lily novak frazer, simon c. lovell and raymond t. o’keefe ... · 7/20/2009 · lily novak...

Analysis of synthetic lethality reveals genetic interactions between the GTPase Snu114p

and snRNAs in the catalytic core of the Saccharomyces cerevisiae spliceosome

Lily Novak Frazer, Simon C. Lovell and Raymond T. O’Keefe*

The University of Manchester, Faculty of Life Sciences, Michael Smith Building, Manchester, United Kingdom, M13 9PT

Genetics: Published Articles Ahead of Print, published on July 20, 2009 as 10.1534/genetics.109.107243

Frazer et. al. page 2

Running Title: SNU114 and snRNA genetic interactions

Key words: pre-mRNA splicing, snRNA, Snu114, Saccharomyces cerevisiae

*Corresponding author: Dr. Raymond T. O’Keefe The University of Manchester Faculty of Life Sciences Michael Smith Building Oxford Road, Manchester M13 9PT United Kingdom Tel: 44 161 275 7393 Fax: 44 161 275 5082 Email: [email protected]


ABSTRACT

Conformational changes of snRNAs in the spliceosome required for pre-mRNA splicing are

regulated by eight ATPases and one GTPase Snu114p. The Snu114p guanine state regulates U4/U6

unwinding during spliceosome activation and U2/U6 unwinding during spliceosome disassembly

through the ATPase Brr2p. We investigated 618 genetic interactions to identify an extensive

genetic interaction network between SNU114 and snRNAs. Snu114p G domain alleles were

exacerbated by mutations that stabilize U4/U6 base pairing. G domain alleles were made worse by

U2 and U6 mutations that stabilize or destabilize U2/U6 base pairing in helix I. Compensatory

mutations that restored U2/U6 base pairing in helix I relieved synthetic lethality. Snu114p G

domain alleles were also worsened by mutations in U6 predicted to increase 5’ splice site base

pairing. Both N-terminal and G domain alleles were exacerbated by U5 loop 1 mutations at

positions involved in aligning exons while C-terminus alleles were synthetically lethal with U5

internal loop 1 mutations. This suggests a spatial orientation for Snu114p with U5. We propose

that the RNA base pairing state is directly or indirectly sensed by the Snu114p G domain allowing

the Snu114p C-terminal domain to regulate Brr2p or other proteins to bring about RNA/RNA

rearrangements required for splicing.


INTRODUCTION

Introns in pre-messenger RNA (pre-mRNA) are removed by the spliceosome to yield mature

mRNA which is then exported into the cytoplasm for translation. The process of pre-mRNA

splicing has been reviewed extensively (MOORE et al. 1993; WILL and LÜHRMANN 2006). Splicing

is carried out in the nucleus by the assembly of small nuclear ribonucleoprotein particles (or

snRNPs) onto the pre-mRNA and occurs by two transesterification steps which are carried out

within the spliceosome by a series of RNA:RNA interactions and RNA:protein rearrangements.

Five snRNPs take part in splicing, U1, U2, U4, U5 and U6, of which the last three form a tri-snRNP

in which U4 is base paired with U6 (MOORE et al. 1993). Although it is thought that the RNAs

within snRNPs are responsible for catalyzing the two steps of splicing, it is the many proteins, in

particular the NTPases, which bring about the multiple rearrangements that are required for

catalysis by the snRNAs (SMITH et al. 2008). Key remodelling steps during splicing include

unwinding of U4/U6 helices, formation of U2/U6 interactions, unwinding of U2/U6 helices and re-

establishment of U4/U6 complexes. Remodelling is facilitated by eight ATPases and one GTPase,

Snu114p, one of the key protein factors required for the regulation of spliceosomal dynamics

(reviewed in (FRAZER et al. 2008).

The single GTPase of the spliceosome, Snu114p (U5-116kDa or hSnu114 in humans), is a

protein of the U5 snRNP (FABRIZIO et al. 1997). It shares significant sequence homology with

translation elongation factor 2 (EF-2) and contains five domains similar to EF-2, with domain I

containing the G domain consensus sequence elements G1–G5 that are important for binding and

hydrolysis of GTP. hSnu114 has been found to crosslink to GTP specifically and a mutation in

Snu114p, expected to abolish GTP binding, is lethal, suggesting that GTP binding and hydrolysis

are necessary for Snu114p function in vivo (BARTELS et al. 2003; FABRIZIO et al. 1997). Snu114p

also has a unique acidic amino-terminus (N-terminus) which is implicated, along with the G

domain, in U4/U6 unwinding during spliceosome assembly (BARTELS et al. 2002).


The relationship of Snu114p to other spliceosomal proteins has been investigated using

conditionally lethal alleles to test for genetic interactions with other splicing factors (BRENNER and

GUTHRIE 2005). Genetic interactions of snu114 alleles were found with prp8, brr2, prp28, prp19,

sad1 and snu66 alleles. The genetic interactions of SNU114 with PRP8 and BRR2 were expected

considering the proteins form a stable complex within the U5 snRNP in human cells (ACHSEL et al.

1998). Other genetic interactions were with genes that are all known to act before the first step of

splicing, suggesting an important role for Snu114p during spliceosomal activation (BRENNER and

GUTHRIE 2005). Although genetic interactions do not necessarily indicate direct interactions, two

hybrid studies have confirmed interactions between hSnu114, hPrp8 and hBrr2 (LIU et al. 2006)

and other studies have indicated a requirement for Prp8p and Snu114p for correct U5 snRNP

assembly (BRENNER and GUTHRIE 2006). Whether Snu114p function requires GTP hydrolysis was

addressed by analysis of spliceosome assembly and disassembly, both of which were promoted by

Snu114p and Brr2p (SMALL et al. 2006). The guanine nucleotide state of Snu114p, rather than GTP

hydrolysis, regulated Brr2p activity during snRNA unwinding.

Compared to the number of known Snu114p-protein interactions, little is known about how

Snu114p interacts with snRNAs of the spliceosome. Evidence of direct Snu114p-RNA interactions

include hSnu114 crosslinks to hairpins introduced in pre-mRNA and hSnu114 crosslinks with the

pre-mRNA pyrimidine tract after spliceosome assembly but prior to the second step (CHIARA et al.

1997; LIU et al. 1997). This suggested a role for hSnu114 in bringing the 3’ splice site into the

active site for the second step of splicing. Parallels have been drawn between the function of

Snu114p in the spliceosome and EF-2 in the ribosome (STALEY and GUTHRIE 1998). The

interaction between U5 loop 1 and the 5’-exon during splicing was proposed to be analogous to the

tRNA anticodon–codon interaction during translation, with a conformational change driving

Snu114p to reposition the 5’-exon-bound U5 loop 1 for the second step of splicing (STALEY and

GUTHRIE 1998). Although Snu114p was found to crosslink to the 5’ side of internal loop 1 of U5


snRNA (DIX et al. 1998), a model for Snu114p function based on GTP driven hydrolysis is no

longer supported (SMALL et al. 2006).

As splicing is most likely RNA-catalyzed, it is important to determine any interactions

between Snu114p and the snRNAs involved in catalysis. We assessed synthetic lethal interactions

of SNU114 with the snRNAs that contribute to the catalytic core of the spliceosome, including U4

which dissociates from the spliceosome prior to assembly of the active complex. We found that

snu114 G domain mutations were synthetically lethal with mutations in U4 that stabilize the upper

portion of U4/U6 stem I and only one mutation within stem I. Similarly, snu114 G domain

mutations were synthetically lethal with mutations in U2 and U6 predicted to stabilize or destabilize

U2/U6 base pairing in helix I. Synthetic lethality could be suppressed by compensatory mutations

that restored U2/U6 base pairing in helix I. In addition, synthetically lethal interactions of G

domain mutations of snu114 with U6 snRNA alleles indicated a role for Snu114p during U6 base

pairing interactions with the 5’ splice site. These results suggest that the Snu114p G domain is

important for sensing the integrity of RNA/RNA interactions in the catalytic core of the

spliceosome. The role of Snu114p within the catalytic core was confirmed by genetic analysis with

U5. We found that snu114 N-terminal and G domain mutations were synthetically lethal with U5

loop 1 alleles in positions important for tethering the exons during splicing and that C-terminal

mutations were synthetically lethal with alleles in U5 internal loop 1. These results suggest a

spatial orientation for Snu114p with U5 and a possible model for regulation. The Snu114p G

domain, and to some extent the N-terminal domain, may sense the state of the snRNAs in the

catalytic core and, possibly through a structural rearrangement with U5, transmit this information to

the C-terminal domain to regulate Brr2p or other proteins to bring about the rearrangements in the

spliceosome required for splicing.


MATERIALS AND METHODS

Yeast strains and manipulation

For analysis of SNU114 mutations, diploid yeast strain Y25023 (Table 1) was obtained from

EUROSCARF and transformed with pRS416-Snu114. All transformations were carried out using

the lithium acetate procedure (GIETZ et al. 1992). G418-resistant and Ura+ transformants were

sporulated and tetrads dissected. Haploid progeny were checked for disruption of SNU114 with

kanMX4 by PCR of genomic DNA to identify the strains YSNU114KO1 and YSNU114KO2 (Table

1). SNU114 knockout strain, YSNU114KO1, containing pRS416-Snu114 was transformed with

each snu114 allele on a separate plasmid. Transformants were transferred to 5-fluoro-orotic acid

(5FOA) to select against the URA3 plasmid by plasmid shuffle (BOEKE et al. 1984) and to

determine the viability of snu114 alleles as the sole source of Snu114p. Cells viable on 5FOA were

tested for temperature sensitivity by streaking onto yeast peptone dextrose (YPD) agar and

incubating at 16°, 25°, 30°, 37°, 39° and 40° for 3-10 days.

To test genetic interactions between snu114 and snRNA alleles, double deletion strains were

constructed by mating strain YSNU114KO2 with strain YU5KO (O'KEEFE 2002), YU2KO or

YU6KO (Table 1). Mated diploid strains were transferred to 5FOA to generate diploid strains

lacking pRS416 plasmids that were complementing the SNU114 and snRNA knockouts. Diploid

strains with single SNU114 and individual snRNA deletions were transformed with a pRS416

plasmid containing both WT genes (pRS416-Snu114-U5, pRS416-Snu114-U2 or pRS416-Snu114-

U6). Viable G418-resistant and Ura+ transformants were sporulated and tetrads dissected. The

haploid progeny were analyzed for disruption of SNU114 and the relevant snRNA gene by PCR of

genomic DNA and by plasmid shuffle for the requirement of Snu114p and U5, U2 or U6 to identify

the strains YSNU114/U5KO, YSNU114/U2KO and YSNU114/U6KO (Table 1). YSNU114/U4KO

was constructed by replacing the U4 gene with the hphNT1 resistance gene (JANKE et al. 2004) in

the SNU114 diploid knockout strain Y25023 with a PCR product amplified from pYM24 using

primers U4hphNT1F and U4hphNT1R (Table S1). G418- and hygromycin B-resistant cells were


transformed with pRS416-Snu114-U4, sporulated and tetrads dissected. Haploid progeny were

checked for integration of kanMX4 and hphNT1 by PCR of genomic DNA and in vivo for the

requirement of both Snu114p and U4 to identify the strains YSNU114/U4KO1 and

YSNU114/U4KO2 (Table 1). Strains YSNU114/U5KO, YSNU114/U2KO, YSNU114/U6KO and

YSNU114/U4KO1 (Table 1) were transformed with snu114 and snRNA alleles on different

plasmids and then tested for synthetic lethality by plasmid shuffle.

Plasmid construction and mutagenesis

SNU114 was cloned by PCR amplification using Pfu DNA polymerase (Stratagene) of yeast

genomic DNA (Promega) using primers Snu114F and Snu114B (Table S1) complementary to

regions upstream and downstream of the open reading frame and containing restriction sites for

EcoRI and BamHI. The PCR product was digested with EcoRI and BamHI, then ligated into

pRS416 (SIKORSKI and HIETER 1989) to produce pRS416-Snu114. pRS416 plasmids containing

SNU114 and each snRNA gene were made in the following manner. pRS314-U5 (m571) (O'KEEFE

et al. 1996) was digested with EcoRI/XhoI and the U5 gene fragment was ligated into EcoRI/XhoI

digested pRS416-Snu114 to generate pRS416-Snu114-U5. pRS415-U2 (MCGRAIL et al. 2006) was

digested with BamHI, filled in with T4 DNA polymerase (Roche), then digested with XhoI. The U2

gene fragment was ligated into pRS416-Snu114 digested with HindIII, filled in as above, then

digested with XhoI to generate pRS416-Snu114-U2. pRS413-U6 (MCGRAIL et al. 2006) was

digested with SacI, blunt ended with T4 DNA polymerase, then digested with XhoI. The U6 gene

fragment was ligated into pRS416-Snu114p digested with HindIII, filled in as above, then digested

with XhoI to generate pRS416-Snu114-U6. pRS416-Snu114-U4 was constructed by PCR

amplification of the U4 gene using primers U4GF and U4GR containing restriction sites for BamHI

and KpnI (Table S1). The PCR product was digested with BamHI and KpnI, then ligated into

pRS416-Snu114 to produce pRS416-Snu114-U4. The PCR product was also digested with BamHI


and ligated into a BamHI/HincII digested pRS413 to produce pRS413-U4. The identity of all

plasmids was confirmed by sequencing.

Conditionally lethal snu114 alleles were generated by identifying hydrophobic stretches of

amino acids in an alignment of SNU114 with the yeast elongation factor, EFT2. Amino acids

within hydrophobic regions were mutated individually to aspartic acid. Lethal substitutions

indicated residues that were good candidates for conditional lethality (CHAKSHUSMATHI et al.

2004). To generate conditionally lethal mutations amino acids within the hydrophobic regions of

Snu114p were mutated individually to alanine, serine, lysine or tryptophan. pBK413-Snu114 used

for mutagenesis was constructed by digesting pRS416-Snu114 with EcoRI and BamHI, then

ligating the SNU114 gene fragment into pBK413 (FRAZER and O'KEEFE 2007). Mutagenesis of

pBK413-Snu114 was by the method of Kunkel (KUNKEL 1985), using the primers listed in Table

S1. Plasmids containing snu114 mutations were named according to the amino acid substitution

introduced (e.g. V266P, P860K, see Table 2). The snu114 N-terminal deletion mutation (ΔN),

which lacked the first 128 amino acids of SNU114, was made as described previously (BARTELS et

al. 2002). Mutations snu114-12, -14, -15, -30 and -40 (P216N, L381P, K146I, G646R and M842R

respectively) were described by Brenner and Guthrie (2005). Selected temperature sensitive snu114

mutations in pBK413-Snu114 were transferred to pRS415.

Plasmids pRS314-U5, pRS415-U2, pRS413-U6 and pRS413-U4 were mutagenized as

described above, using the primers listed in Table S1. Plasmids containing mutated U5, U2, U6 or

U4 were named according to the mutation introduced (see Table 3). The identity of all mutations

was confirmed by sequencing.

The three-dimensional model of Snu114p was constructed by comparative modelling. Yeast

elongation factor 2 (Eft2p, pdb code 1n0v) (JORGENSEN et al. 2003) was selected as the template

structure as it was the best sequence match based on BLAST score (ALTSCHUL et al. 1990). The

sequences of Snu114p and Eft2p were aligned using ClustalW (LARKIN et al. 2007). The N-

terminal domain (residues 1-121) and a region from the C-terminus (residues 982-1008) were


omitted from the model as there are no equivalent residues in the crystal structure of Eft2p. The

percentage sequence identity over the aligned region was 25.3%. Regions of buried hydrophobic

amino acids within the structure of Eft2p were identified using Modeller (SALI and BLUNDELL

1993) and corresponding homologous regions in Snu114p were then identified and targeted for

mutagenesis. Ten three dimensional models were built using Modeller (SALI and BLUNDELL 1993)

and the model with lowest pseudo-energy (that when combined best fit to all input data used to

generate the model) was chosen.

Snu114 protein levels

To analyze protein levels, overnight YPD cultures of cells containing WT or mutated snu114 as the

only source of Snu114p were used to inoculate YPD cultures to an OD600 of 0.5 and grown for at

least three generations at permissive (30°) or restrictive (39°) temperatures. Cells were sedimented

by centrifugation and washed with water prior to being lysed in SDS-loading buffer with acid-

washed glass beads (Sigma) by vigorous vortexing. Cells were boiled for five minutes prior to

centrifugation to remove cell debris and extracts removed then separated on 12% acrylamide

PAGEr gels (Lonza). Levels of Snu114p were detected by western blots with rabbit anti-Snu114p

antibodies generated against a peptide encompassing the first 15 amino acids of Snu114p and

standardized by probing with mouse anti-Zwf1p antibodies (Sigma).

Primer extensions

To determine whether snu114 strains exhibited splicing defects, total RNA was extracted with hot

phenol (KOHRER and DOMDEY 1991) from strain YSNU114KO1 in the presence of pBK413-based

WT or mutated snu114 after incubation in YPD for 16 h at the restrictive temperature of 39°. Equal

amounts of total RNA was hybridized to a 32P end-labelled oligonucleotide primer, U3snoRNA-RT

(Table S1), and primer extensions were carried out as described previously (DOBBYN and O'KEEFE

2004).


Genetic analysis of synthetic lethality

YSNU114/U5KO was transformed with mutated pBK413-Snu114 and pRS414-U5 and

transformants selected on synthetic defined (SD) medium (SD-Ura-His-Trp) (BIO 101 Systems).

YSNU114/U2KO was transformed with mutated pRS413-Snu114 and pRS415-U2,

YSNU114/U6KO with mutated pRS415-Snu114 and pRS413-U6 and YSNU114/U4KO1 with

mutated pRS415-Snu114 and pRS413-U4 and the transformants selected on SD-Ura-His-Leu.

Transformants were then tested for synthetic lethality by plasmid shuffle on 5FOA. Synthetic

lethality was scored as the lack of growth after 3 days at 30°. Synthetic sickness was scored as

growth of smaller or fewer colonies compared to control sectors after 3 days at 30°.

Compensatory analysis

To determine whether the synthetic lethality observed between snu114 and snRNA mutations could

be alleviated, synthetically lethal combinations were transformed with a plasmid containing an

snRNA mutation that restored base pairing in U4/U6 stem I or U2/U6 helices. This was achieved

by producing a vector with MET15 as the selection marker. A 2491 base pair fragment containing

MET15 (COST and BOEKE 1996) was amplified by PCR from yeast genomic DNA with primers

Met15F and Met15B (Table S1). The Met15F primer contained AfeI and the Met15B primer

contained BamHI and PstI restriction enzyme recognition sites. The PCR product was digested

with BamHI and cloned into pRS414 digested with HincII and BamHI. pRS414 plasmids

containing the MET15 fragment were tested for complementation of MET15 in YSNU114/U4KO2.

The MET15 was released from a complementing plasmid with AfeI and PstI, then cloned into

pRS413 where most of the HIS3 gene had been removed by digestion with AfeI and NsiI to produce

the plasmid pRS413MET15. Mutations in the U2, U4 and U6 snRNAs were transferred into this

plasmid.


To assess suppression of Snu114-U2 synthetic lethality, YSNU114/U2KO was transformed

with synthetically lethal mutation combinations of pBK413-Snu114 and pRS415-U2, as well as

pRS413MET15 containing U6 mutations to restore U2/U6 base pairing in the presence of wild type

U6. To assess suppression of Snu114-U4 synthetic lethality, YSNU114/U4KO2 was transformed

with synthetically lethal combinations of pRS415-Snu114 and pRS413-U4, as well as

pRS413MET15 containing a U6 mutation to restore base pairing in U4/U6 in the presence of wild

type U6. To assess suppression of Snu114-U6 synthetic lethality, YSNU114/U6KO was

transformed with synthetically lethal mutation combinations of pRS415-Snu114 and pRS413-U6, as

well as pRS413MET15 containing U2 mutations to restore U2/U6 base pairing or U4 mutations to

restore U4/U6 base pairing in the presence of wild type U2 or U4. Transformants were selected on

SD-Ura-His-Leu-Met agar. Transformants were then tested for growth by plasmid shuffle on 5FOA

alongside the synthetically lethal combinations and controls. Suppression of synthetic lethality was

scored after 3 days at 30°.


RESULTS

Identification of additional snu114 mutations that are temperature sensitive and show splicing

defects at the first step of splicing

Forty-nine single and four double amino acid substitution mutations were generated within

five predicted hydrophobic regions of Snu114p: L234-I239, C264-I267, I311-A314 in domain I,

I561-I564 in domain II, and P860-I861 in domain IVa. Nineteen mutations were lethal and of the

viable alleles five were temperature sensitive (Ts) at 39° (V238D, V266P, F313Q, L563P and

P860K) and one was Ts at 37° (L563F) (Table 2). The Ts mutations mapped to different locations

within Snu114p (Figure 1A). The V238D mutation was proximal to the putative salt bridge at

D233 (with R488) and the V266P mutation was near the critical (N/T)(K/Q)XD G4 motif. This

motif is required for GTP binding and hydrolysis (FABRIZIO et al. 1997) and stabilizing residues in

the G1 motif (BOURNE et al. 1991). The F313Q mutation was adjacent to the (G/T)SAL G5 motif,

which stabilizes residues in the G4 motif (BOURNE et al. 1991), L563F was within domain II and

P860K was in domain IVa (Figure 1A). Previously published Ts snu114 alleles (K146I, P216N,

L381P, G646R and M842R) (BRENNER and GUTHRIE 2005) are positioned in different locations

within the predicted structure from the Ts alleles generated in this study (Figure 1B). An N-

terminal snu114 deletion mutation lacking the first 128 amino acids (ΔN) (BARTELS et al. 2002)

was also produced (Table 2). For clarity, snu114 alleles are annotated with their putative positions

within Snu114p, for example, V266P (G4) signifies the mutation is in G domain motif G4 and

P860K (IVa) signifies the mutation is in domain IVa.

To determine whether the temperature sensitivity of snu114 alleles was due to defects in

protein expression or in pre-mRNA splicing, a SNU114 knockout strain carrying WT or snu114

allele on a plasmid was grown at the permissive and restrictive temperatures. Four strains chosen

for analysis were V266P (G4), F313Q (G5), L563F (II) and P860K (IVa) (Figure 2A) as these were

the most reliable Ts mutations that spanned the predicted three dimensional structure of Snu114p

(Figure 1B). Protein levels in non-Ts (WT and V266K (G4)) and Ts strains were equivalent at the


permissive temperature as demonstrated by western blotting (Figure 2B, lanes 1-7). When cells

were grown at the restrictive temperature for at least three generations, protein levels remained

constant in WT (Figure 2B, lane 8), non-Ts (Figure 2B, lane 9) and Ts strains (Figure 2B, lanes 10-

11, 13-14). The exception was the mutation L563P (II) (Figure 2B, lanes 5 and 12), which

displayed a slight decrease in Snu114p levels compared to WT at both permissive and restrictive

temperatures so was excluded from further investigations. Thus the Ts phenotype observed in

snu114 mutant strains was not due to aberrant levels of protein expression. To assess whether the

temperature sensitivity of snu114 mutant strains was due to a defect in pre-mRNA splicing, total

RNA was extracted from strains grown overnight at the restrictive temperature. The levels of

spliced and unspliced U3A and U3B snoRNAs were analyzed by primer extension with a primer

complementary to their common exon 2. In WT extracts, only a small amount of unspliced U3A or

U3B precursor was detectable (Figure 2C, lane 3). In extracts from snu114 mutant strains, there

was a significant accumulation of unspliced U3A and U3B precursor (Figure 2C, lanes 4-7)

compared to levels in WT extracts, indicating that splicing was inhibited and that the block in

splicing was prior to the first step. Note that a second step block could not be detected by primer

extension in this manner.

snu114 mutations display synthetic lethality with U4 snRNA mutations that alter U4/U6 base

pairing

The interactions of U4 with U6 serve to regulate the U6 snRNA during assembly and

activation of the spliceosome. Unwinding of U4 from U6 during spliceosome assembly is catalyzed

by the DExD/H-box helicase Brr2p (LAGGERBAUER et al. 1998; RAGHUNATHAN and GUTHRIE

1998) which is regulated by the guanine nucleotide state of Snu114p (SMALL et al. 2006). It is not

clear whether the regulation of Brr2p activity by Snu114p is through direct or indirect interactions

with Brr2p, another protein, or the U4 or U6 snRNAs. Genetic interactions were investigated

between snu114 alleles and mutations in U4 positions that base pair with U6 snRNA in stem I and


II of the U4/U6 complex and U4 mutations adjacent to stem I (HU et al. 1995; LI and BROW 1996;

SHANNON and GUTHRIE 1991) as shown in Figure 3. A mutation adjacent to U4 stem I, U4-cs1

(A66U,A67U,A68G), stabilizes U4/U6 stem I by increasing base pairing with the U6 ACAGAGA

region thereby blocking U4/U6 dissociation early in splicing (KUHN et al. 1999; LI and BROW

1996). Genetic analysis revealed that K146I (G1), V266P (G4) and F313Q (G5) demonstrated

synthetic lethality with U4-cs1 (Figure 3 and Table 4). Interestingly, P216N (G2) and L381P (G’’)

were not synthetically lethal but noticeably sick with U4-cs1 (Figure 3 and Table 4). In order to

analyze the effects of other mutations that extend base pairing in the same region of stem I as U4-

cs1, mutations U4-U64C, U4-G65A and U4-U64C,G65A were tested for synthetic lethality with

snu114 mutations. Genetic analysis revealed that K146I (G1) was synthetically lethal with the

double mutation U4-U64C,G65A and synthetically sick with U4-U64C and U4-G65A (Figure 3 and

Table 4). Therefore, it appears that extending U4/U6 base pairing in stem I results in synthetic

lethality with snu114 G domain mutations.

Although many positions in U4 stem I have been shown to be tolerant to mutation, only U4-

G58 has been shown to have any phenotypic effects (HU et al. 1995). Analysis of genetic

interactions between mutations in snu114 and U4 stem I (U57A, G58A, C59U, U60C, U64G)

revealed that only K146I (G1) was synthetically lethal with U4-G58A (Figure 3 and Table 4). No

other snu114 alleles were synthetically lethal with U4-G58A (Table 4) although V266P (G4),

F313Q (G5), G646R (III) and M842R (IVa) were synthetically sick (Figure 3 and Table 4). While

the U4-U57A, U4-G58A, U4-C59U, U4-U60C and U4-U64G mutations are all predicted to disrupt

base pairing between U4 and U6, U4-G58A is the only mutation that disrupts stem I leading to a Ts

phenotype (HU et al. 1995) and displays synthetic lethality and sickness with snu114 mutations in

the G domain and domains III and IVa. These results indicate that only mutation at this nucleotide

of U4 in stem I exacerbates snu114 mutations.

The U4 stem II position G14 is responsible for U4 snRNA functions early in splicing (HU et

al. 1995). Analysis of genetic interactions between snu114 alleles and U4-G14C, which disrupts


U4/U6 stem II base pairing, thus destabilizing the U4/U6 complex (SHANNON and GUTHRIE 1991;

WERSIG and BINDEREIF 1990), revealed that no snu114 mutation displayed synthetic lethality with

this U4 mutation (Table 4).

snu114 mutations display synthetic lethality with U6 snRNA mutations important for base

pairing with the 5’ splice site and that alter U2/U6 base pairing

The role of U6 snRNA is pivotal in the spliceosome as it is involved in three important base

pairing interactions during splicing: with U4 in the U4/U6 di-snRNP (MADHANI et al. 1990), with

the 5’ splice site via a conserved single-stranded ACAGAGA (A47-A53) sequence following tri-

snRNP incorporation into the spliceosome (MADHANI et al. 1990; SAWA and SHIMURA 1992;

SONTHEIMER and STEITZ 1993) and with U2 snRNA in helix I in the active site before the first step

of splicing (MADHANI and GUTHRIE 1992). As Snu114p regulates Brr2p activity and, as shown

above, interacts genetically with mutations in U4 that perturb the U4/U6 complex we determined

whether SNU114 interacts genetically with U6.

Genetic interactions were investigated between snu114 alleles and mutations in the U6

ACAGAGA sequence at positions A47, G50 and G52. The analysis revealed synthetic lethality

between V266P (G4) and U6-G50U and U6-G52U as well as synthetic lethality between F313Q

(G5) and U6-A47G, U6-G50U and U6-G52U (Figure 4 and Table 5). Although L563F (II) and

M842R (IVa) were synthetically sick with U6-A47G, and K146I was synthetically sick with U6-

G50U and U6-G52U, no other snu114 mutations were synthetically lethal/sick with U6

ACAGAGA mutations (Table 5). Interestingly, U6-G50U and U6-G52U both potentially increase

the base pairing of U6 with the conserved 5’ splice site sequence. Therefore, it appears that

stabilization of the interaction of U6 with the 5’ splice site exacerbates snu114 G domain mutations.

Although U6-A53 is included in the ACAGAGA sequence, the mutation U6-A53U is

expected to extend the base pairing between U2 and U6 in helix I. Genetic interactions with this U6

mutation showed that K146I (G1), V266P (G4) and F313Q (G5) were synthetically lethal and


L381P (G’’) was synthetically sick (Figure 4). This suggests that extending U2/U6 helix Ia

exacerbates snu114 G domain mutations.

Genetic interactions were also investigated between snu114 mutations and U6 insertion

mutations at positions A53/U54 and G55/A56 to determine whether extending the distance between

the ACAGAGA sequence and U4/U6 stem I or U2/U6 helix I had any effects. No synthetic

lethality was observed between snu114 alleles and U6 mutations Ins 1U A53/U54 and Ins 1U

G55/A56 (Table 5). Substitution mutations at U6-G55 and U6-A56 are expected to weaken stem I

by disrupting base pairing between U4 and U6 snRNAs (LI and BROW 1996) and insertion

mutations at U6-G55/A56 have been shown to progressively inhibit the first step of splicing

(MCGRAIL et al. 2006). Synthetic lethality was observed between snu114 mutations and

substitution mutations at U6-U54 and U6-G55 which would be expected to extend the base pairing

at the top of U4/U6 stem I or disrupt the base pairing in U2/U6 helix I. In particular, K146I (G1)

was synthetically lethal with U6-U54C, K146I (G1) and V266P (G4) with U6-G55A and K146I

(G1) and V266P (G4) with U6-U54C,G55A (Figure 4). In addition, F313Q (G5) was synthetically

sick with all three substitution mutations U6-U54C, U6-G55A and U6-U54C,G55A (Table 5). This

suggests that extending the base pairing of U4/U6 at the top of stem I or disrupting the base pairing

of U2/U6 helix Ia exacerbates snu114 G domain mutations. These results substantiate the results

obtained with U4-U64C and U4-G65A mutations, which also extend the base pairing of U4/U6

stem I and result in synthetic lethality with snu114 G domain mutations (see previous section) and

the results obtained with U2/U6 helix Ia mutations (see following section).

Further analysis revealed synthetically lethal interactions between snu114 alleles and U6

mutations in the region that mutually exclusively base pairs with U4 in stem I prior to spliceosome

activation or with U2 in helix I. Double mutations at U6-A56,U57 are conditionally lethal but can

be suppressed by U2 mutations which restore helix Ia base pairing (MADHANI and GUTHRIE 1992).

Our analyses revealed that K146I (G1) and V266P (G4) were synthetically lethal with U6-

A56C,U57C (Figure 4 and Table 5). The ΔN, P216N (G2), F313Q (G5), L381P (G’’) and M842R


(IVa) mutations were also synthetically sick with U6-A56C,U57C (Figure 4), but no other snu114

alleles were synthetically lethal with U6-A56C,U57C (Table 5). In addition, the only other genetic

interaction in this U6 region was a synthetically sick interaction between V266P (G4) and U6-C58U

(Table 5).

Mutations in snu114 and the U6-AGC triad that base pairs with U4 to form stem I in the

U4/U6 complex or with U2 to form helix Ib in the U2/U6 complex were investigated for synthetic

lethality. In yeast, substitutions in U6-G60 and U6-C61 result in defects in 5’ splice site cleavage

and substitutions in U6-A59 cause defects in exon ligation and inhibit the second step of splicing

(FABRIZIO and ABELSON 1990; MCGRAIL et al. 2006). Synthetic lethality was observed between

both V266P (G4) and F313Q (G5) and U6-A59C (Figure 4 and Table 5). In addition, ΔN and

K146I (G1) were synthetically sick with U6-A59C (Figure 4 and Table 5). Further analysis

revealed that all snu114 G domain mutations, as well as ΔN and M842R (IVa), were synthetically

lethal with U6-C61G (Figure 4 and Table 5). No other snu114 alleles were synthetically lethal with

U6-C61G (Table 5). Overall, it appears that snu114 mutations in the N-terminus and the G domain

are exacerbated by mutations in U6 that disrupt U2/U6 base pairing within helices Ia and Ib.

Finally, genetic interactions between snu114 and U6-A62C were investigated to assess

whether disrupting the base pairing with U4-U57 at the base of U4/U6 stem I or with U6-C85 in U6

internal stem loop (ISL) or whether extending the base pairing in helix Ib with U2-G20 caused

synthetic lethality. There was no observed synthetic lethality between U6-A62C and any snu114

mutation (Table 5). This supports the lack of synthetic lethality of snu114 G domain mutations

with U4-U57A and suggests that Snu114p does not interact genetically with U6-ISL at position U6-

A62. These data also suggest that although extending the base pairing of U2/U6 helix Ia with U6

mutations exacerbated snu114 G domain mutations, extending the base pairing of U2/U6 helix Ib

does not have the same effect.


snu114 mutations display synthetic lethality with U2 snRNA mutations that alter U2/U6 base

pairing

Two important base pairing interactions of the U2 snRNA are with the branchpoint

sequence (BPS) early in spliceosome assembly (MOORE et al. 1993) and with U6 to form helix Ia

and Ib in the spliceosome active site (MADHANI and GUTHRIE 1992). The U2/U6 helices serve to

bring the 5’ exon together with the BPS adenosine ready for the first step of splicing (MADHANI and

GUTHRIE 1992). We have shown that mutations in the N-terminal and G domains of Snu114p

display synthetic lethality with mutations in U6 that increase base pairing with the 5’ splice site and

disrupt or extend U2/U6 base pairing. Next we determined whether there were genetic interactions

between mutations in snu114 and the U2 snRNA. Mutations in snu114 were tested for synthetic

lethality with U2 helix Ib mutations at positions U2-C22 and U2-U23 (MADHANI and GUTHRIE

1992). U2-C22 forms intramolecular base pairing interactions with U6-AGC triad nucleotide U6-

G60 of helix Ib. U2-U23 is implicated in 5’ splice site recognition, base pairs with U6-A59 of helix

Ib before the second step of splicing, crosslinks to the 3’ exon in the 3’ exon-lariat intermediate and

is important for the second step of splicing (LUUKKONEN and SÉRAPHIN 1998; MADHANI and

GUTHRIE 1992; MADHANI and GUTHRIE 1994; NEWMAN et al. 1995). Our analyses revealed

synthetic lethality between V266P (G4) and both mutations at U2-C22 and U2-U23, while F313Q

(G5) was synthetically sick with both mutations at U2-U23 (Figure 5 and Table 6). No other

snu114 alleles were synthetically lethal with these U2 mutations (Table 6). Interestingly, there was

no synthetic lethality between any snu114 alleles and several mutations in the bulge, positions U2-

U24 and U2-A25 which link U2/U6 helix Ia and Ib (Table 6). These results suggest, similar to that

found with U6 helix Ib mutations, that snu114 G domain mutations display synthetic lethality with

U2 mutations that disrupt U2/U6 base pairing.

Genetic interactions were also investigated between snu114 alleles and U2-A27C,U28C

positions involved in base pairing with U6 in helix I. A second U2 mutation in this region, U2-Ins

1U U28/C29, is located in a position where increasing insertions block the second step of splicing


(MCGRAIL et al. 2006; MCPHEETERS and ABELSON 1992). Genetic analysis revealed that both

V266P (G4) and F313Q (G5) were synthetically lethal with U2-A27C,U28C (Figure 5 and Table

6). In addition, V266P (G4) was also synthetically lethal with U2-Ins 1U U28/C29 (Figure 5 and

Table 6). No other snu114 alleles were synthetically lethal with these U2 mutations (Table 6).

These results suggest that snu114 G domain mutations are exacerbated by mutations in U2 that

disrupt U2/U6 helix Ia. Further analysis revealed that there was no synthetic lethality between any

snu114 alleles and insertion mutations U2-Ins 1U A30/A31 and U2-Ins 1U G32/U33, which are

situated between helix I and the BPS (Table 6). However, synthetic lethality was observed between

V266P (G4) and U2-A31U and L381P (G’’) and U2-A31U, which extends the base pairing of

U2/U6 helix Ia (Figure 5). These results support the idea that mutations in U2 that disrupt or extend

U2/U6 base pairing display synthetic lethality with snu114 G domain mutations.

snu114 mutations display synthetic lethality with mutations in U5 snRNA loop 1 and internal

loop 1

U5 snRNA contributes to the catalytic core of the spliceosome through U5 loop 1 which is

required for tethering the 5’ and 3’ exons for ligation during the second step of splicing (NEWMAN

and NORMAN 1992; O'KEEFE and NEWMAN 1998). Protein interactions between Snu114p, Prp8p

and Brr2p are well known (BRENNER and GUTHRIE 2005; LIU et al. 2006) but the only known direct

interaction of Snu114p with an snRNA is a crosslink with U5-C79 of U5 internal loop 1 (IL1),

found at the base of the stem that carries the important U5 loop 1 (DIX et al. 1998). To determine

whether Snu114p displays synthetic lethality with any mutation in U5, genetic analyses were

carried out with snu114 alleles and twenty-five mutations that spanned the entire structure of the U5

snRNA.

Ten mutations in U5 loop 1 were tested for genetic interactions with nine snu114 alleles

(Figure 6A and Table 7). Our analyses revealed that V266P (G4) was synthetically lethal with

deletion, substitution and insertion mutations in U5 loop 1, while ΔN was synthetically lethal only


with U5 loop 1 double deletion mutations (Figure 6B and Table 7). In particular both V266P (G4)

and ΔN were synthetically lethal with U5-ΔC94,C95 and U5-ΔU96,U97, nucleotides important for

tethering and aligning the 5’ and 3’ exons during splicing (NEWMAN and NORMAN 1992; NEWMAN

et al. 1995; O'KEEFE and NEWMAN 1998; SONTHEIMER and STEITZ 1993). V266P (G4) was also

synthetically lethal with U5-A100U,C101G, and U5-Ins 1U G93/C94, positions opposite each other

in U5 loop 1 (Figure 6A and Table 7). No other snu114 alleles displayed synthetic lethality with

any of the U5 loop 1 mutations tested (Table 7). Thus, snu114 N-terminal and G domain mutations

are exacerbated by mutations in U5 loop 1.

As Snu114p was shown to crosslink to U5 IL1 (DIX et al. 1998), genetic interactions

between mutations in snu114 and U5 IL1 were investigated. Included were eight substitution and

deletion mutations that spanned positions U5-C79 and U5-G80. Increasingly larger deletions on the

5’ side of U5 IL1 at or near position U5-C79 were progressively synthetically sick with M842R

(IVa) (Figure 6C and Table 7).

As mutations on the 5’ side of U5 IL1 did not display synthetic lethality with mutations in

snu114, mutations on the 3’ side of U5 IL1 were investigated. Three deletion mutations on the 3’

side of IL1, U5-ΔC111, U5-ΔC112,G113 and U5-ΔC111-G113 were produced. The U5-ΔC111-

G113 mutation was lethal and not studied further (data not shown). The analysis revealed that only

M842R (IVa) was synthetically lethal with U5-ΔC111 but G646R (III), M842R (IVa), P860K

(IVa), as well as ΔN, were synthetically lethal with the U5 IL1 allele, U5-ΔC112,G113 (Figure 6D

and Table 7). Significantly, no snu114 G domain mutations displayed synthetic lethality with any

mutations in U5 IL1 (Table 7). Thus, our results indicate that snu114 mutations in the N-terminal

domain and domains III and IV exacerbate mutations in the 5’ and 3’ side of U5 IL1 whereas

snu114 mutations in the N-terminal domain and G domain exacerbate mutations in U5 loop 1.


Compensatory mutations that restore base pairing in U2/U6 helix I suppress synthetic

lethality with snu114 mutations

One prediction of the hypothesis that Snu114p senses the state of important RNA/RNA

interactions in the spliceosome is that where synthetic lethality was caused by disruption of base

pairing, restoration of base pairing with a compensatory mutation would suppress the synthetic

lethality. This would support the theory that the synthetic lethality was caused by disruption of base

pairing and not an additional effect of the mutations. To test whether compensatory mutations

could suppress synthetic lethality observed between snu114 and snRNA mutations predicted to

disrupt base pairing, a plasmid containing a compensatory snRNA mutation designed to restore base

pairing was transformed with the synthetic lethal combinations. The compensatory snRNA

mutation was introduced in the presence of the wild-type copy of that gene.

A number of synthetic lethal combinations found between snu114 and snRNA mutations

predicted to disrupt base pairing were not suppressed by compensatory mutations designed to

restore base pairing. Synthetic lethality between K146I (G1) and U4-G58A was not suppressed by

restoring U4/U6 stem I with U6-C61U (data not shown). Synthetic lethality between snu114

mutations and U6-A56C,U57C was not suppressed by restoring U4/U6 stem I with U4-G62,U63G

(Figure 7A). Synthetic lethality of V266P (G4) and F313Q (G5) with U6-A59C was not suppressed

by restoring U4/U6 stem I with U4-U60G (Figure 7B). Synthetic lethality between snu114

mutations and U6-C61G was not suppressed by restoring U4/U6 stem I with U4-G58C or restoring

U2/U6 helix Ib with U2-G21C (data not shown). Synthetic lethality of V266P (G4) with U2-C22A

and U2-C22G was not suppressed by restoring U2/U6 helix Ib with U6-G60U or U6-G60C,

respectively (data not shown). Synthetic lethality of V266P (G4) with U2-U23C and U2-U23G was

not suppressed by restoring U2/U6 helix Ib with U6-A59G or U6-A59C, respectively (data not

shown). Synthetic lethality of snu114 mutations with U2-A27C,U28C was not suppressed by

restoring base pairing in U2/U6 helix Ia with U6-A56G,U57G (data not shown). Taken together,

these results indicate that synthetic lethality between some snu114 and snRNA mutations is not


solely due to the disruption of base pairing and may suggest an additional function for these snRNA

positions besides base pairing.

In contrast to the examples given above a number of synthetic lethal combinations were

suppressed by compensatory mutations. Synthetic lethality between snu114 mutations and U6-

A56C,U57C was suppressed when base pairing in U2/U6 helix Ia was restored with U2-

A27G,U28G (Figure 7A). Synthetic lethality of V266P (G4) and F313Q (G5) with U6-A59C was

suppressed by restoring base pairing in U2/U6 helix Ib with U2-U23G (Figure 7B). These results

demonstrate that the Snu114p G domain can sense the state of base pairing in U2/U6 helix Ia and

Ib.


DISCUSSION

Our aim was to determine the genetic interactions of SNU114 with the snRNAs of the spliceosome.

Snu114p is an integral spliceosomal protein that is present throughout the splicing cycle and is

known to regulate important snRNA interactions required for activation and disassembly of the

spliceosome. By producing new conditional snu114 alleles and combining them with known

conditional snu114 alleles, we have identified an extensive genetic interaction network between

SNU114 and numerous conditional mutations in the U2, U4, U5 and U6 snRNAs. We propose that

the G-domain of Snu114p senses the state of important RNA/RNA interactions formed by the

spliceosome (Figure 8).

Synthetic lethality of SNU114 with mutations that extend U4/U6 stem I confirms its role in

U4/U6 unwinding

Brr2p dependent unwinding of U4/U6 base pairing during spliceosome activation is

regulated by the guanine nucleotide state of Snu114p (SMALL et al. 2006), therefore, we first

investigated genetic interactions between snu114 and various U4 snRNA mutations. Synthetically

lethal interactions of snu114 mutations in the G domain were found with the U4 mutations, U4-cs1

and U4-U64C,G65A, which both extend the U4/U6 stem I. In addition, synthetic lethality was

observed with reciprocal mutations in U6-U54 and U6-G55 which also extend the base pairing with

U4 in stem I of the U4/U6 complex. These patterns of synthetic lethality suggest that G domain

residues in Snu114p are involved in U4/U6 unwinding. This supports work which implicates the

guanine nucleotide state of Snu114p in Brr2p regulation (SMALL et al. 2006) and work with a

snu114 GTP binding mutation within G domain motif G4 that blocks U4/U6 unwinding (BARTELS

et al. 2003). That the snu114 N-terminal deletion mutation did not interact genetically with U4-cs1

or U4-U64C,G65A was surprising since this snu114 allele has been shown to block U4/U6

unwinding at elevated temperature (BARTELS et al. 2002). Intriguingly, in another study no genetic

interactions were identified between snu114 G domain alleles and the cold sensitive BRR2 allele


brr2-1, which decreases U4/U6 unwinding activity of Brr2p and is synthetically lethal with U4-cs1

(BRENNER and GUTHRIE 2005; KUHN and BROW 2000; RAGHUNATHAN and GUTHRIE 1998).

Rather, the brr2-1 allele is synthetically lethal with a C-terminal deletion mutation of snu114

(BRENNER and GUTHRIE 2005), an interaction which has been substantiated by two hybrid assays

(LIU et al. 2006). Therefore, as known genetic and physical interactions of Snu114p with Brr2p

appear to be outside the G domain of Snu114p, our genetic interactions of Snu114p with U4 suggest

that the G domain may sense the state of the U4/U6 helix and signal to Brr2p when to unwind it.

This signal may be transmitted through the domains of Snu114p and Brr2p that have been shown to

interact (BRENNER and GUTHRIE 2005; LIU et al. 2006) or through another protein mediator, such as

Prp8p. Many mutations throughout Prp8p suppress the U4-cs1 mutation (KUHN and BROW 2000;

KUHN et al. 1999) and prp8-1 and prp8-brr mutations impair formation of U5 snRNP and tri-

snRNPs (BROWN and BEGGS 1992; COLLINS and GUTHRIE 1999). Snu114p has been shown to

interact genetically with these prp8 mutations through its C-terminal domain (BRENNER and

GUTHRIE 2005). This suggests that Snu114p may directly or indirectly sense U4/U6 base pairing

with its G domain and signal the state of the U4/U6 helix through its C-terminus to Brr2p. This

signalling to Brr2p may require a structural rearrangement between the G domain and C-terminus of

Snu114p and occur either directly or through Prp8p. Recent in vitro evidence suggests that the C-

terminus of Prp8p is required for the ATP-dependent unwinding of U4/U6 by Brr2p (MAEDER et al.

2009) and electron microscopy localisation of Snu114p to a hinge region of the yeast tri-snRNP

suggests Snu114p may promote rearrangements in the tri-snRNP (HACKER et al. 2008).

snu114 mutations are exacerbated by only one U4/U6 stem I mutation

Whereas mutations that extend the U4/U6 complex are synthetically lethal with snu114 G

domain mutations, the U4-G58A mutation, which is Ts and shows a block in splicing before or at

the first step of splicing (Hu et al., 1995), is synthetically lethal or sick with snu114 mutations in the

G domain, domain III and domain IVa. The compensatory mutation U6-C61U that could restore


base pairing in U4/U6 stem I with U4-G58A did not rescue synthetic lethality. This suggests that in

addition to base pairing with U6-C61 in stem I, U4-G58 has another role during splicing. This role

may be in steps prior to tri-snRNP assembly or in steps following tri-snRNP assembly but prior to

spliceosome activation, since the U4-G58C mutation leads to accumulation of the B complex (Hu et

al., 1995). Other mutations in U4 stem I predicted to disrupt base pairing with U6 do not display

synthetic lethality with snu114 mutations. Further work is required to understand the specific

sensitivity of only position U4-G58A in U4 stem I and the reason for its genetic interactions with

SNU114.

Snu114p senses the state of U6-5’ splice site and U2/U6 base pairing

In addition to sensing the state of U4/U6, we show that Snu114p interacts genetically with

key positions in the U6-ACAGAGA that base pairs with the 5’ splice site. This suggests that

successive interactions of Snu114p with U4 and U6 may facilitate the transitions that occur during

spliceosome activation. Notably, Prp8p also interacts with the U6-ACAGAGA sequence: both

Prp8p and U6-A51 crosslink to the 5’ splice site before both first and second steps of splicing (KIM

and ABELSON 1996; REYES et al. 1999; SONTHEIMER and STEITZ 1993). However, since prp8

mutations only partially suppress U6-A51 mutations which cause 5’ splice site misalignment and

result in defective exon ligation (COLLINS and GUTHRIE 1999), Snu114p, together with Prp8p, may

coordinate U6 at the 5’ splice site. That snu114 alleles interact genetically with mutations of U6-

G52 but not with U2-A25 suggests that the Snu114p genetic interactions with U6-G52 occur during

U6-5’ splice site base pairing rather than during formation of a tertiary interaction with U2 for the

second step of splicing (MADHANI and GUTHRIE 1994). Additional mutations in U6 or the pre-

mRNA designed to stabilize the U6-5’ splice site interactions combined with snu114 G-domain

mutations would provide further support for the hypothesis that the G-domain senses U6-5’ splice

site interactions.


Brr2p dependent unwinding of U2/U6 base pairing during spliceosome disassembly is also

regulated by the guanine nucleotide state of Snu114p (SMALL et al. 2006), therefore, we

investigated genetic interactions between snu114 and various U2 and U6 snRNA mutations that

influence U2/U6 base pairing. Formation of U2/U6 helix I is important for promoting the first and

second chemical steps of splicing (MADHANI and GUTHRIE 1992; MEFFORD and STALEY 2009;

RYAN and ABELSON 2002) and unwinding of the U2/U6 helix I is required for disassembly of the

spliceosome (SMALL et al. 2006). Synthetic lethality is only observed between N-terminus and G

domain snu114 mutations in combination with mutations in U2 and U6 that are predicted to disrupt

or stabilize U2/U6 helix I base pairing. The synthetic lethality patterns observed between Snu114p

with U6 and U2 helix I mutations suggest that Snu114p senses the state of U2/U6 base pairing

interactions. This hypothesis is supported by the suppression of synthetic lethality by compensatory

mutations that restore base pairing in U2/U6 helix I. Therefore, at least for the U2/U6 helix I it is

clear that synthetic lethality was solely due to the disruption of base pairing between U2 and U6 in

this region. The lack of suppression of synthetic lethality by compensatory mutations at other

positions predicted to restore base pairing does not preclude a role for Snu114p in sensing base

pairing at these positions. Rather it suggests a possible additional role, besides base pairing, for the

positions where synthetic lethality can not be suppressed by compensatory mutations.

The specificity of genetic effects of U6 mutations to the U2/U6 conformation is confirmed

by the lack of genetic interactions between Snu114p and U4 in U4 stem I. Although positions in

U4 stem I which participate in the U4/U6 complex opposite U6-A56,U57 were not assayed, three

other U4 stem I alleles displayed no synthetic lethality with snu114 alleles. This suggests that

SNU114-U6 genetic interactions are related to U6 in the U2/U6 conformation rather than the U4/U6

conformation. That synthetically lethal effects are specific to disruption of U2/U6 helix I is

supported by the lack of synthetic lethality with mutations and deletions at U2-U24 and U2-A25

which form a non-base paired bulge in U2/U6 helix I.


Three differences are observed between U2 and U6 synthetically lethal and compensatory

interactions. First, the snu114 N-terminus deletion mutation is synthetically lethal with mutations

in U6 that disrupt base pairing whereas there is no synthetic lethality of the N-terminus deletion

mutation with U2 mutations that disrupt base pairing. Second, insertion mutations in opposite sides

of helix Ia display different genetic effects with SNU114. An insertion mutation of U6 in helix Ia

(U6-Ins 1U A53/U54) is viable with all snu114 mutations tested. On the other hand, an insertion

mutation of U2 in helix Ia (U2-Ins 1U U28/A29) displays synthetic lethality with snu114 G domain

mutation V266P (G4). It is known that insertions at this location in U6 inhibit the first step of

splicing whereas insertions at this location in U2 inhibit the second step of splicing (MCGRAIL et al.

2006; MCPHEETERS and ABELSON 1992). Third, suppression of synthetic lethality by compensatory

mutations was successful with mutations in U2 that restored base pairing with U6 mutations in helix

Ia and Ib, however, mutations in U6 that restored base pairing with U2 mutations in helix Ia and Ib

were not found to suppress synthetic lethality with snu114 mutations. Overall, these differences

suggest additional roles for nucleotides in U2 that form helix Ia and Ib during splicing. These roles

could be manifested when U2 and U6 are not base paired either before formation of U2/U6 helix I,

after formation of U2/U6 helix I or when U2/U6 helix I is destabilized by Prp16p between the

catalytic steps of splicing (MEFFORD and STALEY 2009).

Snu114p interactions and orientation with U5 snRNA

As Snu114p is an integral protein of the U5 snRNP, we investigated synthetic lethality

between snu114 and twenty-five U5 mutations. Mutations in U5 displayed synthetic lethality with

the snu114 N-terminal deletion mutation and G domain mutations. U5 was the only snRNA to

show synthetic lethality with the snu114 C-terminal P860K (IVa) mutation. The snu114 G domain

mutation V266P (G4) was synthetically lethal with U5 loop 1 mutations while the snu114 C-

terminal domain mutations G646R (III) and P860K (IVa) were synthetically lethal with a U5 IL1

mutation. This suggests an orientation of Snu114p with U5 within the spliceosome such that U5


links the G domain to the other domains of Snu114p. We observed spatially distinct genetic

interactions between the C-terminus of Snu114p, which displayed synthetic lethality with U5 IL1,

and the G domain which did not. This orientation suggests that U5 and Snu114p may work

together to transmit information from the G domain to the C-terminal domain which has been

shown to interact with other splicing factors (BRENNER and GUTHRIE 2005; LIU et al. 2006). It is

possible that the state of the snRNAs sensed through the G domain of Snu114p is transduced

through the C-terminal end of Snu114p, perhaps through a structural change/domain shift, which is

the mechanism of function of other regulatory GTPases (JORGENSEN et al. 2003).

The SNU114 N-terminal domain also interacts genetically with U5 IL1 which suggests that

it may also be involved in contacting U5 in this region along with U5 loop 1. This is the first

evidence that suggests a conformation for the N-terminal domain of Snu114p (Figure 8), which

cannot be modelled on EF-2. Whether the N-terminal domain interacts with U5 loop 1 and U5 IL1

at the same or at different times is unknown. For both the N- and C-terminal domains of Snu114p

to interact with U5 IL1 suggests a long range intramolecular interaction such that the N-terminus

and domain IV come into close proximity with U5 IL1 if these interactions are simultaneous. These

interactions may also involve Prp8p since it has been found to crosslink to position C112 in U5 IL1

(DIX et al. 1998). The only other genetic interactions found with the snu114 ΔN mutation were

with mutations in the U6 snRNA. The fact that no U2 or U4 synthetically lethal interactions were

found with the snu114 ΔN mutation suggests that the Snu114p N-terminus may be involved in U5

and U6 interactions at the 5’ splice site.

We have identified an extensive genetic interaction network between the spliceosomal

GTPase Snu114p and the snRNAs of the spliceosome (Figure 8). By combining four new snu114

mutations with five previously known mutations we have distinguished different roles for distinct

regions of Snu114p and propose an orientation for Snu114p with U5 snRNA. It appears that

Snu114p recognizes the state of important RNA/RNA interactions within the spliceosome (Figure

8). Snu114 G domain mutations exacerbate mutations in U4 that change U4/U6 and U2/U6 base


pairing. Snu114 G domain mutations also exacerbate mutations in U6 that increase base pairing

with the 5’ splice site. Mutations in the snu114 G domain and N-terminal domain exacerbate

mutations in U5 at positions which align the exons in the catalytic core. Thus the Snu114p G

domain may have a significant role in signalling the state of the snRNAs throughout splicing

(Figure 8). Additionally, snu114 mutations in the N-terminal domain and domains III and IV

exacerbate mutations in U5 internal loop 1 which could serve to link the N- and C-terminal domains

of Snu114p functionally. These genetic interactions with U5 also provide a structural link between

the G domain and C terminal domain, and therefore a link between the proposed sensing and

transducing regions of Snu114p. Thus by analysis of the genetic interactions between distinct

domains in Snu114p and the snRNAs of the spliceosome we have defined a functional and spatial

network of interactions that we propose is essential for spliceosome function. Further work is

required to test the new snu114 mutations with alleles in proteins that have been found to interact

with Snu114p previously (BRENNER and GUTHRIE 2005). This work would establish how the

genetic interactions we found between Snu114p and snRNAs fit into the network of Snu114p-

protein interactions in the spliceosome and would provide an insight into whether these genetic

interactions represent direct or indirect physical interactions with Snu114p. Conversely, genetic

interactions between snRNA mutations and other splicing protein alleles should be investigated to

discover how the Snu114p-snRNA interactions fit in with other potential snRNA-protein networks.


SUPPLEMENTARY DATA

Supplementary data are available at the Genetics journal online.


ACKNOWLEDGEMENTS

We thank Riz Alvi for constructing the U5KO, U2KO and U6KO strains, Moses Tandiono for

constructing the YSNU114/U4KO strains and pRS416-Snu114-U4 plasmid and members of the

O’Keefe lab for critical reading and advice on the manuscript. This work was funded by a grant to

RTO by the Wellcome Trust (079536).


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TABLE 1. Yeast strains used in this study

Strain Genotype Reference Y25023 MATa/α; his3Δ1/ his3Δ1; leu2Δ0/leu2Δ0;

lys2Δ0/LYS2; MET15/met15Δ0; ura3Δ0/ura3Δ0; YKL173w::kanMX4/YKL173w

EUROSCARF

YSNU114KO1 MATa; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; YKL173w::kanMX4; pRS416-Snu114

This study

YSNU114KO2 MATα; his3Δ1; leu2Δ0; ura3Δ0; YKL173w::kanMX4; pRS416-Snu114

This study

YU5KO (YROK2) MATa; his3Δ200; leu2Δ1; trp1Δ63; ura3-52; snr7::kanMX4; pRS416-U5

O’KEEFE, 2002

YU2KO MATa; snr20::kanMX4; pRS416-U2 This study YU6KO MATa; his3Δ1; leu2Δ0; lys2Δ0; met15Δ0;

snr6::kanMX4; pRS416-U6 This study

YSNU114/U5KO MATα; his3Δ200; leu2Δ0; trp1Δ63; ura3Δ0; YKL173w::kanMX4; snr7::kanMX4; pRS416-Snu114-U5

This study

YSNU114/U2KO MATα; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; YKL173w::kanMX4; snr20::kanMX4; pRS416-Snu114-U2

This study

YSNU114/U6KO MATa; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; YKL173w::kanMX4; snr6::kanMX4; pRS416-Snu114-U6

This study

YSNU114/U4KO1 MATα; his3Δ1; leu2Δ0; ura3Δ0; YKL173w::kanMX4; snr14::hphNT1; pRS416-Snu114-U4

This study

YSNU114/U4KO2 MATα; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; YKL173w::kanMX4; snr14::hphNT1; pRS416-Snu114-U4

This study


TABLE 2. snu114 mutations used in this study snu114 mutation Location Viability Temperature

sensitivitya Reference

ΔN Deletion of N-terminal domain Viable 40 BARTELS et al., 2002 K146I snu114-15, Domain I (G domain) Viable 37 BRENNER and GUTHRIE, 2005 P216N snu114-12, Domain I (G domain) Viable 37 BRENNER and GUTHRIE, 2005 V235D Domain I, G domain Viable 40 This study L236D/P Viable 40 This study L236E Viable This study V238D Viable 39 This study V238E/P Viable This study C264D Viable 40 This study F265D Lethal ND This study V266D/H/W Lethal ND This study V266A/Y Viable This study V266F/K/S Viable 40 This study V266P Viable 39 This study I312D Viable 40 This study I312E/P Viable This study F313D/H/K/R/S Lethal ND This study F313A/E Viable 40 This study F313Q Viable 39 This study F313T/W Viable This study L381P snu114-14, Domain I (G” domain) Viable 39 BRENNER and GUTHRIE, 2005 V562D/K/S/W Domain II Lethal ND This study V562A Viable 40 This study L563D/H/Y Lethal ND This study L563A/M Viable This study L563F Viable 37 This study L563P Viable 39 This study L563K/S/W Viable 40 This study V562L563K/S/W Lethal ND This study V562AL563A Viable 40 This study G646R snu114-30, Domain III Viable 37 BRENNER and GUTHRIE, 2005 M842R snu114-40, Domain IVa Viable 37 BRENNER and GUTHRIE, 2005 P860D Domain IVa Viable 40 This study P860E Viable This study P860K Viable 39 This study a Blank entry indicates that the mutant was not temperature sensitive up to 40°; ND indicates that temperature sensitivity was not assayed.


TABLE 3. snRNA mutations used in this study Mutation Location Reference U5 C79A Invariant positions of internal loop 1 – 5’ side This study G80C This study G80U This study G80A This study ΔC79 This study ΔC79G80 This study ΔC79-A81 This study ΔA78-A81 This study G113U Invariant positions of internal loop 1 – 3’ side This study C112A This study C111U This study ΔC111 This study ΔC112G113 This study ΔA41-U74 Variable stem loop O’KEEFE et al., 1996 U5 short form O’KEEFE et al., 1996 A100G Loop 1 This study A100UC101G This study ΔG93 O’KEEFE and NEWMAN, 1998 ΔC94C95 O’KEEFE and NEWMAN, 1998 ΔU96U97 O’KEEFE and NEWMAN, 1998 ΔA100 O’KEEFE and NEWMAN, 1998 ΔC101 O’KEEFE and NEWMAN, 1998 Ins 1U U97/U98 O’KEEFE and NEWMAN, 1998 Ins 1U G93/C94 MCGRAIL et al., 2006 Ins 1U C94/C95 MCGRAIL et al., 2006 U2 G20A/U 5’ to helix Iba This study C22A/G Helix Iba MADHANI and GUTHRIE, 1992 U23C/G MADHANI and GUTHRIE, 1992 U24A/C/G Bulge between helix Ia and Iba MADHANI and GUTHRIE, 1994 ΔU24 MADHANI and GUTHRIE, 1994 A25C/G/U MCPHEETERS and ABELSON, 1992 ΔA25 MCPHEETERS and ABELSON, 1992 A27C, U28C Helix Iaa MADHANI and GUTHRIE, 1992 Ins 1U U28/A29 MCPHEETERS and ABELSON, 1992 Ins 1U A30/A31 3’ to helix Iaa MCGRAIL et al., 2006 A31U This study Ins 1U G32/U33 MCGRAIL et al., 2006 U4 C14G Stem IIb LI and BROW, 1996 U57A Stem Ib This study G58A MADHANI et al., 1990 C59U MADHANI et al., 1990 U60C MADHANI et al., 1990 U64G This study U64C This study G65A This study U64C,G65A This study A66UA67UA68G (cs-1)

3’ to stem I LI and BROW, 1996 LI and BROW, 1996

U6 A47G Base-paired with pre-mRNA MADHANI et al., 1990 C48A This study A49C This study G50U 5’ to helix Iaa MADHANI et al., 1990 G52U MADHANI et al., 1990 A53U This study


Ins 1U A53/U54 This study U54C Helix Iaa This study G55A This study U54C,G55A This study Ins 1U G55/A56 MCGRAIL et al., 2006 A56C, U57C MADHANI and GUTHRIE, 1992 C58U MADHANI and GUTHRIE, 1992 A59C Helix Iba MADHANI and GUTHRIE, 1992 C61G MADHANI et al., 1990 A62C 3’ to helix Ib This study a U2:U6 complex; b U4:U6 complex


TABLE 4. Summary of the genetic interactions of snu114 and U4 snRNA mutations snu114 mutations

U4 mutations

WT ΔN K146I (G1) (114-15)

P216N (G2) (114-12)

V266P (G4)

F313Q (G5)

L381P (G’’) (114-14)

L563F (II)

G646R (III) (114-30)

M842R (IVa) (114-40)

P860K (IVa)

WT + + + + + + + + + + + G14C + + + + + + + + + + + U57A + + + + + + + + + + + G58A + + - + +/- +/- + + +/- +/- + C59U + + + + + + + + + + + U60C + + + + + + + + + + + U64C + + +/- + + + + + + + + U64G + + + + + + + + + + + G65A + + +/- + + + + + + + + U64C,G65A + + - + + + + + + + + A66U,A67U,A68G (U4-cs1)

+ + - +/- - - +/- + + + +

- indicates synthetically lethal interaction; +/- indicates synthetically sick interaction, and + indicates WT growth after 3 days at 30°.



U6 mutations

WT ΔN K146I (G1) (114-15)

P216N (G2) (114-12)

V266P (G4)

F313Q (G5)

L381P (G’’) (114-14)

L563F (II)

G646R (III)

(114-30)

M842R (IVa) (114-40)

P860K (IVa)

WT + + + + + + + + + + + A47G + + + + + - + +/- + +/- + G50U + + +/- + - - + + + - + G52U + + +/- + - - + + + - + A53U + + - + - - +/- + + + + Ins 1U A53/U54 + + + + + + + + + + + U54C + + - + + +/- + + + + + G55A + + - + - +/- + + + + + U54C,G55A + + - + - +/- + + + + + Ins 1U G55/A56 + + + + + + + + + + + A56C,U57C + +/- - +/- - +/- +/- + + +/- + C58U + + + + +/- + + + + + + A59C + +/- +/- + - - + + + + + C61G + - - +/- - - - + + - + A62C + + + + + + + + + + + - indicates synthetically lethal interaction, +/- indicates synthetically sick interaction, and + indicates WT growth after 3 days at 30°.



U2 mutations

WT ΔN K146I (G1) (114-15)

P216N (G2) (114-12)

V266P (G4)

F313Q (G5)

L381P (G’’) (114-14)

L563F (II)

G646R (III)

(114-30)

M842R (IVa) (114-40)

P860K (IVa)

WT + + + + + + + + + + + C22A + + + + - + + + + + + C22G + ND + + - + + + + + + U23C + + + + - +/- + + + + + U23G + ND + + - +/- + + + + + U24A + ND ND ND + + ND + ND + + U24C + ND ND ND + + ND + ND + + U24G + + + + + + + + + + + ΔU24 + + + + + + + + + + + A25C + ND ND ND + + ND + ND + + A25G + + + + + + + + + + + A25U + ND ND ND + + ND + ND + + ΔA25 + + + + + + + + + + + A27C,U28C + + + + - +/- +/- + + + + Ins 1U U28/A29 + + + + - + + + + + + A31U + + + + - + - + + + + Ins 1U A30/A31 + + + + + + + + + + + Ins 1U G32/U33 + + + + + + + + + + + - indicates synthetically lethal interaction; +/- indicates synthetically sick interaction, and + indicates WT growth after 3 days at 30°; ND indicates comparison was not done.


TABLE 7. Summary of the genetic interactions of snu114 and U5 snRNA mutations

U5 mutations

snu114 mutations WT ΔN K146I

(G1) (114-15)

P216N (G2) (114-12)

V266P (G4)

F313Q (G5)

L381P (G’’) (114-14)

L563F (II)

G646R (III) (114-30)

M842R (IVa) (114-40)

P860K (IVa)

WT + + + + + + + + + + + C79A + + + + + + + + + + + G80C + ND ND ND + + ND + ND + + G80U + + + + + + + + + + + G80A + ND ND ND + + ND + ND + + ΔC79 + + + + + + + + + + + ΔC79,G80 + ND ND ND + + ND + ND + + ΔC79-A81 + ND ND ND + + ND + ND +/- + ΔA78-A81 + + + + + + + + + +/- + C111U + + + + + + + + + + + ΔC111 + + + + + + + + + - + C112A + + + + + + + + + + + G113U + + + + + + + + + + + ΔC112,G113 + - + + + + + + - - - ΔA41-U74 + + + + + + + + + + + U5 short form + + + + + + + + + + + A100G + + + + + + + + + + + A100U,C101G + + + + - + + + + + + ΔA100 + + + + + + + + + + + ΔC101 + + + + + + + + + + + ΔG93 + + + + + + + + + + + ΔC94,C95 + - + + - + + + + + + ΔU96,U97 + - + + - + + + + + + Ins 1U U97/U98 + + + + + + + + + + + Ins 1U G93/C94 + + + + - + + + + + + Ins 1U C94/C95 + + + + + + + + + + + - indicates synthetically lethal interaction; +/- indicates synthetically sick interaction, and + indicates WT growth after 3 days at 30°; ND indicates comparison was not done.


FIGURE LEGENDS

FIGURE 1. Location of snu114 mutations. A. Linear diagram of Snu114p based on sequence

homology to yeast elongation factor (Eft2p). Domains are labelled in Roman numerals. The N-

terminal domain is unique to Snu114p. Within domain I, the G domain, the GTPase conserved

motifs are labelled 1-5. The G domain also contains a G’’ element which is unique to Snu114p and

Eft2p. Ts snu114 mutations used to analyze genetic interactions with snRNAs are displayed above

the linear diagram and mutations from a previous study (BRENNER and GUTHRIE 2005) are shown

below the linear diagram. B. Three dimensional predicted structure of Snu114p based on sequence

homology with Eft2p. The N-terminal domain (residues 1-121) and a region from the C-terminus

(residues 982-1008) were omitted. Colors correspond to the linear diagram of Snu114p. Yellow

spheres indicate location of amino acids mutated in this study and red spheres indicate amino acids

mutated by Brenner and Guthrie (2005).


FIGURE 2. Growth defects, protein expression levels and splicing defects of snu114 mutants. A.

Growth of WT and snu114 strains. Serial dilutions of WT and snu114 strains grown at 16° for 10

days or 25°, 30°, 37° and 39° for 3 days. snu114 mutant L563F exhibits a growth defect at 37°

whereas the remaining snu114 mutants exhibit a growth defect at 39°. B. Protein expression levels

of Snu114p monitored at permissive (30°) and restrictive temperatures (39°). Protein levels in WT

and snu114 strains remained constant after a shift to 39° compared to the loading control, yeast

glucose 6-phosphate dehydrogenase (Zwf1p). C. snu114 mutations inhibit pre-mRNA splicing.

RNA was extracted from WT or snu114 strains grown at the restrictive temperature and analyzed

by primer extension of pre-U3 snoRNA. Pre-U3A, pre-U3B and mature U3 snoRNA are indicated

to the right of the panel.


FIGURE 3. Genetic interactions between snu114 and U4 snRNA mutations. Strain

YSNU114/U4KO1 containing the WT SNU114 and U4 snRNA alleles on a URA3 plasmid was

transformed with WT or snu114 and U4 snRNA alleles on separate plasmids. Transformants were

transferred to 5FOA to evict the URA3 plasmid containing the wild type genes and determine the

synthetic lethality between mutant alleles. The annotated structure of base paired U4/U6 in the

conformation within the tri-snRNP is displayed above the panels. Positions of substitutions are

underlined. The U4-cs1 mutation is A66U,A67U,A68G.


FIGURE 4. Genetic interactions between snu114 and U6 snRNA mutations. Strain

YSNU114/U6KO containing the WT SNU114 and U6 snRNA alleles on a URA3 plasmid was

transformed with WT or snu114 and U6 snRNA alleles on separate plas

lily novak frazer, simon c. lovell and raymond t. o’keefe ... · 7/20/2009 · lily novak...

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