Synthetic Lethal Mutations Research Articles

Role of synthetic lethal mutations for the immune response: Exploratory studies of two non-small cell lung cancer cohorts and two pan-cancer cohorts.

e21137 Background: Synthetic lethality (SL) is a phenomenon in which mutations in either of two genes do not affect cell survival, but abnormalities in both genes lead to cell death. However, it is little known whether synthetic lethality affects the response of immunotherapy. Methods: Synthetic lethal gene pairs come from the SynLethDB database, which provides a webserver to calculate the confidence scores for each SL pair by integrating individual scores derived from different evidence sources. The analysis included two immunotherapy cohorts of non-small cell lung cancer (Chan, 2015 and Hellmann, 2018) and two solid tumor cohorts (Morris, 2019 and Berger, 2020) in the published literature. Synthetic lethal gene pairs were defined as genes with a score > 0.5. The patients with synthetic lethal gene pairs were divided into one subgroup (SL group), and the other patients were divided into another subgroup (non-SL group). Kaplan-Meier analysis was used to determine progression-free (PFS) and overall survival (OS). Results: Overall, four public cohorts with 12271 patients were included in the analysis. In the cohort of Chan 2015, a total of 34 patients with non-small cell lung cancer (NSCLC) received immunotherapy. Among them, 21 patients were in the SL group and 13 were in the non-SL group, with the former having a significantly longer PFS (p = 0.0345). In the other cohort of immunotherapy (Hellmann, 2018), 90 (37.5%) patients with NSCLC were in SL group. PFS for patients with SL group was significantly longer than those in non-SL group (p = 0.0261). In cohorts with pan-cancer, patients in Morris 2019 cohort (n = 1661) received immunotherapy and patients in Berger 2020 cohort (n = 10336) received other kinds of treatment. A better prognosis was observed in patients with the immunotherapy in SL group than non-SL group (p < 0.0001). In contrast, patients with other kinds of treatment had a significantly worse prognosis in SL group than non-SL group (p < 0.0001). Conclusions: Our results suggested that synthetic lethal gene pairs containing scores greater than 0.5 may be associated with higher progression-free survival in patients with various types of tumors. The specific reasons for the impact still need to be further explored.

Investigation of synthetic lethality gene pairs in the DNA damage response pathway in response to immunotherapy in pan-cancer.

2567 Background: The concept of synthetic lethality (SL) refers to the phenomenon where the simultaneous loss of function of two genes leads to cell death, while the loss of function of either gene alone has no effect. PARP inhibitors target poly ADP ribose polymerase (PARP) enzymes, which are involved in DNA repair, are the successful cases of utilizing synthetic lethality-based therapy to treat cancers with DNA damage response (DDR) deficiency. The prognosis for cancer patients already has a combination of synthetic lethal mutations especially in DDR pathway can vary and it is not yet fully understood how these mutations may impact the response to immunotherapy. Methods: SL gene combinations in the DDR pathway were screened out from SynLethDB database (http://histone.sce.ntu.edu.sg/SynLethDB/). The immunotherapy cohort (Samstein 2019, n=1661) and non-immunotherapy cohort (Zehir 2017, n=10336) with solid tumor were used to Kaplan-Meier analyze the correlation between SL gene combinations and prognosis. TMB scores were compared between DDR co-mutation group and non DDR co-mutation group. For investigating the associations between immune infiltrates and genetic features, TIMER 2.0 database ( http://timer.cistrome.org/ ) was conducted to estimation of immune infiltration levels on DDR mutation genes in pan-cancer. Results: 430 pairs of DDR mutation combinations were used to subgroup stratification, the DDR co-mutation group was associated with worse prognosis in non-immunotherapy cohort (HR=1.305, p<0.0001) but better in immunotherapy cohort (HR=0.741,p<0.0061). Interestingly, the TMB score in the DDR co-mutation group was significantly higher than non DDR co-mutation group (mean 50.03 vs 8.868, p<0.0001) in immunotherapy cohort. The high-frequency mutation combinations (>10%) were ATM-POLE(14.40%), ATM-ATR (13.60%), ATR-BRCA2 (13.60%), ATM-BRCA2 (12.00%) and BRCA2-RECQL4 (10.40%), respectively. Comparing common immune component with statistically significant under different DDR mutant genes, the ATM-POLE co-mutation tumor tended to have more CD4+ T cell infiltration while the ATM-ATR co-mutation tumor appeared to have more myeloid dendritic cell infiltration. Conclusions: The combinations of DDR SL mutation tumor might result to worse prognosis whereas these patients might benefit from immunotherapy. Our results will help to shed light on the complex interplay for the combinations of SL mutation in DDR pathway between cancer cells and the immune system and provide new insights into the development of effective cancer treatments.

Synthetic lethal mutations in the cyclin A interface of human cytomegalovirus

Generally, the antagonism between host restriction factors and viral countermeasures decides on cellular permissiveness or resistance to virus infection. Human cytomegalovirus (HCMV) has evolved an additional level of self-imposed restriction by the viral tegument protein pp150. Depending on a cyclin A-binding motif, pp150 prevents the onset of viral gene expression in the S/G2 cell cycle phase of otherwise fully permissive cells. Here we address the physiological relevance of this restriction during productive HCMV infection by employing a cyclin A-binding deficient pp150 mutant virus. One consequence of unrestricted viral gene expression in S/G2 was the induction of a G2/M arrest. G2-arrested but not mitotic cells supported viral replication. Cyclin A destabilization by the viral gene product pUL21a was required to maintain the virus-permissive G2-arrest. An HCMV double-point mutant where both pp150 and pUL21a are disabled in cyclin A interaction forced mitotic entry of the majority of infected cells, with a severe negative impact on cell viability and virus growth. Thus, pp150 and pUL21a functionally cooperate, together building a cell cycle synchronization strategy of cyclin A targeting and avoidance that is essential for productive HCMV infection.

Systematic identification of synthetic lethal mutations with reduced-genome Escherichia coli: synthetic genetic interactions among yoaA, xthA and holC related to survival from MMS exposure.

Reduced-genome Escherichia coli strains lacking up to 38.9% of the parental chromosome have been constructed by combining large-scale chromosome deletion mutations. Functionally redundant genes involved in essential processes can be systematically identified using these reduced-genome strains. One large-scale chromosome deletion mutation could be introduced into the wild-type strain but not into the largest reduced-genome strain, suggesting a synthetic lethal interaction between genes removed by the deletion and those already absent in the reduced-genome strain. Thus, introduction of the deletion mutation into a series of reduced-genome mutants could allow the identification of other chromosome deletion mutations responsible for the synthetic lethal phenotype. We identified a synthetic lethality caused by disruption of nfo and xthA, two genes encoding apurinic/apyrimidinic (AP) endonucleases involved in the DNA base excision repair pathway, and two other large-scale chromosome deletions. We constructed temperature-sensitive mutants harboring quadruple-deletion mutations in the affected genes/chromosome regions. Using these mutants, we identified two multi-copy suppressors: holC, encoding the chi subunit of DNA polymerase III, and yoaA, encoding a putative DNA helicase. Addition of the yoaA disruption increased the methyl methanesulfonate (MMS) sensitivity of xthA single-deletion or xthA nfo double-deletion mutants. This increased MMS sensitivity was not suppressed by the presence of multi-copy holC. These results indicate that yoaA is involved in MMS sensitivity and suggest that YoaA functions together with HolC.

Synthetic lethals in HIV: ways to avoid drug resistance : Running title: Preventing HIV resistance.

BackgroundRNA viruses rapidly accumulate genetic variation, which can give rise to synthetic lethal (SL) and deleterious (SD) mutations. Synthetic lethal mutations (non-lethal when alone but lethal when combined in one genome) have been studied to develop cancer therapies. This principle can also be used against fast-evolving RNA-viruses. Indeed, targeting protein sites involved in SD + SL interactions with a drug would render any mutation of such sites, lethal.ResultsHere, we set up a strategy to detect intragenic pairs of SL and SD at the surface of the protein to predict less escapable drug target sites. For this, we detected SD + SL, studying HIV protease (PR) and reverse transcriptase (RT) sequence alignments from two groups of VIH+ individuals: treated with drugs (T) or not (NT). Using a series of statistical approaches, we were able to propose bona fide SD + SL couples. When focusing on spatially close co-variant SD + SL couples at the surface of the protein, we found 5 SD + SL groups (2 in the protease and 3 in the reverse transcriptase), which could be good candidates to form pockets to accommodate potential drugs.ConclusionsThus, designing drugs targeting these specific SD + SL groups would not allow the virus to mutate any residue involved in such groups without losing an essential function. Moreover, we also show that the selection pressure induced by the treatment leads to the appearance of new mutations, which change the mutational landscape of the protein. This drives the existence of differential SD + SL couples between the drug-treated and non-treated groups. Thus, new anti-viral drugs should be designed differently to target such groups.ReviewersThis article was reviewed by Neil Greenspan Csaba Pal and István Simon.Electronic supplementary materialThe online version of this article (doi:10.1186/s13062-015-0044-y) contains supplementary material, which is available to authorized users.

Parallel universes of Black Six biology.

Creation of lethal and synthetic lethal mutations in an experimental organism is a cornerstone of genetic dissection of gene function, and is related to the concept of an essential gene. Common inbred mouse strains carry background mutations, which can act as genetic modifiers, interfering with the assignment of gene essentiality. The inbred strain C57BL/6J, commonly known as “Black Six”, stands out, as it carries a spontaneous homozygous deletion in the nicotinamide nucleotide transhydrogenase (Nnt) gene [GenBank: AH009385.2], resulting in impairment of steroidogenic mitochondria of the adrenal gland, and a multitude of indirect modifier effects, coming from alteration of glucocorticoid-regulated processes. Over time, the popular strain has been used, by means of gene targeting technology, to assign “essential” and “redundant” qualifiers to numerous genes, thus creating an internally consistent “parallel universe” of knowledge. It is unrealistic to suggest phasing-out of this strain, given the scope of shared resources built around it, however, continuing on the road of “strain-unawareness” will result in profound waste of effort, particularly where translational research is concerned. The review analyzes the historical roots of this phenomenon and proposes that building of “parallel universes” should be urgently made visible to a critical reader by obligatory use of unambiguous and persistent tags in publications and databases, such as hypertext links, pointing to a vendor’s strain description web page, or to a digital object identifier (d.o.i.) of the original publication, so that any research done exclusively in C57BL/6J, could be easily identified.ReviewersThis article was reviewed by Dr. Neil Smalheiser and Dr. Miguel Andrade-Navarro.

Isolation of synthetic lethal mutations in combination with spnab2 of fission yeast

spNab2 is a fission yeast, Schizosaccharomyces pombe, homologue of the budding yeast Nab2 protein that is an essential poly(A)+ RNA-binding protein required for both nuclear export of mRNA to cytoplasm and poly(A)+ tail length control. Here we performed a synthetic lethal genetic screen in the fission yeast to isolate mutants that are genetically linked to spnab2. We isolated three mutants that showed synthetic lethality under the repressed condition of the spnab2 expression. These mutants defined in different complementation groups. All the mutants exhibited the accumulation of poly(A)+ RNA in the nucleus under the restricted condition. In addition, the growth defects of one mutant (SLnab2) were complemented partially by some genes (mlo3 and rae1) required for mRNA export, while those of the rest (SLnab1 and SLnab3) were not complemented by any S. pombe genes we tested, which were known to be involved in mRNA export. These results suggest that the isolated mutants might harbor mutations in novel genes functionally linked to the spnab2 gene.

Interfering With Tumor Pathways That Augment Viral Oncolysis

Interfering With Tumor Pathways That Augment Viral Oncolysis

Isolation of synthetic lethal mutations in the rsm1-null mutant of fission yeast

To identify mutations in genes that are genetically linked to rsm1, we performed a synthetic lethal genetic screen in the fission yeast, Schizosaccharomyces pombe. Four mutations that showed synthetic lethality in combination with the rsm1null allele were isolated from approximately 320,000 colonies and defined in three complementation groups. One mutant (SLrsm1) exhibited a significant accumulation of poly(A)(+) RNA in the nucleus under synthetic lethal conditions, while the rest had no mRNA export defects. In addition, some genes (spmex67, rae1, or mlo3) required for mRNA export complemented the growth defects of the identified mutants. These results suggest that the isolated mutants contain mutations in genes that are involved in mRNA export and/or pre-mRNA retention.

Ordered assembly of Sld3, GINS and Cdc45 is distinctly regulated by DDK and CDK for activation of replication origins

Initiation of chromosome DNA replication in eukaryotes is tightly regulated through assembly of replication factors at replication origins. Here, we investigated dependence of the assembly of the initiation complex on particular factors using temperature-sensitive fission yeast mutants. The psf3-1 mutant, a GINS component mutant, arrested with unreplicated DNA at the restrictive temperature and the DNA content gradually increased, suggesting a defect in DNA replication. The mutation impaired GINS complex formation, as shown by pull-down experiments. Chromatin immunoprecipitation assays indicated that GINS integrity was required for origin loading of Psf2, Cut5 and Cdc45, but not Sld3. In contrast, loading of Psf2 onto origins depended on Sld3 and Cut5 but not on Cdc45. These results suggest that Sld3 functions furthest upstream in initiation complex assembly, followed by GINS and Cut5, then Cdc45. Consistent with this conclusion, Cdc7-Dbf4 kinase (DDK) but not cyclin-dependent kinase (CDK) was required for Sld3 loading, whereas recruitment of the other factors depended on both kinases. These results suggest that DDK and CDK regulate distinct steps in activation of replication origins in fission yeast.

Bimodal interaction between replication-protein A and Dna2 is critical for Dna2 function both in vivo and in vitro.

We have previously shown that replication- protein A (RPA), the heterotrimeric single-stranded DNA binding protein of eukaryotes, plays a role in Okazaki fragment processing by acting as a molecular switch between the two endonucleases, Dna2 and Fen1, to ensure the complete removal of primer RNAs in Saccharomyces cerevisiae. The stimulation of Dna2 endonuclease activity by RPA requires direct protein-protein interaction. In this report we have analyzed genetically and biochemically the interaction of Dna2 with RPA. RFA1, the gene encoding the large subunit of RPA, displayed allele-specific interactions with DNA2 that included synthetic lethality and intergenic complementation. In addition, we identified physical and functional interactions between these proteins and found that RPA binds Dna2 predominantly through its large subunit, Rpa1. Consistent with the mapping of synthetic lethal mutations, robust interaction localizes to the C-termini of these proteins. Moreover, the N-terminal domains of Dna2 and Rpa1 appear to be important for a functional interaction because the N-terminal domain of RPA1 was required to maximally stimulate Dna2 endonuclease activity. We propose that a bimodal interaction of Dna2 with Rpa1 is important for Dna2 function both in vivo and in vitro. The relevance of each interaction with respect to the function of the Dna2 endonuclease activity is discussed.

Schizosaccharomyces pombe spPABP, a homologue of Saccharomyces cerevisiae Pab1p, is a non-essential, shuttling protein that facilitates mRNA export.

Poly(A)-binding proteins play important roles in mRNA metabolism in eukaryotic cells. We examined the role of the Schizosaccharomyces pombe homologue of the Saccharomyces cerevisiae poly(A)-binding protein, Pab1p, in cellular growth and mRNA export. In contrast to PAB1, the sppabp gene is not essential for cellular viability. Like the human hPABP1 protein, spPABP is cytoplasmically localized and can shuttle between the nucleus and the cytoplasm. We found that a spPABP-GFP fusion protein expressed from a multicopy plasmid could suppress the growth and mRNA export defect of rae1-16 7 nup184-1 synthetic lethal mutations. However, about 20-25% of cells in the population exhibited a pronounced nuclear accumulation of poly(A)(+) RNA. The same cells also localized the spPABP-GFP fusion to the nucleus, suggesting that the shuttling ability of spPABP is related to its function in mRNA export. When a heterologous nuclear export activity from spMex67p was fused to spPABP-GFP fusion protein, it overcame the nuclear retention but did not increase nuclear mRNA export. We discuss the implications of these observations in relation to how spPABP could function in mRNA export. Published in 2002 by John Wiley & Sons, Ltd.

Synthetic lethal interactions suggest a role for the Saccharomyces cerevisiae Rtf1 protein in transcription elongation.

Strong evidence indicates that transcription elongation by RNA polymerase II (pol II) is a highly regulated process. Here we present genetic results that indicate a role for the Saccharomyces cerevisiae Rtf1 protein in transcription elongation. A screen for synthetic lethal mutations was carried out with an rtf1 deletion mutation to identify factors that interact with Rtf1 or regulate the same process as Rtf1. The screen uncovered mutations in SRB5, CTK1, FCP1, and POB3. These genes encode an Srb/mediator component, a CTD kinase, a CTD phosphatase, and a protein involved in the regulation of transcription by chromatin structure, respectively. All of these gene products have been directly or indirectly implicated in transcription elongation, indicating that Rtf1 may also regulate this process. In support of this view, we show that RTF1 functionally interacts with genes that encode known elongation factors, including SPT4, SPT5, SPT16, and PPR2. We also show that a deletion of RTF1 causes sensitivity to 6-azauracil and mycophenolic acid, phenotypes correlated with a transcription elongation defect. Collectively, our results suggest that Rtf1 may function as a novel transcription elongation factor in yeast.

Two mutant forms of the S1/TPR-containing protein Rrp5p affect the 18S rRNA synthesis in Saccharomyces cerevisiae.

The genetic depletion of yeast Rrp5p results in a synthesis defect of both 18S and 5.8S ribosomal RNAs (Venema J, Tollervey D. 1996. EMBO J 15:5701-5714). We have isolated the RRP5gene in a genetic approach aimed to select for yeast factors interfering with protein import into mitochondria. We describe here a striking feature of Rrp5p amino acid sequence, namely the presence of twelve putative S1 RNA-binding motifs and seven tetratricopeptide repeats (TPR) motifs. We have constructed two conditional temperature-sensitive alleles of RRP5 gene and analyzed them for associated rRNA-processing defects. First, a functional "bipartite gene" was generated revealing that the S1 and TPR parts of the protein can act independently of each other. We also generated a two amino acid deletion in TPR unit 1 (rrp5delta6 allele). The two mutant forms of Rrp5p were shown to cause a defect in 18S rRNA synthesis with no detectable effects on 5.8S rRNA production. However, the rRNA processing pathway was differently affected in each case. Interestingly, the ROK1 gene which, like RRP5, was previously isolated in a screen for synthetic lethal mutations with snR10 deletion, was here identified as a high copy suppressor of the rrp5delta6 temperature-sensitive allele. ROK1 also acts as a low copy suppressor but cannot bypass the cellular requirement for RRP5. Furthermore, we show that suppression by the Rok1p putative RNA helicase rescues the 18S rRNA synthesis defect caused by the rrp5delta6 mutation.

A screen for dynein synthetic lethals in Aspergillus nidulans identifies spindle assembly checkpoint genes and other genes involved in mitosis.

Cytoplasmic dynein is a ubiquitously expressed microtubule motor involved in vesicle transport, mitosis, nuclear migration, and spindle orientation. In the filamentous fungus Aspergillus nidulans, inactivation of cytoplasmic dynein, although not lethal, severely impairs nuclear migration. The role of dynein in mitosis and vesicle transport in this organism is unclear. To investigate the complete range of dynein function in A. nidulans, we searched for synthetic lethal mutations that significantly reduced growth in the absence of dynein but had little effect on their own. We isolated 19 sld (synthetic lethality without dynein) mutations in nine different genes. Mutations in two genes exacerbate the nuclear migration defect seen in the absence of dynein. Mutations in six other genes, including sldA and sldB, show a strong synthetic lethal interaction with a mutation in the mitotic kinesin bimC and, thus, are likely to play a role in mitosis. Mutations in sldA and sldB also confer hypersensitivity to the microtubule-destabilizing drug benomyl. sldA and sldB were cloned by complementation of their mutant phenotypes using an A. nidulans autonomously replicating vector. Sequencing revealed homology to the spindle assembly checkpoint genes BUB1 and BUB3 from Saccharomyces cerevisiae. Genetic interaction between dynein and spindle assembly checkpoint genes, as well as other mitotic genes, indicates that A. nidulans dynein plays a role in mitosis. We suggest a model for dynein motor action in A. nidulans that can explain dynein involvement in both mitosis and nuclear distribution.

Rig2, a RING finger protein that interacts with the Kin28/Ccl1 CTD kinase in yeast.

Kin28/Cell, a cyclin-dependent kinase, is essential for the in vivo phosphorylation of the C-terminal domain of the largest subunit of RNA polymerase II in Saccharomyces cerevisiae. In a search for mutations co-lethal with a thermosensitive kin28 mutation, we have identified genes whose products interact functionally with Kin28. In the present work, we have studied a new complementation group of synthetic lethal mutations. The corresponding gene, RIG2, encodes a predicted RING finger protein. Rig2 is likely to be a homolog of MAT1 of higher eukaryotes which forms a ternary complex with MO15(cdk7) and cyclin H. Our genetic data suggest that Rig2 is a component of transcription factor TFIIH. Transcription activity in a rig2-ts mutant is impeded at restrictive temperature. However, none of the rig2-ts mutants obtained was UV sensitive, suggesting that Rig2 is dispensable for nucleotide excision repair.

Sls1p, an Endoplasmic Reticulum Component, Is Involved in the Protein Translocation Process in the Yeast Yarrowia lipolytica

Signal recognition particle-dependent targeting of secretory proteins to the endoplasmic reticulum membrane is predominant in the yeast Yarrowia lipolytica. A conditional lethal mutant of the SCR2-encoded 7S RNA provided the first in vivo evidence for involvement of this particle in cotranslational translocation (He, F., Beckerich, J. M., and Gaillardin, C. M. (1992) J. Biol. Chem. 267, 1932-1937). In order to identify partners of 7S RNA or signal recognition particle in their function, we selected synthetic lethal mutations with the 7S RNA mutation (sls). The SLS1 gene, cloned by complementation of the sls1 mutant growth defect, encodes a 426-amino acid polypeptide containing a NH2-terminal signal peptide and a COOH-terminal endoplasmic reticulum (ER) retention motif. The SLS1 gene product behaves as a lumenal protein of the ER. Sls1p was sedimented with membrane-rich organelles and was resistant to protease degradation without prior membrane solubilization. Immunofluorescence microscopy showed a typical endoplasmic reticulum perinuclear staining. Co-immunoprecipitation revealed that Sls1p resides close to the major translocation apparatus component, Sec61p. Deletion of the SLS1 gene led to a temperature-sensitive growth phenotype. Synthesis of several secretory proteins was shown to be specifically reduced in delta sls1 cells. We propose that Sls1p acts in the preprotein translocation process, interacting directly with translocating polypeptides to facilitate their transfer and/or help their folding in the ER.

Characterization of an essential Orc2p-associated factor that plays a role in DNA replication.

The Saccharomyces cerevisiae Orc2 protein is a subunit of the origin recognition complex, ORC, which binds in a sequence-specific manner to yeast origins of DNA replication. With screens for orc2-1 synthetic lethal mutations and Orc2p two-hybrid interactors, a novel Orc2p-associated factor (Oaf1p) was identified. OAF1 is essential, its gene product is localized to the nucleus, and an oaf1 temperature-sensitive mutant arrests as large budded cells with a single nucleus. The mutant oaf1-2, isolated in the synthetic lethal screen, loses plasmids containing a single origin of DNA replication at a high rate, but it maintains plasmids carrying multiple potential origins of DNA replication. In addition, the OAF1 gene product tagged with the hemagglutinin antigen epitope binds to a DNA affinity column containing covalently linked tandem repeats of an essential origin element. These results suggest a role for OAFI in the initiation of DNA replication. Mutant alleles of cdc7 and cdc14 were also isolated in the orc2-1 synthetic lethal screen. Cdc7p, like Oaf1p, also interacts with Orc2p in two-hybrid assays.

Synthetic lethal mutations suggest interactions between U5 small nuclear RNA and four proteins required for the second step of splicing.

To investigate the function of the U5 small nuclear ribonucleoprotein (snRNP) in pre-mRNA splicing, we have screened for factors that genetically interact with Saccharomyces cerevisiae U5 snRNA. We isolated trans-acting mutations that exacerbate the phenotypes of conditional alleles of the U5 snRNA and named these genes SLU, for synergistically lethal with U5 snRNA. SLU1 and SLU2 are essential for the first catalytic step of splicing, while SLU7 and SLU4 (an allele of PRP17 [U. Vijayraghavan, M. Company, and J. Abelson, Genes Dev. 3:1206-1216, 1989]) are required only for the second step of splicing. Furthermore, slu4-1 and slu7-1 are lethal in combination with mutations in PRP16 and PRP18, which also function in the second step, but not with mutations in factors required for the first catalytic step, such as PRP8 and PRP4. We infer from these data that SLU4, SLU7, PRP18, PRP16, and the U5 snRNA interact functionally and that a major role of the U5 snRNP is to coordinate a set of factors that are required for the completion of the second catalytic step of splicing.

Synthetic lethal mutations suggest interactions between U5 small nuclear RNA and four proteins required for the second step of splicing.

To investigate the function of the U5 small nuclear ribonucleoprotein (snRNP) in pre-mRNA splicing, we have screened for factors that genetically interact with Saccharomyces cerevisiae U5 snRNA. We isolated trans-acting mutations that exacerbate the phenotypes of conditional alleles of the U5 snRNA and named these genes SLU, for synergistically lethal with U5 snRNA. SLU1 and SLU2 are essential for the first catalytic step of splicing, while SLU7 and SLU4 (an allele of PRP17 [U. Vijayraghavan, M. Company, and J. Abelson, Genes Dev. 3:1206-1216, 1989]) are required only for the second step of splicing. Furthermore, slu4-1 and slu7-1 are lethal in combination with mutations in PRP16 and PRP18, which also function in the second step, but not with mutations in factors required for the first catalytic step, such as PRP8 and PRP4. We infer from these data that SLU4, SLU7, PRP18, PRP16, and the U5 snRNA interact functionally and that a major role of the U5 snRNP is to coordinate a set of factors that are required for the completion of the second catalytic step of splicing.