2011

MECHANISMS OF CYTOPLASMIC mRNA TURNOVER IN EUKARYOTES

Kim Keeling, Ph.D.

kkeeling@uab.edu

Room 456, Bevill Bldg

975-6585

1. General mRNA turnover pathways- Play key role in controlling basal gene expression levels

2. Aberrant RNA turnover pathways- Recognize and degrade aberrant mRNAs or mRNAs that are translated inefficiently- Increases the quality control of mRNA biogenesis and gene expression

3. Specialized mRNA turnover pathways - mRNA turnover acts as a site for regulation in response to specific signals (hormones, cell cycle, viral infection, differentiation, nutrient availability, stress)- AU-rich element mediated (ARE)

4. Specialized sites of cytoplasmic mRNA turnover- Processing bodies (P bodies)- Exosome granules for ARE-mediated mRNA turnover

Lecture Overview

Determining mRNA Stability Experimentally

1. Shut off transcription: thiolutin, actinomycin D, promoters that can be repressed (such as copper or tetracycline regulated promoters).

2. Isolate mRNA at several time points after transcriptional shut-off.

3. Determine the percent of mRNA remaining at each time point after transcriptional shut-off.

4. Graph the level of RNA remaining for each time point on a semi-log graph. The slope = the rate of mRNA decay. The time at which half of the mRNA is degraded is the half-life of the mRNA.

Keeling et al. (2006) Mol Cell Biol 26:5237

0 10 20 30 4010

20

30

405060708090

100

t1/2 =tpa124 min

t1/2 =wild type12 min

% ACT1 remaining

Time (m)

time (m)

WT tpa1∆

0 10 20 40 0 10 20 40

inhibit expression

Northern blot of ACT1 mRNA

mRNAs Are Modified During Nuclear Processing to Promote Cytoplasmic Stability

1. Addition of 5’ cap and the CAP binding protein complex

2. Addition of the 3’ poly(A) tail and multiple poly(A) binding proteins

3. Circularization of mRNA by bridging the 5’ CAP and the 3’ poly(A) tail via eIF4G

Goldstrohm & Wickens (2008) Nat Rev Mol Cell Biol 9:337

Nucleus

Cytoplasm

1) General mRNA turnover pathways

The Major Eukaryotic mRNA Turnover Pathways

5’ 3’ pathway 3’ 5’ pathway

Biochem. Soc. Trans. (2006) 34:35

CAP hydrolysis

The Major Eukaryotic mRNA Turnover Pathways

5’ 3’ pathway 3’ 5’ pathway

Biochem. Soc. Trans. (2006) 34:35

CAP hydrolysis

Mammalian Deadenylation Occurs in Two Phases

Muhlemann (2005) Nat Struct Mol Biol 12:1024

Slow

Fast

PAN2 complex

CCR4 complex

Deadenylase Machinery

2) CCR4 complex:- Second phase- Fast, processive- Inhibited by PAB- Independent function as a transcription factor

1) PAN complex:- First phase- Slow, distributive- Stimulated by PAB- Also trims poly(A) tails in nucleus

3) PARN complex:- Used for specialized rapid deadenylation (ARE-mediated decay) in mammalian cells

Parker & Song (2004) Nat Struct Mol Biol 11:121

Determining Deadenylation Rates Experimentally

RNase H Northern blot

Control RNA

Transcriptionalshut off

Northern blot

AAAAAAAAAA

RNase H

Probe

TTTTTTTTTT

DNA oligo

+ oligo dT poly(A)-

PTETOFF

slow synchronous

fastasynchronous

Yamashita et al. (2005) Nat Struct Mol Biol 12:1054

PAN complex CCR4 complex

PAN and CCR4 Sequentially Deadenylate mRNAs

Yamashita et al. (2005) Nat Struct Mol Biol 12:1054

No differenceDelayed 1st phase

Delayed2nd phase

Enhanced2nd phase

Enhanced 1st phase No difference

Hosoda et al (2003) JBC 278:38287

Deadenylation Is Coupled to Translation Termination

- eRF3 is a GTPase that mediates translation termination.

- The N-terminal domain of eRF3 interacts with the Pab1p C-terminal domain.

- This interaction inhibits the ability of Pab1p molecules to oligomerize efficiently on the poly(A) tail. This increases the exposure of the poly(A) tail to deadenylases and turnover.

Hoshino et al. (1999) Biochem (Mosc) 64:1367

eRF3 Binds to Pab1p and Inhibits Its Oligomerization

- Gel shift assay: radio-labeled RNA is incubated with increasing amounts of Pab1p protein.

- A shift in the mobility of the RNA indicates Pab1p protein binding.

- Addition of the eRF3 N-terminal domain to the binding assay inhibits the ability of Pab1p to oligomerize on the RNA.

The eRF3-Mediated Inhibition of Pab1p Results in Deadenylaton and mRNA Turnover

Hosoda et al (2003)JBC 278:38287

- Expression of a mutant eRF3 where the N-terminal domain is deleted so that it can no longer interact with Pab1p results in longer poly(A) tail length. This mutant also leads to an increased mRNA half-life = decreased turn- over rate.

- This suggests that normally, eRF3 mediates mRNA turnover by decreasing Pab1p oligomerization on poly(A) tails which leads to deadenylation and mRNA decay. This represents one way to mediate general mRNA decay.

The Major Eukaryotic mRNA Turnover Pathways

5’ 3’ pathway

Biochem. Soc. Trans. (2006) 34:35

Pan2, Ccr4

CAP hydrolysis

Decapping Enzymes

- Dcp2p = catalytic subunit - Dcp1p = stimulates Dcp2p - Hedls = also needed to stimulate Dcp2p activity in mammals

- In yeast, Dcp1p and Dcp2p can directly interact with each other to stimulate decapping

- Dcp2p is differentially expressed in mammalian tissues

- Another decapping enzyme was identified in the cytoplasm of mammalian cells called Nudt16. No ortholog found in yeast, C. elegans or Drosophila.

- Nudt16 and Dcp2 appear to each work on subsets of cellular transcripts.

Yeast interactions

Human interactions

Nudt16

Dcp1

Dcp2

Hedls

Taylor & Peculis (2008) NARS 36:6021

Simon et al. (2006) TIBS 31:241

1. Poly(A) tail status - Poly(A) binding protein inhibits decapping by physically interacting with the 5’ cap complex (via eIF4G) to stabilize the cap and enhance translation.

2. CAP status - The CAP protein complex must dissociate from the cap prior to decapping.

3. Translation status - A transition must take place from a translation competent state to a state where translation does not take place (change in mRNP structure).

4. Assembly of the decapping complex - Specific factors are required for the localization, assembly, and activation of the decapping complex.

Control of the Decapping Reaction

Decapping Activators

Pat1p - Interacts with both poly(A) [+] and poly(A) [-] transcripts - Seeds decapping complex onto mRNA - Interacts with Dcp1p, Dcp2p, Lsm1-7p, Dhh1p, Xrn1p - Along with the Lsm complex, represses translation

Lsm1-7p - 7 ring complex interacts with poly(A) [-] mRNA - Facilitates the assembly of the decapping complex in cytoplasmic and nuclear decapping - Interacts with Dcp1p, Dcp2p, Dhh1p, Pat1p, Xrn1p

Dhh1p - ATP dependent helicase - Binds directly to mRNA substrate - Interacts with Dcp1p, Dcp2p, Lsm1-7p, Ccr4p, Pop2p, Edc1p, Edc2p

Edc1/Edc2 - Binds directly to mRNA substrate - Enhances decapping activity - Interacts with Dcp1 and Dcp2 - Enhances alterations in mRNA stability in response to nutrient changes

Parker & Song (2004) Nat Struct Mol Biol 11:121

The Major Eukaryotic mRNA Turnover Pathways

5’ 3’ pathway

Biochem. Soc. Trans. (2006) 34:35

Pan2, Ccr4

Dcp2, Nudt16

CAP hydrolysis

The Major Eukaryotic mRNA Turnover Pathways

5’ 3’ pathway

Biochem. Soc. Trans. (2006) 34:35

Pan2, Ccr4

Dcp2, Nudt16

CAP hydrolysisXrn2/Rat1= nuclear

exonuclease

The Major Eukaryotic mRNA Turnover Pathways

Biochem. Soc. Trans. (2006) 34:35

Pan2, Ccr4

CAP hydrolysis

Parker & Song (2004) Nat Struct Mol Biol 11:121

Exosome ComponentsCore Subunits: all essentialRrp4pRrp40pCsl4p Rrp41p/Ski6pRrp42pRrp43pRrp44p/Dis3pRrp45pRrp46pMtr3p

Associated factors:Mtr4p essential ATP-dependent helicaseSki2p non-essential ATP-dependent helicaseSki3p non-essential TPR repeatSki8p non-essential WD repeatSki7p non-essential GTPase

Nuclear subunits:Rrp6p non-essential nuclear subunit

The Exosome Gains Access to mRNAs for Degradation Via Ski7p and the SKI Complex

Araki et al. (2001) EMBO J 20:4684

- Ski7p bridges an interaction between the mRNA-bound SKI complex and the exosome.

- In the nucleus, a group of factors called the TRAMP complex is the bridge between mRNAs and the exosome.

The Major Eukaryotic mRNA Turnover Pathways

Biochem. Soc. Trans. (2006) 34:35

Pan2, Ccr4

Dcp2, Nudt16

CAP hydrolysis

Final Step of mRNA Decay: Degrading the 5’ Cap

Parker & Song (2004) Nat Struct Mol Biol 11:121

- The scavenger decapping enzyme hydrolyzes di- and tri-phosphorylated CAPs to the mono form.

- Substrates are less than 10 nucleotides in length.

Summary of the General Eukaryotic mRNA Decay Pathways

Meyer et al. (2004)Crit Rev Biochem Mol Biol 39:197

Nuclear

Yes

Yes

Xrn2p/Rat1p

TRAMP

Nuclear mRNA Decay Also Utilizes Components of the 5' 3' and 3' 5' mRNA Turnover Pathways

TRAMP

Xrn2p

Moore (2002) Cell 108:431

2) Aberrant mRNA Decay Pathways

A. Nonsense-mediated mRNA decay (NMD) - Degrades mRNAs with premature stop codons

B. Nonstop mRNA decay (NSD) - Degrades mRNAs without a stop codon

C. No-go mRNA decay (NGD) - Degrades mRNAs that have a stalled ribosome

D. Ribosome extension-mediated decay (REMD) - Degrades mRNAs where ribosome translates past the stop codon and into the 3’ UTR

Nonsense-Mediated mRNA Decay- Specialized pathway that degrades mRNAs that contain premature translation termination signals

- Protects the cell from translating mRNAs that might produce truncated peptides that could lead to harmful dominant negative effects- Occurs in all eukaryotes.- 30% of disease-generating mutations result in premature stop codons- Up to 10-20% of the transcriptome is regulated by NMD - PTC-containing transcripts caused by point mutations, frameshift mutations, mRNAs with faulty alternative splicing, pre-mRNAs that escape nuclear retention, mRNAs that contain upstream open reading frames, mRNAs that carry introns in 3´ untranslated regions, or mRNAs with long 3´ untranslated regions

Czapllinski et al. (1999)Bioeassay 21:685

Current NMD Models

Splicing-dependent NMD

Splicing-independent NMD

Normal Termination

Aberrant Termination

Wen & Brogna (2008) Biochem Soc Trans 36:514

Splicing-Dependent NMD

NMD Factors Associate With the EJC

core NMD components

Core NMD Components:

UPF3: associates with the EJC in the nucleus

UPF2: perinuclear and binds to Upf3 as the mRNA is exported

UPF1: associates at the stop codons in mRNAs during translation

Regulation of NMD byUpf1p Phosphorylation

Kashima et al. (2006) Gene Dev 20:355

1. The SURF complex composed of SMG1, UPF1, and the eRF1 and eRF3 release factors forms. This complex recognizes stop codons.

2. If the SURF complex interacts with a downstream EJC complex that includes UPF2, UPF3, and Y14 (the DECID complex), then SMG-1 phosphorylates UPF1. This marks the stop codon as premature.

3. UPF1 phosphorylation induces a change in the mRNP structure that recruits SMG5, SMG6, SMG7, and the PP2A phosphatase to dephosphorylate UPF1. This signals for the mRNA to be degraded.

Aberrant Translation Termination Mediates NMD

Muhlemann (2008) Biochem Soc Trans 36:497

Nonstop mRNA Decay

- Pathway to degrade mRNAs that do not contain any stop codons

- Typically caused by the presence of cryptic poly(A) addition sites that leads to polyadenylation of transcripts upstream of the termination signal

- Utilizes Ski7p, the SKI complex, and the exosome to degrade nonstop messages

Maquat (2002) Science 295:2221

No Go mRNA Decay

- Degrades messages with translation elongation stalls (i.e. hairpin structures, pseudoknots, rare codons)

- Requires two factors that bind to the A site of the stalled ribosome -- Dom34p (eRF1-like homologue) -- Hbs1p (eRF3-like homologue)

- Promotes ribosomal subunit dissociation and peptidyl tRNAs to remove them from translation elongation stalls and recycle them

- mRNA cleaved at stalled position by Dom34p which has endonuclease activity and then degraded by the exosome and/or Xrn1p

Tollervey (2006) Nature 440:425

Many of the Specialized mRNA Decay Pathways Recruit Similar Proteins to the Ribosomal A Site to Trigger different mRNA Decay Mechanisms.

Clement & Lykke-Anderson (2006) Nat Struct Mol Biol 13:299

NMD eRF1 eRF3

NGD DOM34 HSB1

NSD ? SKI7

A site binder GTPasePathway

Chen et al. (2010) Nat Struct Mol Biol 17:1233

Ribosome Extension-Mediated mRNA Decay

- Constant Spring (CS) mutation in -globin changes the UAA stop codon at the end of the mRNA into a CAA (glutamine) codon

- Mutation is the most prevalent non-deletion mutation that causes -thalassemia

- Ribosome continues to translate 31 codons in the 3’ UTR until a stop codon is encountered (UAA)

- Level of mRNA is severely decreased due to this mutation

- CS mutation causes a decrease in the mRNA half-life due to rapid induction of deadenylation

- Mechanism behind this rapid deadenylation is still unknown

- REMD is cell-type restrictedKong & Liebhaber (2007) NSMB 14:670

Summary of mRNA Surveillance Pathways

Doma & Parker (2007) Cell 131:660

3) Specialized mRNA turnover pathways- ARE-mediated mRNA decay

ARE-Mediated mRNA Turnover

- AU rich elements (50-150nts) AUUUA; UUAUUUA(U/A)(U/A); or U-rich- cis-acting element located in 3' UTRs of mRNAs - transcripts that encode proteins that require rapid changes in response to stimuli such changes in the cell cycle, growth factors, response to microorganisms, inflammatory stimuli, and environmental factors- 10% of mammalian mRNAs contain AREs

- A diverse set of trans-acting proteins bind to AREs. These proteins can mediate other protein interactions that modulate mRNA stability. Various ARE-associated proteins can promote rapid mRNA turnover by promoting enhanced decapping, deadenylation, exosome recruitment, endonucleolytic cleavage or combinations of these. Alternatively, some proteins that bind to AREs can stabilize the mRNA.

Goldstrohm & Wickens (2008) Nat Rev Mol Cell Biol 9:337

ARE

Various Triggers Alter the Balance of Effectors to Modulate ARE-mediated mRNA Decay

Eberhardt et al. (2007) Pharm Ther 114:56

Example of ARE-Mediated Change in mRNA Levels Before and After the DNA Damage Response

Cevhar & Kleiman (2010) WIRE 1:193

Under non-damage conditions:- AUF1 competes with the PABP for poly(A) tail binding, exposing it to PARN; TTP (tristetraproline) and KSRP (KH-type splicing regulatory protein) recruit PARN and CCR4 to deadenylate prior to degradation by the exosome.

Under DNA damage conditions:- Genes involved in the DNA damage response pathway are up-regulated. HuR is up-regulated and competes with AUF1 for binding to the same ARE region. Loss of AUF1 binding stabilizes PABP association with the poly(A) tail. HuR also competes with TTP and KRSP to prevent recruitment of the deadenylases and exosome.

4) Specialized sites of cytoplasmic mRNA turnover- Processing bodies- Exosome granules

RNA Processing Bodies (P bodies)

- Discrete cytoplasmic granular structures that contain a reservoir of 5’ 3’ mRNA decay factors; NMD factors; RNA-induced silencing complex

- Found in all eukaryotes

- Size and number of P bodies depend upon the amount of RNA to be degraded

Conditions that promote P body formation include: - Glucose deprivation- Osmotic stress- UV light- Decreased translation initiation rates- Non-translating mRNA

Note that all of these conditions involve moving mRNAs from a translatable pool that is ribosome-associated to a non-translatable pool that is not ribosome- associated

Conditions that reduce P bodies include:- Inhibition of translation elongation (ribosome can’t dissociate from mRNA)- RNase A treatment (RNA degraded)- Increased rates of translation initiation (increase in ribosome-bound mRNA)

Identification of Factors That Reside in P Bodies

Factors Not Found in P Bodies- Translation initiation factors- Ribosomal subunits- SKI proteins & exosome

Kulkarni et al. (2010) Biochem Soc Trans 38: 242Kulkarni et al. (2010) Biochem Soc Trans 38: 242

RNA Decay IntermediatesLocalize to P Bodies

- PolyG tract (18) in 3’ UTR blocks Xrn1p to create a mRNA decay intermediate

- MS2 = bacteriophage coat protein binding site

- MS2-GFP protein will bind to MS2 sites in mRNA and allows its localization by visualization of the green fluorescence protein (GFP)

Sheth & Parker (2003) Science 300:805

P Bodies Disassemble After Translation Restoration

- Polysome profile (ribosomes fractionated through a sucrose gradient)

- Active translation = (+) polysomes- Inactive translation = (-) polysomes

- P body-associated mRNAs can return to the translatable pool

Brengues et al. (2005) Science 310:486.

Exosome Granules Are Distinct From P Bodies and Contain ARE mRNAs and PARN

Lin (2007) JBC 282:19958

exosomeprotein

P bodyprotein

merge

AREmRNA

PARN exosomeprotein

merge

1. General mRNA turnover pathways - 5’3 & 3’5’ - Deadenylases - Decapping complex - Xrn1, exosome, DcpS

2. Aberrant RNA turnover pathways - Premature stop codons: nonsense-mediated mRNA decay (NMD) - No stop codons: non-stop mRNA decay (NSD) - Elongation stall: no-go mRNA decay (NGD) - Translation into the 3’ UTR: ribosome extension-mediated mRNA decay (REMD

3. Specialized mRNA turnover pathways - ARE-mediated mRNA turnover: AU-rich elements in the 3’ UTR are bound by proteins that modulate the stability of mRNAs in response to regulatory signals

4. Locale of mRNA turnover- P bodies: contain 5’3’ mRNA turnover machinery and degrade mRNAs

that are no longer available for translation- Exosome granules: contain PARN and exosome and participate in ARE-

mediated decay

Lecture Overview