exon-4 and exon-5. The resulting semi-nested

PCR product (8 ll) was then subjected to electrophoresis

on a 1.5% (w/v) agarose gel, stained with ethidium bro-

mide and photographed on a UV transilluminator.

Subcloning and sequencing

The PCR amplified products were electrophoresed on 1.5%

(w/v) agarose gels. Anticipated ethidium-bromide-stained

bands were cut from the gels and DNA was recovered

using a Qiaquick PCR gel purification kit. The purified

DNA was subcloned using the plasmid cloning vector. E.

coli JM109-competent cells were transformed. Trans-

formed colonies were grown overnight at 37�C and plas-

mid DNA was purified for sequencing. Plasmid containing

the insert was sequenced using an automatic sequencer

using either M13 forward or reverse primers.

Bioinformatics tools and databases used

Homology and similarity searches of the nucleotide

sequences were performed using the BLASTN non-red-

undant database (http://www.ncbi.nlm.nih.gov/BLAST).

Gene/Exon finding tools were used at HMMgene (http://

www.cbs.dtu.dk/services/HMMgene), Genescan (http://genes.

mit.edu/GENSCAN.html), GeneSplicer (http://cbcb.umd.

edu/software/GeneSplicer/), FGENESH (http://www.soft

berry.com/berry.phtml). Alignment analysis was carried

out using the Gene stream Align tool (http://www2.igh.cnrs.

fr/bin/align-guess.cgi) and ClustalW tool available at www.

ebi.ac.uk/clustalw. Promoter regions were characterized

using tools such as TFSEARCH (http://www.cbrc.jp/

research/db/TFSEARCH.html), TFBIND (http://tfbind.hgc.

jp/), SIGSCAN (http://www-bimas.cit.nih.gov/molbio/

signal/), CpG Island Searcher (http://www.uscnorris.com/

cpgislands2/cpg.aspx) and CpGPlot (http://www.ebi.ac.uk/

Tools/emboss/cpgplot/index.html). Expressed sequence tag

search was done at DNA bank of Japan (DDBJ) using

BLASTN program (http://blast.ddbj.nig.ac.jp/top-e.html).

Results

Four transcripts of mouse Prkar1a gene that differ

in the 50 UTR exons

The 50 UTR of mouse regulatory type 1 alpha gene is

complex having multiple upstream non-coding exons.

Using 50-RACE followed by cloning and subsequent

sequencing, we found four unique clones having the het-

erogeneous structure of non-coding exons present upstream

of first coding exon (designated as E2). Sequence analysis

indicated that all of these clones contain a common

Mol Cell Biochem

123

sequence from exon E2. Aligning these sequences with the

available genomic DNA sequence of the RIa gene down-

loaded from MGI database (MGI: 104878, Fig. 1a), con-

firmed that these sequences are part of the genomic DNA

of the RIa gene representing its 50 UTR. Three exons

designated as E1a, E1b, and E1c were located upstream of

first coding exon E2 according to the organization in DNA

sequence of RIa gene (Fig. 1a). The non-coding exon, E1c

corresponded to the non-coding exon of previously

described transcript available in pubmed with GenBank

accession no. NM_021880. Further analysis revealed that

the four clones are the result of alternate exons in the 50

UTR and presence of multiple transcription start sites.

However, one of the newly identified transcript contains

Fig. 1 Schematic diagram of genomic structure of the mouse Prkar1a

gene and mRNA transcripts expressed in adult mouse brain, heart,

liver, and skeletal muscle as determined by 50-RACE, RT-PCR and

sequencing. a The exon–intron organization on gene and position of

gene on chromosome 11. Boxes refer to exons (size of boxes indicate

relative sizes of exons) and interconnecting lines as introns. The 50

UTR of the Prkar1a gene consists of at least three different exon 1

variants: E1a, E1b, and E1c (size and genomic positions relative to

ATG at position ?1 are shown). The small starches of nucleotides

below indicate the nucleotides present at exon–intron boundaries.

b These exon 1 variants are spliced (alone or in combination) to a

common exon 2 splice acceptor region (position –6, relative to ATG

at position ?1). ORF is located in exon 2. c Four transcripts were

detected in brain, heart, liver, and skeletal muscle of mouse: T1, T2,

T3, and T4. In transcript isoform T2, exon E1a is spliced with exon

E1b and then to the common splice acceptor region of exon 2. d 50

RACE of mouse mRNA. 50 Rapid amplification of cDNA ends was

independently performed three times using RIa-subunit encoding

gene-specific internal primer and RNA isolated from mouse tissues.

The products were resolved on a 1.5% agarose gel and stained with

ethidium bromide. M indicates DNA size marker, ?RT indicates

reverse transcriptase was added, and -RT indicates no reverse

transcriptase was added to the reaction mixture. Excised products

were subcloned into TOPO-TA plasmid and sequenced. Same band

pattern was reproduced from all four tissues. e, f RT-PCR and semi-

nested PCR amplified products electrophoresed on 1.5% agarose gel

using exon-specific forward primers. Lane 1 indicates marker (I kb

ladder). Lanes 2, 3, and 4 represent amplified products obtained using

forward primers for exons E1a, E1b, and E1c, respectively. Second

lane shows two bands, above one represents transcript T2 and below

one transcript T3. Anticipated sizes of the products obtained in RT-

PCR are 796 bp and 612 bp (lane 2), 719 bp (lane 3), and 643 bp

(lane 4) using exon-specific forward primers PF1, PF2, and PF3,

respectively with reverse PR1. Internal reverse primer PR2 was used

for semi-nested PCR and bands of anticipated size were obtained.

Arrows indicate size of marker and corresponding product size. RT-

PCR products were subcloned in TOPO-TA plasmid and both strands

were sequenced using M13 forward and M13 reverse primers.

g Alignment of nucleotide sequence of four transcripts. The 50 UTR

exons are indicated with the name of exon. The accession numbers

are given at the end of each transcript variant. The sequence shown is

up to the sixth exon. Sequence from exon-2 onward is the same in all

the transcripts. Translational initiation codon is bold and underlinedin exon-2

Mol Cell Biochem

123

Fig. 1 continued

Mol Cell Biochem

123

two non-coding exons in which E1a is spliced with E1b

and then spliced to the common acceptor site in exon 2

(Fig. 1b, c). The new transcripts were named as T2, T3,

and T4 considering published transcript as T1 as seen in 50

RACE experiment (Fig. 1d).

In order to further validate the presence of these newly

identified exons with transcripts, RT-PCR was done using

forward primers specific to these 50 UTR exons and reverse

primers from downstream known exons to maximize the

length of transcript. Three bands (lanes 2 and 3 marked

with arrows) were obtained at indicated sizes using the

forward primers FP1 (corresponding to the exon E1a) and

FP2 (corresponding to the E1b), with reverse primer PR1

(Fig. 1e—lanes 2 and 3). As a control, we used the forward

primer from exon E1c (FP3) with the same reverse primer

PR1, and found a single band of the anticipated size

(Fig. 1e, lane 4). Two bands were obtained in lane 2 with

forward primer FP1 as expected, one corresponding to

transcript T2, and other corresponding to transcript T3

validating the presence of splicing event between exons

E1a and E1b. The new transcript T2 was the largest one

containing both E1a and E1b upstream exons. The semi-

nested PCR also proved the presence of these transcripts.

The expected products were obtained using another reverse

primer (PR2) internal to the previous reverse primer

(Fig. 1f). The presence of these new transcripts having

different exon structures at the 50 end was confirmed by

three independent RT-PCR experiments performed using

different RNA preparations, and by cloning and sequencing

the products. The cDNA sequences of these new transcripts

were submitted in the GenBank and the assigned acces-

sions numbers are T2 (HQ412666), T3 (HQ412667), and

T4 (HQ412668).

The two new exons E1a and E1b have TG/GT locus for

splicing donor sites indicating minor variation from con-

sensus splice donor site, like AG/GT (Fig. 1a). However,

the first two and last two bases in the intron between E1a

and E1b are GT and AG, respectively. Also, a splice

acceptor site at the 50 end of first coding exon is always

CAGA which indicated that the same protein product is

generated from all the three transcripts. This is also clear

from multiple sequence alignment of four transcripts which

showed 100% sequence homology from exon 2 (Fig. 1g).

Mouse Prkar1a gene is expressed from three distinct

promoter regions

Using an alternative approach of bioinformatics tools, three

different promoter regions were characterized in the 50

UTR of Prkar1a gene designated as P1, P2, and P3

(Fig. 2a). Earlier it has been established that promoter

regions of the RIb and RIIb subunit genes of PKA are

TATA less and G/C rich, and initiation of transcription

occurs at multiple sites [15, 16]. Using CpG island detec-

tion tools such as CpG Island Searcher and CpGPlot, we

found that no CpG island is present in or near upstream of

exon E1a; however, there is the presence of CpG islands in

and near upstream of exon E1b and E1c (Fig. 2b). These

CpG islands have an average GC content of about 65–70%

and observed/expected CpG ratio [60%. It is noteworthy

that all the three promoter regions are TATA less, but have

CAAT window upstream of transcription start site that

matches with conventional CAAT box. The position of

CAAT window varies upstream of transcription start site of

exons E1a, E1b, and E1c at positions -346, -414, and-

273, respectively. These findings supported the occurrence

of transcript T4 having first exon as E1b. An interesting

fact is that E1b exon has E-box consensus sequence of

CACGTG spanning from position ?40 to ?45 downstream

of transcription start site. This E-box may act either as an

enhancer or a silencer depending on the binding protein. It

is well established that Max/Myc dimer binding with E-box

activates transcription whereas the Max/Mad dimer sup-

presses transcription of these genes [17]. The transcription

from P2 promoter might also be regulated by Max/Myc

dimer.

In order to visualize the transcription regulation from

promoter regions, in silico analysis was performed by using

tools like TFSEARCH, TFBIND, and SIGSCAN for find-

ing transcription factor-binding consensus sequence motifs

(Table 1). Analysis has identified a complex array of rec-

ognition sequences that are involved in different cellular

processes while responding to variety of signals in and out

of a cell. The most important observation was that all three

promoters have some binding sites in common, e.g., for

Sp1 (steroidogenic factor 1) which is expressed in all

mammalian cells and is involved in regulating the tran-

scriptional activity of genes implicated in most cellular

processes [18, 19], CREB (cAMP response element-bind-

ing) which decreases or increases transcription [20], GR

(glucocorticoid receptor) responds to cortisol, and other

glucocorticoids [21], LF-A1 (Liver factor A1) binds pro-

moter regions of several liver-specific genes [22], NF-I

(Nuclear factor I) recognizes the sequence CAAT box and

is implicated in eukaryotic transcription, as well as DNA

replication [23], etc. However, there are different binding

sites for different transcription factors specific to each

promoter, e.g., AP-2 (Activator protein-2), which is known

to have its roles in vertebrate development, apoptosis, and

cell-cycle control [24], can bind to promoter P3; TFIID

(Transcription factor II D), is one of several general

transcription factors that make up the RNA polymerase II

pre-initiation complex [25], can bind to promoter P1; and

F2F (Footprint II-binding factor) is believed to be a

Mol Cell Biochem

123

transcriptional repressor [26] can bind to promoter P2.

These observations clearly describe the diverse regulations

of Prkar1a gene transcription.

Differential expression of Prkar1a gene in mouse heart,

brain, skeletal muscle, and liver

To give some insights into the regulatory behavior of

alternate promoters, the expression patterns of the alter-

native transcript isoforms were analyzed in four major

organs (heart, brain, skeletal muscle, and liver) by RT-PCR

at different postnatal development stages of mouse like

0.5 day, 3 day, 15 day, and 60 day named as PN0.5, PN3,

PN15, and PN60, respectively. The identity of the resulting

amplicons was confirmed by DNA sequencing (not shown).

Only few tissues from early and late postnatal stages were

studied because it was beyond the scope of the study pre-

sented here to study all tissues at different developmental

stages of mouse. The RT-PCR analysis showed ubiquitous

pattern across these organs in adult stages of development.

However, earlier stages of postnatal development showed

variation in their expression patterns (Fig. 3). Under our

experimental conditions, at PN60 all four transcripts were

amplified from the RNAs isolated from heart, brain, skel-

etal muscle, and liver. The transcript T3 showed the most

apparent expression at all postnatal stages which may be

because its transcription was under the promoter P1 that

possesses binding site for transcription factor TFIID. Dur-

ing the developmental stages, the transcript T1 was found

to be very weakly expressed at PN0.5 and PN3 stages;

however, in brain and liver at stage PN15, it showed rel-

atively higher expression. In adult, at stage PN60, all four

tissues examined, i.e., heart, brain, skeletal muscle, and

liver, showed higher expression of transcript T1. Interest-

ingly, at early postnatal stages, transcript T4 was amplified

from muscle tissue only. The transcript T2 was not

detected from early postnatal stages. However, both T2 and

T4 transcripts were detected at late postnatal stage PN60 in

all four tissue types examined (Fig. 3). This indicated that

the splicing event between exons E1a and E1b occurs

mostly in adult mice, the consequence of which is

unknown. The absence of CpG Islands in promoter P1 may

be the reason behind the ubiquitous type of expression

patterns of transcripts expressed from this promoter. In

Fig. 2 a Schematic

representation of promoters

upstream of 50 exons.

Transcription start sites (TSS)

are depicted as ?1. All three

promoters have CAAT window

located at position -346,-414

and -273 upstream of TSS of

exons E1a, E1b and E1c,

respectively. Hanging linesshow E box sequence in exon

E1b spanning from ?40 to ?45

downstream of TSS.

b CPGPLOT generated by CpG

Plot tool available at EBI used

for CpG Island prediction.

Query sequence of 2021 bp was

used as input covering genomic

nucleotide sequence from

promoter P1 to exon E1c.

Double headed arrows show

relative position of promoter

regions. CpG Islands are present

in regions of promoter P2 and

P3 with observed/expected

ratio [0.60 and G?C

percentage [55.00

Mol Cell Biochem

123

order to further identify tissue and developmental stage-

specific expression, ESTs were searched for all four tran-

script isoforms using DDBJ blast program. We retrieved

several ESTs that showed considerable match with these

transcript isoforms. The accession numbers of ESTs, along

with their presence in tissue types and respective devel-

opmental stages are given in Table 2. The EST data sup-

ported our observations of differential and ubiquitous type

of Prkar1a gene expression in temporal manner. However,

due to lack of EST coverage from all tissues, it was not

conclusive to correlate data comparatively. No EST was

found against transcript T3 belonging to embryonic

developmental stages which may be due to the absence of

or very rare expression of T3 transcript during embryonic

development. In brain, the role of R1a subunit might be

more diverse and complex because all four transcripts were

expressed in adult mice brain tissue types (visual cortex,

cerebellum, lateral ventricle wall, corpora quadrigemina,

etc.). One of the most important observations from EST

data was that the splicing event between exon E1a and E1b

occurs not only in adult mice, but also in embryo. Among

all four transcripts, a large number of ESTs with many

tissue types were retrieved for transcript T1 which clearly

indicated its abundant expression. These facts evidently

suggest complex regulation of transcription of Prkar1a

from multiple promoter regions that we envisaged during

in silico analysis of these promoter regions.

Discussion

Our results confirmed the presence of four RIa transcript

variants in different tissues of adult mouse like brain, liver,

heart and skeletal muscle. These transcripts of RIa gene

arise due to alternate promoter usage and alternative RNA

splicing of three distinct non coding exons in 50 UTR. The

consensus sequences at the splice-donor/acceptor site are

believed to be the structural features that determine whe-

ther a specific splice site is used although these consensus

sequences may not be always present [27]. Analysis of the

exon–intron junction region of all new exons indicated

structural conservation however, splice donor site varies

slightly from consensus where A is replaced by T (TG/GT

instead of AG/GT). The presence of more than 75% con-

sensus sequences in the splice-donor/acceptor site provides

evidence for the described splicing events. Thus, our data

indicated that there are four possible transcript variants for

mouse Prkar1a gene including one reported earlier, which

are expressed in heart, liver, brain, and skeletal muscle.

Interestingly, all four transcript variants differ only in the

Fig. 3 Relative RT-PCR products of mouse heart, brain, liver, and

skeletal muscle mRNA at postnatal stages PN0.5, PN3, PN15, and

PN60. Primers used were same as given in ‘‘Methods’’ section with

forward primer exon specific and same reverse primer from internal

exon. 1.5% agarose gels in row represent tissue and, in column,

represent postnatal stage. Each lane across column indicates same

RT-PCR product. Names of exon and respective transcript (given in

curved bracket) amplified are shown above each lane. At PN60 [lane

E1a, E1a ? E1b (T3, T2)] two products representing transcripts T2

and T3 are amplified using forward primer PF1 (from exon E1a).

However, at PN0.5, PN3, and PN15, only one band appeared that

represent transcript T3. Few lanes have unexpected extra bands that

are due to non-specific binding of primers. Marker (1 kb DNA ladder)

was always run to correlate the amplified product size (not shown

here)

Mol Cell Biochem

123

non-coding exons. In all variants, coding exons are present

unchanged encoding identical proteins. The 50 RACE and

bioinformatics analysis has revealed multiple transcription

sites controlled by distinct promoter regions. The initiation

of transcription at several sites, generating transcripts dif-

fering in their 50 UTR could render the expression of this

important gene less vulnerable to mutations which could

modify its expression. Also the sequence composition in

the upstream region for promoters were found TATA less

and G/C rich, which is in accordance with the promoter

regions of the genes for mouse RIb, and mouse/rat RIIb as

described previously [15, 16]. However, all three promot-

ers have varying GC content ratio making P1 as non-CpG-

related promoter while P2 and P3 as CpG-related pro-

moters. The presence of CpG islands may play an impor-

tant role in gene regulation, for example, in causing gene

silencing or transcriptional repression by hypermethylation

of the CpG Island [28, 29]. It is noteworthy that in silico

study revealed characteristic signature sequences in all the

three promoter regions which are potential binding sites for

different transcription factors. This supports the existence

of multiple promoters for Prkar1a gene. The existence of

multiple promoters may be due to the need for the

expression of this gene in many but not all cell types and/or

at different times during embryogenesis. The individual

promoters can be regulated independently so that the gene

can be transcribed in different and overlapping cell types or

in response to different signaling systems. In silico analysis

of the promoter elements together with expression profiling

have shown diagnostic differences; however, in vitro

analysis is needed to sort out and establish consensus over

the significance of these differences. Another consequence

of using different promoters is the addition of a different 50

UTR sequence to the mRNA. The sequences present in

E1a, E1b, and E1c could also potentially play a very

selective role in translational regulation of Prkar1a gene

and/or may contain recognition sites for RNA binding

proteins involved in mRNA translocation, degradation, or

stability [30]. Particularly, the transcript T2 that contains

two 50 exons spliced to each other could play role in post-

transcriptional regulation of translation, functions that rely

on a combination of primary and secondary structures of

the RNA, such as containing a susceptible site for miRNA

or hairpin formation [31].

RIa is the only regulatory subunit which is crucial for

the progression of embryogenesis in the mouse [32]. We

have examined the expression profile of these transcripts at

different postnatal development stages. The results showed

variation in expression of these transcripts across tissues at

different postnatal development stages. For example, the

transcript T3 containing exon E1a was almost expressed

ubiquitously at all postnatal development stages. However,

Table 1 Comparative in silico

prediction of different

transcription factor binding sites

of promoter regions and their

position relative to transcription

start sites using TFSEARCH,

TFBIND, SIGSCAN tools

Signs (-,?) indicates position

upstream or downstream of

transcription start site

Transcription factor Target sequence P1 P2 P3

AP-2 TGGGGA -92

ATF TGACGT -9

CAC-binding-pro GGTGG -71,-378 -95,?33

EBP45 GTTTGCTTT -198

GATA-1 TTATCT -256,-82

GATA-2 AGATAA -193

CREB TGACG -189 -378 -148,-10

F2F TAAAAT -527

GR CAGAG -229,?83 -25,?19

GR TGTCCC -519

GR GACACA -296

H4TF-1 GATTTC -428,-207

LF-A1 GGGCA -237,-29 -350 -177

MEP-1 TGCGCTC -103

NF-1 GCCA -46

NF-1 TCCA -284 ?278

NF-1/L TGGCA -108, -386

NF-E CTGTC -303 -394,?23

PEA3 AGGAAA -148 -236

Sp1 CCGCCC -41 -34

Sp1 GGGCGG -236

TFIID TTCAAA -364

Mol Cell Biochem

123

transcripts like T1, T2, and T4 showed differential

expression where as T2 was heavily expressed in adult

mice (Fig. 3). In later stages, expression of all four tran-

scripts was ubiquitous that appeared unchanged. These

results suggest developmental stage-specific expression of

Prkar1a gene. Expressed sequence tags matching these

transcripts also supported our RT-PCR analysis. However,

EST data revealed that transcript T2 is also expressed in

embryo and that transcript T1 is abundantly present at

different embryonic developmental stages. These findings

Table 2 Accession numbers,

tissues and developmental

stages of expressed sequence

tags matching transcript variants

Transcripts EST Accession No. Tissues (cell types) Developmental stages

T1 CX238268 Lateral ventricle wall Adult

CN676072 Embryonic stem (ES) cell Embryo

BY350563 Whole joints Adult

BY325193 Synovial fibroblasts Adult

BY295910 Visual cortex Adult

BY171043 Bone marrow macrophage Adult

BY099397 Kidney Adult

BU922352 Retina 14.5 days embryo

BE850859 Mammary gland Adult

BB665656 Oviduct 2 days pregnant adult female

BB654792 Whole embryo 9 days

CJ182565 Rathke’s pouches 14.5 days embryo

CA894890 Neural stem cell Differentiated

BY782095 Whole body 17.5 days embryo

BY747531 Thymus thymic cells 2 days neonate

BY736666 Blastocyst Embryo

BY726571 Corpora quadrigemina Adult

BY705244 Liver Adult

BB610002 Lung Adult

BY350231 Whole joints Adult

BY333602 Synovial fibroblasts Adult

CN663942 Whole embryo 13.5 days embryo

CA540754 Whole Embryo 7.5 days embryo

BY229339 Olfactory sensory neurons Adult

BY064378 Kidney 17 days embryo

BB850806 Inner ear Adult

BB864160 B lymphocyte (B cells) Adult

BY036616 Liver 14 days embryo

CF167794 Germ Cell Embryonic

T2 BB610165 Cerebellum Adult

BY132531 Whole body 17.5 days embryo

BY153772 Pancreas islet cells Adult

BB567873 Head 12 days embryo

T3 DN174114 Lateral Ventricle Wall Adult

BY129491 Brain Adult

BB610165 Cerebellum Adult

T4 BU709493 Whole brain Develop. stage: embryo 15.5

CB194067 Embryonic limb, maxilla

and mandible (pooled)

Embryo day 12.5, 13.5,

14.5, and 15.5

BI657582 Tumor, gross tissue 5 months

BY267453 Visual cortex Adult

BY026492 Mammary gland Adult

BY153772 Pancreas islet cells Adult

Mol Cell Biochem

123

further support the importance of RIa gene for progression

of embryogenesis in mouse.

In human RIa, gene initiation at different transcription

start sites lead to three distinct, alternatively spliced

transcripts coding for identical proteins [12]. Multiple

sequence alignment of human and mouse R1a gene tran-

scripts showed that 50 UTR exons of mouse and human are

different and share less homology (Fig. 4). However, all of

these transcripts have almost 100% homology from exon 2.

This observation indicated two possibilities that either 50

UTR of human and mouse R1a gene are less conserved in

sequence or that the 50 UTR exons that we reported here

have not been identified in human and vice versa. The pair-

wise alignment of human R1a 50 UTR exons with genomic

DNA of mouse and that of mouse R1a 50 UTR exons with

genomic DNA of human using NCBI BLAST program

showed no significant match at exon level. This fact

excluded the possibility of unreported transcripts in human

of the same type of sequence that we found in mouse. Also,

the 50 UTR of human and mouse R1a gene showed 68%

homology at nucleotide level which indicates less conser-

vation of nucleotide sequence. It was also found that no

splicing event is reported between human R1a 50 UTR

exons similar to that of mouse where such an event pro-

duces transcript T2.

These data suggest complex regulation of R1a subunit

gene transcription in mouse and human to produce the

same protein product. The study of transcripts belonging to

mouse and human R1a gene will be helpful while inves-

tigation of diseases and other functional aspects in mouse

as model, in particular to those cases which pertain to

regulation of this gene. This study seeks to understand the

mechanism and significance of splicing event that gives

rise to transcript T2 in mouse and also whether such

splicing event is present in human R1a subunit gene.

Acknowledgments The authors are thankful to the Department of

Biotechnology (DBT), the Ministry of Science and Technology, New

Delhi, India for providing generous funding to project No. BT/PR-

10917/BID/07/254/2008. The necessary facility provided by A.M.U.,

Aligarh, India is also thankfully acknowledged.

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1 23

Molecular Biology ReportsAn International Journal onMolecular and Cellular Biology ISSN 0301-4851 Mol Biol RepDOI 10.1007/s11033-011-1108-4

Differentially expressed three non-codingalternate exons at 5# UTR of regulatorytype I beta subunit gene of mouse

Abdul Rouf Banday, Shafquat Azim &Mohammad Tabish

1 23

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Differentially expressed three non-coding alternate exonsat 50 UTR of regulatory type I beta subunit gene of mouse

Abdul Rouf Banday • Shafquat Azim •

Mohammad Tabish

Received: 23 May 2011 / Accepted: 17 June 2011

� Springer Science+Business Media B.V. 2011

Abstract Prkar1b gene encodes regulatory type I, beta

subunit (RIb) of cAMP dependent protein kinase A in

mouse. Among the various isoforms of regulatory and

catalytic subunits that comprise mammalian PKA, RIbsubunit is considered to be one of the important subunits

for neuronal functions. This is involved in multiple forms

of synaptic plasticity, and influences memory and learning

by maintaining hippocampal long-term potentiation (LTP).

Deficient expression of this gene has been implicated in

autoimmune disease systemic lupus erythematosus (SLE).

We have identified two novel non-coding exons of the

Prkar1b gene (designated as exon 1A and exon 1B), which

are spliced to the canonical exon 2 and constitute the 50

untranslated region giving rise to three alternative tran-

script isoforms. We have also confirmed the expression of

the previously known first exon (designated as exon 1C)

with known transcript published earlier. The transcripts

containing exons 1A, 1B and 1C are differentially regu-

lated during the development and tissue types. In silico

study of more than 20 kb nucleotide sequence upstream of

known translational initiation codon revealed three distinct

promoter regions named as PA, PB, and PC upstream of

the exon 1A, exon 1B and exon 1C respectively. PB is non-

CpG related promoter but PA and PC are CpG related

promoters, however all three promoters are TATA less.

Further analysis showed that these promoters possess

potential signature sequences for common as well as dif-

ferent transcription factors suggesting complex regulation

of Prkar1b gene.

Keywords Alternative splicing � Alternate promoter �Differential expression � Exon � Regulatory type 1 beta

subunit � Transcription factors � RT-PCR

Introduction

In eukaryotic cells, cAMP regulates many different cellular

functions. Its effects are in most cases mediated by cAMP-

dependent protein kinase A (PKA). PKA enzyme is tetra-

mer that contains two catalytic (C) subunits bound to

homo- or heterodimers of either regulatory type I (RI) or

regulatory type II (RII) subunit. At least four regulatory

(RIa, RIb, RIIa, and RIIb) and three catalytic (Ca, Cb and

Cc) subunits have been characterized in mammals. The

activation of PKA is controlled by the regulatory (R) sub-

units of the holoenzyme. Different anatomical distribution

of the regulatory isoforms may contribute to determine the

specificity of diverse effects of cAMP [1].

Signaling events mediated by PKA are critical for many

neuronal functions [2]. PKA is involved in multiple forms

of synaptic plasticity [3–5], synaptic transmission, memory

and learning [6]. Among the various isoforms of regulatory

and catalytic subunits that comprise mammalian PKA,

the regulatory type I beta (RIb) subunit is considered to be

one of the important subunits for neuronal functions. RIbsubunit has been found to be important for mossy fiber

long-term potentiation (LTP) [7], hippocampal long-term

depression and depotentiation [8, 9], inflammation and

sensitization of primary afferent nociceptors [10]. In

autoimmune disease systemic lupus erythematosus (SLE)

25% of subjects show no detectable RIb protein in their T

cells [11]. One of the studies has demonstrated that isoform

specific antagonists of PKA R subunits may be targeted for

the novel drug discovery to be used in many cancer types

A. R. Banday � S. Azim � M. Tabish (&)

Department of Biochemistry, Faculty of Life Sciences,

A.M. University, Aligarh, UP 202002, India

e-mail: tabish.bcmlab@gmail.com

123

Mol Biol Rep

DOI 10.1007/s11033-011-1108-4

Author's personal copy

[12]. Alternative splicing generates variants that are tissue

or developmental stage specific and can be differentially

affected by pharmacological agents to either up regulate or

down regulate the expression as per the requirements of

disease condition [13]. But this is possible only when

spliced variants are known for any given gene. Owing to

this we proposed that there could be alternatively spliced

variants in case of mouse RIb gene which can become

therapeutic targets.

The PKA RIb subunit encoding gene named Prkar1b is

present on chromosome 5 in mouse. To identify the pos-

sible spliced variants arising from RIb subunit encoding

gene, we have studied the mouse Prkar1b gene as mouse is

the best model for correlating gene structure and function

with human. In GenBank, single transcript was reported for

mouse Prkar1b gene. Previously, the promoter region of

mouse Prkar1b gene has been described as TATA-less

flanked by 1.5 kilobases of 50-upstream sequence and 1.8-

kilobase intron [14]. Here, we have demonstrated three

transcripts that arise due to alternative splicing in pre-

mRNA and alternative promoter usage in 50 UTR of

Prkar1b gene. We have also located two more promoter

regions which lead to the expression of three novel tran-

script variants of Prkar1b gene. These variants differ from

the previously reported transcript with respect to first exon.

One of the new exons was found to be part of previously

described promoter (PDP) region [14]. We have also

studied their expression pattern in brain, heart, skeletal

muscle and liver across different postnatal development

stages.

Materials and methods

Materials

Mice (C57BL/6 J) were obtained from NII (National

Institute of Immunology), New Delhi, India and bred in

house. All animals were housed according to the Institu-

tional Animal Care and Use Committee and Guidelines of

the Committee on Care and Use of Laboratory Animal

Resources, National Research Council (Department of

Health, Education and Welfare; National Institutes of

Health). The total RNA extraction kit was purchased from

iNtRON Biotechnology, Inc. (Gyeonggi-do, Korea).

M-MuLV-Reverse Transcriptase, PCR nucleotide mix,

1 kb DNA ladders were purchased from Fermentas Life

Sciences, USA. The TOPO-TA cloning kit was obtained

from Invitrogen Corp. (Carlsbad, CA). The Plasmid DNA

miniprep kit and Qiaquick PCR gel purification kits were

purchased from Qiagen, Inc. (Santa Clarita, CA). The 50

RACE kit was purchased from Clontech (Palo Alto, CA).

Primers were custom synthesized from Sigma-Aldrich

chemicals Pvt. Ltd. (Bangalore, India). All other chemicals

used in the experiments were of molecular biology grade.

Preparation of RNA from different tissues of mouse

Total cellular RNA was isolated from various mouse tis-

sues using RNA extraction kit according to manufacturer’s

instructions. Finally, the RNA was re-dissolved in diethyl

pyrocarbonate-treated water and quantitated spectrophoto-

metrically. Integrity of RNA was confirmed by ethi-

diumbromide staining after denaturing agarose gel

electrophoresis. RNA prepared was either used immedi-

ately or stored at -80�C.

Primers

Primer positions are indicated in parenthesis. The follow-

ing oligonucleotide primers were used:

PF1: 50-CGAA CCT TGA GGG CGA GGC AGA AGA

(from exon 1A)

PF2: 50-ATGGAC TCT GTT TCC ACA GGA GCA

GAT G (from exon 1B)

PF3: 50-CTA AGA GAA GCA AGT GTA ATT GGC

TAG CG (from exon 1C)

PR1: 50-CAC ATA TAC ATC TAC TTC TCC TTG

GTC (from junction of exons 6 and 7)

PR2: 50-ATG TCA CTT CTC TCG TTG TCG TCC

AGG (from junction of exons 4 and 5)

PR3: 50-GTC ATG GTC TTA TAG TCC TTG GGA

ATG (from exon 4)

50 Rapid amplification of cDNA ends (RACE)

50 RACE reactions were performed with 3 lg of total RNA

isolated from mouse brain using a 50 RACE kit. The total

RNA was annealed with gene-specific reverse primer PR3

according to the instructions provided with the kit. 50

RACE products were PCR amplified with cycling condi-

tions as follows: after an initial denaturation for 3 min at

94�C, 30 cycles were performed with denaturation at 94�C

for 30 s, annealing at 66�C for 30 s, extension at 72�C for

30 s followed by a final extension at 72�C for 8 min in an

Amplicator Block (Taurus Scientific, USA). The RACE

product was visualized on 1.3% agarose gels. Several

bands were excised from the gel, purified and subcloned

into the TOPO vector. E. coli JM109-competent cells were

transformed. Transformed colonies were grown overnight

at 37�C. Plasmid DNA was purified using a plasmid puri-

fication kit. Plasmids containing the insert were sequenced

using an automatic sequencer using either M13 forward or

reverse primers.

Mol Biol Rep

123

Author's personal copy

Reverse transcriptase (RT)-PCR

To confirm the RACE data, we performed RT–PCR anal-

ysis in touch down mode. Tissue specific total RNA (2 lg)

was primed with oligo-dT for the synthesis of cDNA using

M-MuLV-Reverse Transcriptase at 42�C for 1 h in a total

volume of 20 ll. Subsequently, 2 ll of the single-stranded

cDNA thus synthesized was amplified by touchdown PCR

with an appropriate set of primers using the PCR master

mix in a total volume of 50 ll. PCR was performed for 30

cycles as; initial denaturation at 94�C for 4 min followed

by denaturation 93�C, 30 s; annealing 70�C, 30 s with a

decrease of 0.3�C per cycle, extension 72�C for 45 s, with

final extension at 72�C for 8 min. The PCR products

obtained were subjected to electrophoresis on a 1.3% (w/v)

agarose gel, stained with ethidium bromide and photo-

graphed on a UV transilluminator.

Semi-nested RT-PCR

To further validate the genuine PCR product, semi-nested

PCR was performed. After the RT reaction, as described

above, PCR amplification was carried out for 30 cycles (as

above) using gene-specific reverse primer (PR1) and first-

exon-specific primers as down- and upstream primers,

respectively. The resulting RT-PCR product (1 ll) was

used as a template for further amplification by PCR for 30

cycles using the same upstream primer but downstream

primer from internal exon (PR2). The resulting semi-nested

PCR product (8 ll) was then subjected to electrophoresis

on a 1.3% (w/v) agarose gel, stained with ethidium bro-

mide and photographed on a UV transilluminator.

Subcloning and sequencing

The PCR amplified products were electrophoresed on 1. 3%

(w/v) agarose gels. Anticipated ethidium-bromide-stained

bands were cut from the gels and DNA was recovered using

a Qiaquick PCR gel purification kit. The purified DNA was

either directly sequenced or subcloned into TOPO vector. E.

coli JM109-competent cells were transformed. Transformed

colonies were grown overnight at 37�C and plasmid DNA

was purified for sequencing. Plasmid containing the insert

was sequenced using an automatic sequencer using either

M13 forward or reverse primers.

Computational analysis

Homology and similarity searches of the nucleotide

sequences were performed using the BLASTN nonredun-

dant database (http://www.ncbi.nlm.nih.gov/BLAST). Gene/

Exon finding tools were used at HMMgene (http://www.cbs.

dtu.dk/services/HMMgene), Genescan (http://genes.mit.edu/

GENSCAN.html), GeneSplicer (http://cbcb.umd.edu/software/

GeneSplicer/), FGENESH (http://www.softberry.com/berry.

phtml). Alignment analysis was carried out using the Gene

stream Align tool (http://www2.igh.cnrs.fr/bin/align-guess.cgi)

and ClustalW tool available at http://www.ebi.ac.uk/clustalw.

Promoter regions were characterized using tools such as

TFSEARCH (http://www.cbrc.jp/research/db/TFSEARCH.

html), TFBIND (http://tfbind.hgc.jp/), SIGSCAN (http://

www-bimas.cit.nih.gov/molbio/signal/), CpG Island

Searcher (http://www.uscnorris.com/cpgislands2/cpg.aspx)

and CpGPlot (http://www.ebi.ac.uk/Tools/emboss/cpgplot/

index.html).

Results and discussion

Diversity at 50 end of mouse Prkar1b transcripts

It has been reported that mouse Prkar1b consists of 11

exons and 10 introns [14]. The structure of this gene

appears obscure at 50 end as there is large segment of DNA

flanking upstream of known transcription start site not

belonging to any gene. This fact prompted us to analyze the

50 transcriptional regulatory region of this gene. To this end

we sought to define the 50 end of Prkar1b encoding tran-

script by applying the 50 RACE as described in material

and method section. Using 50 RACE followed by cloning

and subsequent sequencing, we found three unique clones

having the heterogeneous structure at the 50 end. Sequence

analysis indicated that 50 region of Prkar1b transcripts is

diverse but all of the transcripts contain a common

sequence from exon 2. Aligning these sequences with the

available genomic DNA sequence of the Prkar1b gene

along with 20 kb flanking sequence of nucleotides

upstream of known transcription site downloaded from

MGI database (MGI: 104878, Fig. 1), confirmed that these

sequences are part of the genomic DNA of the Prkar1b

gene representing its 50 UTR. Three non-coding exons

appearing as variants of exon 1 designated as exon 1A,

exon 1B and exon 1C were located upstream of first coding

exon (exon 2) according to the organization in DNA

sequence of Prkar1b gene (Fig. 1). The non-coding exon

1C corresponded to the non-coding exon of previously

described transcript available in pubmed with GenBank

accession number NM_008923 [14]. The new transcripts

were named as Mr1b-A and Mr1b-B considering published

transcript as Mr1b-C according to the presence of 50 exon 1

variant. The translational initiation codon ‘methionine’ lies

in exon 2 in case of all three transcript variants which are

depicted in Fig. 1. The number of nucleotides in 50 UTR of

each transcript varies from 106 bp to 217 bp as shown in

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Fig. 1. The initiation of transcription at several sites, gen-

erating transcripts differing in their 50 UTR could render

the expression of this important gene less vulnerable to

mutations which could modify its expression.

In order to further validate the presence of newly

identified exons with transcripts by 50 RACE (Fig. 2a); RT-

PCR was done using forward primers specific to newly

identified exons from 50 UTR and reverse primers from

downstream known coding exons to maximize the length

of transcripts. As shown in Fig. 2b, using common reverse

primer PR1, two bands were amplified from forward pri-

mer PF2 (lane 2) and one band was amplified from forward

primer PF1 (lane 3). As a positive control, we used the

forward primer PF3 from the 1st exon published earlier

[14] with same reverse primer, and found a single band of

the anticipated 718 bp size (Fig. 2b, lane 1). One band in

lane 2 was of expected size (770 bp) as anticipated from

the result of 50 RACE however, a second band appeared

below showing a smaller variant. Sequencing the second

band revealed that a product of 688 bp size was amplified

because one more transcript variant existed in which a part

of exon 1B was skipped off. This transcript variant was

named as Mr1b-B’ as being a variant of transcript Mr1b-B.

In order to remove PCR artifacts, we performed semi-

nested PCR as described in materials and methods section.

The semi-nested PCR reaction also resulted in the ampli-

fication of expected size products that were obtained by

using another reverse primer (PR2) internal to the previous

reverse primer PR1 (Fig. 2c). The presence of these new

transcripts having different exon structure at the 50 end was

confirmed by three independent RT-PCR experiments

performed using different RNA preparations, and by

cloning and sequencing the products. The sequences of

these new transcripts were submitted to the GenBank and

were assigned accessions numbers: Mr1b-A (GenBank

accession no. JF720023), Mr1b-B (GenBank accession no.

JF720024) and Mr1b-B’ (GenBank accession no.

JF720025). Multiple sequence alignments of all four tran-

scripts of Prkar1b gene including one published earlier [14]

showed 100% sequence homology from exon 2 (Fig. 2d).

The transcripts of Prkar1b gene arise due to alternate

promoter usage and alternative RNA splicing of three

distinct non coding exons in 50 UTR. The nucleotide

sequences present in exon 1A, exon 1B and exon 1C could

also potentially play a very selective role in translational

regulation of Prkar1b gene and/or may contain recognition

Fig. 1 Schematic diagram of genomic structure of the mouse Prkar1b

gene and mRNA transcripts as determined by 50 RACE, RT-PCR and

sequencing. The exon–intron organization on gene and position of

gene on chromosome 5. Boxes refer to exons (size of boxes indicate

relative size of exons) and interconnecting lines as introns. The

sequence of Prkar1b gene in GenBank runs from 139493258 bp to

139605706 bp on chromosome 5 whereas we have identified the

sequence runs further upstream to 139473009 bp. The 50 UTR of the

Prkar1b gene consists of at least 3 different variants of first exon

namely exon 1A, exon 1B and exon 1C (size and genomic positions

relative to ATG at position ?1 are shown). PA, PB and PC are

promoter regions upstream of exon 1A, exon 1B and exon 1C

respectively. Each of the first exon could splice to a common exon 2

splice acceptor region (position –21, relative to ATG at position ?1)

generating multiple transcripts. Translational initiation codon ‘ATG’

is located in exon 2. Four transcripts were detected in brain namely

Mr1b-A, Mr1b-B, Mr1b-B’ and Mr1b-C. At the 30 end of exon 1B a

sequence of 82 nucleotides was skipped off which resulted in a

shorter variant Mr1b-B’. Previously described promoter (PDP) is

shown in dotted box. The region upstream of position -3505 was

newly identified part of Prkar1b gene in mouse

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sites for RNA binding proteins involved in mRNA trans-

location, degradation or stability [15].

Analysis of the exon–intron junction region of exon 1A

and exon 1B for splicing donor–acceptor sites indicated the

presence of conserved consensus sequences (Fig. 3). The

two new exons 1A and 1B have consensus AG/GT locus

for splicing donor sites (Fig. 3). Also, a splice acceptor site

at the 50 end of first coding exon is always GGAG which

indicated that the same protein product could be generated

from all the three transcripts. The presence of consensus

Fig. 2 50 RACE, RT–PCR and semi-nested PCR. a 50 Rapid

amplification of cDNA ends was independently performed three

times using Prkar1b gene-specific internal primer (PR3) and RNA

isolated from mouse brain. The products were resolved on 1.3%

agarose gel and stained with ethidium bromide. M indicates DNA size

marker, ?RT indicates reverse transcriptase was added and -RT

indicates no reverse transcriptase was added to the reaction mixture.

Excised products were subcloned into TOPO-TA plasmid and

sequenced. b RT-PCR amplified products were electrophoresed on

1.3% agarose gel using exon specific forward primers. These

amplified products were obtained by using forward primers for exon

1A, exon 1B and exon 1C respectively. Two bands were obtained

with forward primer FP2 (exon 1B, lane 2), upper one represented

transcript Mr1b-B and lower one represented transcript Mr1b-B’.

Anticipated sizes of the products obtained in RT-PCR are 635 bp

(lane 3), 770 bp and 688 bp (lane 2) and 718 bp (lane 1) using exon

specific forward primers PF1, PF2 and PF3 respectively with reverse

primer PR1. Second band in lane 2 appears very weak. In lane 4, 1 kb

DNA ladder was run as marker. c Semi-nested PCR analysis was

performed as described above. Using internal reverse primer PR2 all

four bands were obtained with anticipated size. Arrows indicate size

of marker and corresponding product size. RT-PCR products were

subcloned in TOPO-TA plasmid and both strands were sequenced

using M13 forward and M13 reverse primers. d Jalview of Multiple

sequence alignment of partial sequence of four transcripts. The

sequence from exon 2 at position 215 bp onwards is same in all the

transcripts

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sequences in the splice-donor/acceptor site provides evi-

dence for the described splicing events. This is also clear

from multiple sequence alignment of four transcripts which

showed 100% sequence homology from exon 2 (Fig. 2d).

Three distinct promoter regions and expression

of mouse Prkar1b gene

Earlier it was found that regulation of mouse Prkar1b gene

is controlled by a sequence of genomic DNA comprised of

a TATA-less promoter flanked by 1.5 kb of 50-upstream

sequence and 1.8 kb downstream sequence [14]. Align-

ment between the nucleotide sequences of newly found

exons and PDP revealed that exon 1B belongs to PDP as

shown in Fig. 1. Using an alternative approach of

bioinformatics tools the immediate upstream regions of

exon 1A, exon 1B and exon 1C were characterized as

promoter regions designated as PA, PB and PC respec-

tively. Thus PDP contains PB, exon 1B and PC sequence

regions. Using CpG island detection tools like CpG Island

Searcher and CpG Plot, no CpG island was established in

or near upstream of exon 1B, however there was presence

of CpG islands in and near upstream of exon 1A and exon

1C (Fig. 4). These CpG islands have an average GC con-

tent of about 65–70% and observed/expected CpG ratio

greater than 60%. The presence of CpG islands may play

an important role in gene regulation, for example, in

causing gene silencing or transcriptional repression by

hypermethylation of the CpG Island [16, 17]. There was an

E-box consensus sequence of CACGTG at position -538

Fig. 3 DNA sequence of newly defined 50 region of Prkar1b gene.

Exons are indicated in uppercase nucleotides and promoters/introns in

lowercase nucleotides. Transcription start sites are identified by

inverted arrow (;) and translational initiation codon is indicated in

bold letters with a bent arrow on it. TF binding sequence motifs are

shown in blue color and overlapped regions for more than one TF are

underlined with each TF over its binding site. The intron–exon

junctions are also underlined. The part of exon 1B which was skipped

in a shorter transcript is boxed. The previously published sequence is

up to start of PB [14]. Previously, whole region of PB, exon 1B and

PC was considered as a single promoter region. An intron of more

than 18 kb was identified between exon 1A and PB. This intron was

first such large intron of its kind identified in 50 region of cAMP

dependent protein kinase A subunit genes. This newly defined 50 UTR

sequence was submitted to the GenBank and the provided accession

number was JF720022

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upstream of transcription start site of exon 1B. This E-box

may act either as an enhancer or a silencer depending on

the binding protein. It is well established that Max/Myc

dimer binding to E-box activates transcription whereas the

Max/Mad dimer binding to E-box suppresses transcription

of the genes [18]. Thus, in silico studies indicated that

Max/Myc or Max/Mad dimer can regulate transcription of

Prkar1b gene from promoter PB.

As three promoter regions lead to the expression of the

transcript encoding same protein, there may be possibility

of differences in their transcription factor (TF) binding

sites so that they can respond to variety of signals in and

out of the cell. For such analysis we performed in silico

analysis using tools and databases like TFSEARCH,

TFBIND and SIGSCAN. Analysis revealed a complex

array of recognition sequence motifs for different types of

TFs (Fig. 3). It was interesting to observe that the three

promoters PA, PB and PC show discrepancy for TFs

responsible for constitutive and conditional expression of

Prkar1b gene. All three promoters have some binding

sites in common for some of the TFs though number vary

for example the general TF Sp1 (steroidogenic factor 1)

and NF-I (Nuclear factor I) remain constitutively active.

Sp1 is expressed in all mammalian cells and is involved

in regulating the transcriptional activity of genes impli-

cated in most cellular processes [19, 20]. NF-I is involved

in eukaryotic transcription as well as DNA replication

[21]. However, there are different binding sites for dif-

ferent transcription factors specific to each promoter e.g.

signal dependent TF CREB (cAMP response element-

binding) which decreases or increases transcription [22]

has binding site in promoter region PA and this site may

be involved in auto-regulation of transcription of Prkar1b

gene by its catalytic subunit counterpart; GR (glucocor-

ticoid receptor) TF which is a conditionally active and

responds to cortisol and other glucocorticoids [23] has

number of binding sites in promoter region PB; AP-2

(Activator protein-2) which is known to have role in

vertebrate development, apoptosis and cell-cycle control

[24], can bind to promoter PB and PC. The PB promoter

seems to be more regulatory in nature as it has number of

different TF binding sites than PA and PC. The promoter

Fig. 4 CpG Plot generated by tools available at EBI used for CpG

Island prediction. The predicted promoter region sequence was used

for all three promoters. CpG Islands were present in the regions of

promoter PA and PC with observed/expected ratio [0.60 and G ? C

percentage [55.00. However, the putative CpG Islands were found in

PC only

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PB has binding sites for GATA, PIT-1, Hox etc. which

take part in developmental and cell specific expression

and once expressed require no additional activation. This

fact prompted us to find out whether expression from PB

was differentially regulated or not. To this end we studied

the expression patterns of 50 UTR exons by RT–PCR

using exon specific forward primer and reverse primer

(PR2) in four major organs namely heart, brain, skeletal

muscle and liver at three different postnatal development

stages of mouse like 3 day, 15 day, 60 day namely PN3,

PN15, PN60 respectively (Fig. 5). Only few tissues from

early and late postnatal stages were studied because it was

beyond the scope of the work presented here to study all

tissues at different developmental stages of mouse. As

envisaged from in silico analysis the expression from

exon 1B was found to be differentially expressed. The

exon 1B containing transcript was only expressed in brain

at all developmental stages. It was interesting to note that

the transcripts having exon 1B was found in all tissues

tested from PN60 stage mouse. However, the exon 1A

and exon 1C were found to be expressed in all tissues at

all postnatal development stages. Also the expression

patterns revealed that once PB gets activated it then tends

to be constitutively expressed as seen in PN3, PN15,

PN60 stages of brain tissue (Fig. 5). These observations

further support the role of R1b subunit in neuronal tissues

and thus per se addresses why this gene was earlier

considered as neuron specific [14, 25].

Conclusion

Our results confirmed the presence of four Prkar1b tran-

script variants in different tissues of adult mouse like

brain, liver, heart and skeletal muscle where transcript

variants Mr1b-A and Mr1b-C were ubiquitously expressed

however, the transcript variants Mr1b-B and Mr1b-B’

were expressed in brain at PN3 and PN15 only and

expressed in all tested tissues at PN60. The study of

transcripts belonging to mouse will be helpful while

investigation of diseases and other functional aspects in

mouse as model, in particular to those cases which pertain

to regulation of this gene or cloning experiments in

expression vectors with one of the three putative promoter

regions required as per the factors under which expression

has to be investigated.

Fig. 5 RT-PCR products of mouse heart, brain, liver and skeletal

muscle mRNA at postnatal stages PN3, PN15 and PN60. Primers used

were same as given in methods section with forward primer exon

specific and reverse primer RP2 from internal exon. 1.3% agarose

gels in row represent postnatal stage and in column represent tissues.

Each lane across column indicates same RT-PCR product. Exon 1B

containing transcript was amplified only in brain at early PN stages

however, at PN60 (adult mice) exon 1B was expressed in all tissues.

Exon 1A and exon IB were expressed in all tissues at all PN stages

examined. Few lanes have unexpected extra bands that were due to

non specific binding of primers as confirmed by sequencing. Marker

(1 kb DNA ladder) was always run to correlate the amplified product

size

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Acknowledgments The authors are thankful to the Department of

Biotechnology (DBT), Ministry of Science and Technology, New

Delhi, India for providing generous funding to project No. BT/PR-

10917/BID/07/254/2008. The necessary facilities provided by

A.M.U., Aligarh, India are thankfully acknowledged.

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ORIGINAL PAPER

Alternatively Spliced Three Novel Transcripts of gria1in the Cerebellum and Cortex of Mouse Brain

Shafquat Azim • Abdul Rouf Banday •

Mohammad Tabish

Received: 22 July 2011 / Revised: 30 August 2011 / Accepted: 8 September 2011

� Springer Science+Business Media, LLC 2011

Abstract Glutamate receptor type 1 (GluR1) subunit of

a-amino-3-hydroxy-5-methyl-4-isoxazole propionate recep-

tors plays an important role in the expression of long-term

potentiation and memory formation. GluR1 is encoded by

gria1 gene containing 16 exons and 15 introns in mouse.

Previous studies have reported two alternatively spliced

variants of this subunit. These flip and flop variants differ

enormously in their properties as well as expression. In our

studies, we report the presence of three new transcripts of this

gene present in the cerebellum and cortex of mouse brain

produced by alternative splicing at 50 end. Four new exons are

reported; N1 is located in 50 untranslated region, N2 is located

in the 1st intronic region while N3 and N4 are located in the

2nd intronic region. The properties of these new exons

encoding N-terminal variants are highly diverse. N1, N3 and

N4 are coding while N2 is a non-coding exon and results in a

truncated transcript. The existence of N2 exon containing

transcript is further supported by the presence of an Expressed

Sequence Tag from the database. The translated amino acid

sequences of these transcripts differ in the presence of signal

peptide as well as in their phosphorylation and acetylation

pattern. The differences in their properties might be involved

in receptor modulation.

Keywords Glutamate � a-amino-3-hydroxy-5-methyl-4-

isoxazole propionate receptors � Cerebellum � Cortex �Alternative splicing � Flip and flop variants

Introduction

The a-amino-3-hydroxy-5-methyl-4-isoxazole propionate

receptors (AMPARs) are of fundamental importance in the

brain as they are responsible for the majority of fast

excitatory synaptic transmission, synapse formation and

stabilization and synaptic plasticity. AMPARs are com-

prised of four subunits (GluR1–GluR4). Amino acid

sequences for these subunits share approximately 70%

sequence homology. The genes encoding these subunits

may undergo alternative splicing in two distinct regions,

resulting in long or short C-termini, and flip or flop variants

in an extracellular domain [1]. Two variants designated as

flip and flop are produced and they differ in 38 amino acids

that lie between the third and fourth putative transmem-

brane domain [2]. These variants differ in their channel

kinetics, pharmacological properties, sensitivity to certain

allosteric modulators and exhibit regional and cell-specific

expression, developmental regulation, and modification by

physiological insults, lesions and disease [3–11]. AMPARs

are homomers of identical subunits or heteromers of dif-

ferent subunits. The N-terminal is important for receptor

assembly and receptor targeting [12, 13]. Subunits com-

patible only at the NTD interacts with each other and are

functional only if there is compatibility in the C-terminal

half of the subunits. Such hetero-oligomerization produces

functional receptors. This suggests that there might be

diversity in the N-terminus of the subunits which facili-

tates the various combinations of the subunits. Moreover,

AMPARs have widespread and varied distribution in the

brain [14].

The presence of flip-flop variants suggests that alterna-

tive splicing plays an important role in generating diversity

which facilitates their differential expression and varied

subunit combinations. The other family of glutamate

S. Azim � A. R. Banday � M. Tabish (&)

Department of Biochemistry, Faculty of Life Sciences,

Aligarh Muslim University, Aligarh, UP 202002, India

e-mail: tabish.bcmlab@gmail.com

123

Neurochem Res

DOI 10.1007/s11064-011-0599-7

receptors includes NMDARs. The NR2B subunit encoding

gene undergoes alternative splicing to produce 5 variants

with different N-termini [15–17]. These subunits belonging

to the different families of the glutamate receptor share

considerable sequence homology. This provided us the

impetus to study the N-terminal variants of gria1 subunit of

AMPAR. In this study we have analyzed the potential new

50 end exons which can splice with the internal exons to

produce new transcript variants of gria1 gene in mouse

brain. The novel exons of these new transcripts might play

a role in differential targeting of the receptor in different

areas of the brain. We have identified three new transcripts

of gria1 gene that arise due to alternative splicing of four

new exons encoding GluR1 subunit with three different

N-termini. These new exons N1, N2, N3 and N4 were

identified from the 50 untranslated region (50UTR), 1st

intron and 2nd intron. They can splice with each other or

internal exons to produce three transcript variants T1, T2

and T3.

Materials and Methods

Materials

Mice (A/J) were bred in house according to the Institu-

tional Animal Care and Use Committee and Guidelines of

the Committee on Care and Use of Laboratory Animal

Resources, National Research Council (Department of

Health, Education and Welfare; National Institutes of

Health). The total RNA extraction kit was purchased

from iNtRON Biotechnology, Inc. (Gyeonggi-do, Korea).

M-MuLV-Reverse Transcriptase, PCR nucleotide mix,

100 bp PCR DNA ladders were purchased from GenScript

(USA). The TOPO-TA cloning kit II was obtained from

Invitrogen Corp. (Carlsbad, CA). The Plasmid DNA

miniprep kit and Qiaquick PCR gel purification kits were

purchased from Qiagen, Inc. (Santa Clarita, CA). Primers

were custom synthesized from Sigma Aldrich Chemicals

Pvt. Ltd., India. All other chemicals used in the experi-

ments were of molecular biology grade.

RNA Preparation

Total cellular RNA from cerebellum and cortex of

2 months old A/J mouse was prepared using the easy

spinTM (DNA free) Total RNA Extraction Kit (iNtRON

Biotechnology, INC) according to the manufacturer’s

instructions. The eluted RNA was quantitated spectropho-

tometrically and RNA integrity was checked by ethidium

bromide staining on denaturing agarose gel electrophoresis.

Primers

Genomic sequence of gria1 gene was downloaded from

NCBI with accession number GenBank ID NM001113325.

Primers were designed using the downloaded sequence.

The following oligonucleotides primers were used:

E1 specific forward

primer

MCTRGRIA1 GCC GTA CAT CTT

TGC CTT TTT CTG

CAC CG

N1 specific forward

primer

MN1GRIA1 CAT TCT ACA GAT

ACA TGC TGA GCT

CAG GG

N2 specific forward

primer

MN2GRIA1 CAT TCC TGG CTT

CCA TCC TCT CAA

CCT G

N3 specific forward

primer

MN3GRIA1 GCT CAG GGC AGA

ATG CAG TTG ATT

GGT C

Reverse primer from

the junction of 5th and

6th exon

MREV1GRIA1 GTC AAT GTC CAT

GAA GCC CAG GTT

GGC

Reverse primer from

5th exon

MREV2GRIA1 GTG GTA CCC GAT

GCC GTT CTT TTC

TAG C

Rapid Amplification of cDNA Ends (50RACE)

The presence of alternatively spliced transcripts was

examined through 50 rapid amplification of cDNA ends

(RACE). 2 lg of total RNA from cerebellum and cortex

were amplified using a 50RACE kit and gria1 specific

reverse primer (MREV1GRIA1) from the junction of exon

5 and exon 6. The amplification was performed according

to the instructions provided with the kit. The RACE

product was fractionated by electrophoresis using 1.2%

agarose gel. Several bands were excised from the gel,

purified and sub-cloned into the TOPO vector, and

sequenced as described later.

Reverse Transcriptase (RT)-PCR

Multiple transcripts encoded by gria1 gene were identified

using RT-PCR. RNA (2 lg) from the cortex and cerebel-

lum were primed with oligo (dT)18 and single-stranded

cDNA was synthesized using the RevertAidTM H-Minus

Reverse Transcriptase at 43�C for 1 h in a total volume

20 ll. 1 ll of the single-stranded cDNA was then added to

PCR system for amplification using appropriate set of

primers in a total vol. 50 ll. PCR product was amplified

using touchdown PCR for 30 cycles. The cycle was per-

formed as; denaturation at 94�C for 4 min, 1 cycle; 93�C,

Neurochem Res

123

30 s; annealing 66�C, 30 s with a decrease of 0.3�C per

cycle, extension 72�C for 45 s, with final extension at 72�C

for 8 min. Final product was subjected to electrophoresis

on a 1.2% agarose gel, stained with ethidium bromide and

photographed on a UV transilluminator.

Semi-Nested RT-PCR

To confirm the results obtained after first round of PCR and

reconfirm the genuine PCR product, semi-nested PCR was

performed. After the RT reaction, as described above, PCR

amplification was carried out for 30 cycles (as above) using

gene-specific reverse primer (MREV1GRIA1 from the

junction of exon 5 and exon 6) and first-exon-specific

primers (based on the sequence obtained from 50RACE

products) as down and upstream primers, respectively. 1 ll

of the resulting PCR product was used as a template for

further amplification by PCR using the same upstream pri-

mer and a downstream primer from exon-5 (MREV2GRIA1

internal to the reverse primer used in 1st PCR). 10 ll of the

semi-nested PCR product was then subjected to electro-

phoresis on 1.2% (w/v) agarose gel, stained with ethidium

bromide and photographed on a UV transilluminator.

Subcloning and Sequencing of RT-PCR Products

The RACE and semi-nested PCR amplified products were

electrophoresed on 1.2% (w/v) agarose gels. Anticipated

ethidium-bromide-stained bands were cut from the gels and

DNA was purified using a PCR gel purification kit. The

purified DNA was subcloned using the plasmid cloning

vector. E. coli JM109-competent cells were transformed.

Transformed colonies were grown overnight at 37�C and

plasmid DNA was purified for sequencing. Plasmids con-

taining the insert were sequenced by automatic sequencer

using either M13 forward or reverse primers [18].

Bioinformatic Analysis

Homology and similarity searches of the obtained nucleo-

tide sequences were performed using the BLASTN non-

redundant database (http://www.ncbi.nlm.nih.gov/BLAST).

Alignment analysis was carried out using the Gene stream

Align tool (http://www2.igh.cnrs.fr/bin/align-guess.cgi) and

ClustalW tool available at www.ebi.ac.uk/clustalw [19].

TFBIND (http://tfbind.hgc.jp/), TFSEARCH (http://www.

cbrc.jp/research/db/TFSEARCH.html) and WWW signal

scan (http://www-bimas.cit.nih.gov/molbio/signal/) were

used to study the transcription factors binding to the

upstream region of the exons. Some of the properties of the

amino acid sequences coded by the new exons were analyzed

using ExPASy tools (http://ca.expasy.org/).

Results and Discussion

Three New and Differentially Expressed Transcripts

of gria1

AMPARs are expressed in various subunit combinations.

The four subunits (GluR1–GluR4) of the receptor assemble

in various stoichiometries to form functional ion channels

[20]. The different subunit composition determines channel

gating kinetics, conductance, vesicular trafficking and tar-

geting to specific synapse [21–23]. In mouse, gria1 gene is

located on chromosome 11 and a large 50UTR of almost

20 kb is present, the 1st intron is 3 kb and the 2nd intron is

150 kb in size as depicted in Fig 1a. The second intron is

unusually large. Mouse database searches revealed that this

subunit is coded by a single gene consisting of 16 exons

and 15 introns. cDNA corresponding to gria1 is present in

the database with Accession number NM001113325 that

encodes for a protein of 907 amino acids. In order to

investigate the presence of possible new splice variants

50RACE was performed. Total RNA isolated from cere-

bellum and cortex was amplified using a 50RACE kit using

the primers spanning exon 5 and exon 6. We were able to

amplify multiple transcripts from the RNA preparations of

mouse brain as shown in Fig 2. Following 50RACE,

sequencing of the products was done as described in

‘‘Materials and Methods’’ section. The cDNA sequences

containing the reverse primer sequence corresponding to

the part of 5th and 6th exon was further analyzed for their

50 ends. We obtained several clones containing new 50 end

sequences. The analysis of the sequencing data revealed the

identification of three new cDNA of gria1 differing at the

50 ends which were not reported earlier. The alignment of

these sequences with the genomic sequence confirmed that

these cDNA were transcribed from gria1 gene and the new

exons were derived from 50UTR, 1st intron and 2nd intron

of the gene. Three new sequences differing in their

N-terminus were produced from the usage of four novel

exons. These exons were designated as N1, N2, N3 and N4

and the resulting transcripts as T1, T2 and T3.

N1 exon derived from 50UTR splices with the 1st

reported exon (E1) producing a variant (T1) which encodes

for an additional 19 amino acid residues to the published

N-terminus of GluR1. N2 exon was found to be non-coding

and splices with the 2nd reported exon (E2) to produce a

variant (T2) which is truncated at the N-terminal by 69

amino acids. N3 exon splices with a novel N4 exon which

then splices with the 3rd reported exon (E3) producing a

different variant (T3) which is 40 amino acid residues

shorter as compared to the reported sequence. N4 exon is

also located in the 2nd intron and is 42,173 bp downstream

of exon N3. These transcripts variants produced through

alternative splicing are depicted in Fig 1b. The presence of

Neurochem Res

123

N2 variant was further supported through Expressed

Sequence Tag (EST). EST with accession number

BY719132 from adult male olfactory brain encodes for a

sequence identical to N2 variant. These analyses were

further confirmed by RT-PCR using upstream and down-

stream primer (given in ‘‘Materials and Methods’’) from

the 1st newly identified exons and from the junction of 5th

and 6th exon (MREV1GRIA1) respectively. The RT-PCR

results revealed bands of expected sizes as predicted from

50RACE sequence data. These results were further con-

firmed through semi-nested PCR using the downstream

primer MREV2GRIA1 (from exon 5th) which was

designed from a sequence internal to the first reverse pri-

mer (MREV1GRIA1). The agarose gel electrophoresis of

the semi-nested PCR revealed bands of expected sizes for

T1 (752 bp), T2 (684 bp) and T3 (573 bp) in both cere-

bellum and cortex of mouse brain (Fig 3). These semi-

nested RT-PCR products were also cloned and sequenced.

Sequencing confirmed that these cDNAs were produced

through alternative splicing of gria1 gene and the tran-

scripts were formed through the use of N1, N2 and N3

exons in combination with the internal exons as discussed

above. The sequences were submitted to the GenBank and

following accession numbers JN180918, JN180919 and

JN180920 were obtained for T1, T2 and T3 respectively.

The study of the structure of these exons showed that the

donor–acceptor residues associated with these new exons

are found to be conserved. The donor ‘‘GT’’ and the

Fig. 1 a Genomic organization of gria1 gene in mouse. The gene is

located on chromosome 11 with a large 50UTR of almost 20 kb, the

1st and 2nd intron are 3 and 150 kb in size, respectively. The

positions of reported exons (E1–E6, E16) as well as new exons (N1,

N2 and N3 and N4) are shown. The positions of the primers are

shown by arrows above the exons. CtrF. N1F, N2F, N3F are exon

specific forward primers and R1 and R2 are reverse primers. b The

transcript variants produced through alternative splicing. Only exons

are shown in mature transcripts. Each rectangle represents an exon,

dark filled rectangles represent newly identified exons while light

filled rectangles are published exons. (i) The published sequence

containing all the exons. (ii) T1 variant produced through the splicing

of a new exon (N1) to the preexisting first exon (E1). The rest of the

sequence is identical to the reported sequence. (iii) A new exon (N2)

replaces E1 and splices with E2 to produce a new transcript variant

T2. (iv) Two new exons N3 and N4 replace E1 and E2 of the reported

sequence and splices with E3 to produce T3 transcript. All these

transcript variants differ in their 50 ends. Sizes of exons are not to

scale

Fig. 2 Agarose gel electrophoresis of 50 Rapid Amplification of

cDNA ends (RACE). a Amplified 50RACE product from cerebellum.

b Amplified 50RACE product from cortex. These amplified products

were electrophoresed on 1.2% gel and stained with ethidium bromide.

In both (a) and (b) Lanes M, -RT, ?RT are 100 bp DNA ladder,

RACE products in absence of reverse transcriptase and RACE

products in presence of reverse transcriptase respectively. Products

from ?RT lane were excised and cloned. Sequencing data confirmed

the presence of transcripts published earlier in addition to the newly

identified transcripts

Neurochem Res

123

acceptor ‘‘AG’’ residues are conserved in all these new

exons (Fig 4). In order to understand the differential

expression of these variants we analyzed the 50 upstream of

these new exons. In silico analysis employing programs

like TFBIND, TFSEARCH and SIGSCAN of the upstream

region of these newly predicted exons showed that a

number of transcription factor (TF) binding sites are

available as depicted in Fig 5. The 50 upstream region of

N1, N2 and N3 exons contain TATA Binding Protein

(TBP) sites. TBP is a key component of eukaryotic tran-

scription initiation machinery [24]. The locations of other

general TFs are also shown in the Fig 5. SP-1 TF binding

site is present in the upstream of all these exons. This TF is

involved in the gene expression in early development of an

organism [25]. Multiple Nuclear Factor-1 (NF-1) binding

sites are present preferably this is associated with the

upstream region of the genes that are highly expressed [26].

Upstream of E1, N1 and N2 exons report Myc-associated

zinc-finger protein (MAZ) TF binding site. MAZ and SP-1

are often found regulating the same gene and may even

bind to the same sites [27]. Activating protein-2 (AP-2) TF

binding site is located upstream of N2 and AP-2 play

important regulatory roles in vertebrate development,

apoptosis and cell-cycle control [28]. Pit-1 TF is a pitui-

tary-specific TF while serum response factor (SRF) is

ubiquitous nuclear protein that stimulates both cell prolif-

eration and differentiation. A number of TFs bind to

CCAAT box such as NF-Y, CP-1, CDP, GATA-1, and

NFE3. Some TFs are tissue specific while others are con-

stitutively expressed. These analyses suggest that these

transcripts may involve various TF activities which might

be responsible for their differential activity in different

regions of the mouse brain.

Comparative Analysis of the New Variants

In order to understand the structure and functions of these

variants we analyzed their conceptually translated amino

Fig. 3 Agarose gel electrophoresis of the semi-nested PCR products

from a cerebellum and b cortex of mouse brain. 10 ll of the semi-

nested PCR product was electrophoresed on 1.2% agarose gel

electrophoresis. The lanes are labeled as M, ctrl, T1, T2 and T3 which

correspond to DNA marker 100 bp ladder and RT-PCR products

obtained using exon specific primers. Ctrl lane corresponds to 679 bp

and is the amplified product obtained from primers designed from

published sequence, T1 corresponds to the first new transcript having

anticipated size 752 bp, T2 is second new transcript with an

anticipated size of 684 bp and T3 is the third new transcript variant

where the band corresponds to the anticipated 573 bp. The expression

levels of these transcripts are different as seen by the intensity of the

band

Fig. 4 Splice donor–acceptor sites of first few exons of the transcripts. The junction residues are conserved in all the exons. The donor ‘‘GT’’

and the acceptor ‘‘AG’’ residues are conserved in all these new exons and are shown by capital italicized alphabets

Neurochem Res

123

Fig. 5 Analysis of the upstream

region of these exons using

web-based programs TFBIND,

TFSEARCH and SIGSCAN.

The upstream region of the

exons is represented by

rectangular boxes and the

binding positions of various

transcription factors (TFs) are

shown. 600 bp upstream

sequences are analyzed to locate

the position of TFs. Analysis

revealed that many brain

specific TFs can potentially bind

to upstream regions of the

newly identified exons

Fig. 6 Multiple sequence

alignment (ClustalW) of the

deduced amino acid sequences

of the published transcripts (E1)

and the newly identified

transcripts (N1 N2 and N3). (*),

colon (:) and dash (–) represents

identical, similar and no residue

respectively. The accession

numbers are provided at the end

of the sequences. All sequences

are identical from third exon

onwards. Accession numbers

are given at the end of each

sequence

Neurochem Res

123

Ta

ble

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uen

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AN

FP

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HA

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EP

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IDIV

NIS

DS

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MT

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ML

SS

GR

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KG

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AC

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LQ

MP

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GF

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MT

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FS

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YE

RR

TV

NM

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SF

CG

AL

HV

CF

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SF

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site

Neurochem Res

123

acid sequences. Multiple sequence alignment employing

web-based ClustalW of these translated sequences revealed

that they differed only in their N-terminal (Fig 6). A

number of properties of these variants were studied and

compared using ExPASy tool. Table 1 compares some of

the properties of these translated exon sequences and pro-

vides a number of difference in the properties of these new

exons. The phosphorylation and acetylation pattern of these

sequences are very different. N1 and N2 contain 3S and 2T

respectively which can be potentially acetylated. However

no such residues are present in E1 and N3 variants.

Although a majority of eukaryotic proteins, almost 50% of

yeast proteins and 80% of the human proteins are N-ter-

minally acetylated (Nt-acetylated), the function of this

modification is largely unknown [29, 30]. For some pro-

teins such as actin, Nt-acetylation has been reported to

affect protein functionality, e.g. non-acetylated actin is less

efficient at assembling microfilaments [31]. Recently the

other role suggested for acetylation is that Nt-acetylation of

a protein can function as a degradation signal (degron)

playing a role in protein turnover and homeostasis [29]. N1

and N2 variants have potential PKC dependent phosphor-

ylation sites. Protein phosphorylation of neuronal gluta-

mate receptors by cyclic AMP-dependent protein kinase

(PKA) and protein kinase C (PKC) may regulate their

function and play a role in some forms of synaptic plas-

ticity [32]. E1 contains a signal peptide while N1 contains

signal anchor. Many integral proteins contain signal non-

cleavable transmembrane signal sequence called signal

anchor sequence which anchors the protein to the mem-

brane [33–35]. N2 contains a mitochondrial targeting

sequence. Glutamate excitotoxicity often leads to mito-

chondrial dysfunctioning associated with apoptotic and

necrotic neuronal cell death [36]. N3 does not contain such

signal sequence. The detailed study of this variant might

provide new pathways to study the regulation in neuronal

cell death. The diverse molecular properties of the variants

might be responsible their differential expression in cere-

bellum and cortex of the mouse brain as shown by the

RT-PCR result in our study.

Our studies provide the 1st experimental evidence for

the presence of multiple alternatively spliced transcripts

variants of the gria1 gene encoding different N-termini.

The new exons are produced from the large intronic

regions of the gene. The large introns are frequently

associated with the evolution of a gene. These transcripts

being differentially expressed in cerebellum and cortex of

mouse brain suggest that the N-termini may be associated

with differential targeting of the receptor to different

regions of the brain. The properties of these variants are

highly heterogeneous and might play a role in providing

functional, structural and molecular diversity to the AMPA

receptors. A detailed study of these variants might throw

light on the mechanism of synaptic plasticity, neuronal

development and various neuropathological conditions

involving AMPARs.

Acknowledgments The authors are thankful to CSIR, New Delhi,

India for providing senior research fellowship to SA. Partial funding

from the Department of Biotechnology (DBT), Ministry of Science

and Technology, New Delhi and necessary facilities provided by

AMU, Aligarh, India is thankfully acknowledged.

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