scholarly journals Synthetic Lethality of Yeast slt Mutations with U2 Small Nuclear RNA Mutations Suggests Functional Interactions between U2 and U5 snRNPs That Are Important for Both Steps of Pre-mRNA Splicing

1998 ◽  
Vol 18 (4) ◽  
pp. 2055-2066 ◽  
Author(s):  
Deming Xu ◽  
Deborah J. Field ◽  
Shou-Jiang Tang ◽  
Arnaud Moris ◽  
Brian P. Bobechko ◽  
...  
Keyword(s):  
Synthetic Lethality ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Small Nuclear Rna ◽  
U2 Snrna ◽  
U6 Snrna ◽  
Two Factors ◽  
Synthetically Lethal ◽  
U5 Snrna ◽  
New Alleles

ABSTRACT A genetic screen was devised to identify Saccharomyces cerevisiae splicing factors that are important for the function of the 5′ end of U2 snRNA. Six slt (stands for synthetic lethality with U2) mutants were isolated on the basis of synthetic lethality with a U2 snRNA mutation that perturbs the U2-U6 snRNA helix II interaction. SLT11 encodes a new splicing factor andSLT22 encodes a new RNA-dependent ATPase RNA helicase (D. Xu, S. Nouraini, D. Field, S. J. Tang, and J. D. Friesen, Nature 381:709–713, 1996). The remaining four sltmutations are new alleles of previously identified splicing genes:slt15, previously identified as prp17(slt15/prp17-100), slt16/smd3-1,slt17/slu7-100, and slt21/prp8-21. slt11-1 andslt22-1 are synthetically lethal with mutations in the 3′ end of U6 snRNA, a region that affects U2-U6 snRNA helix II; however,slt17/slu7-100 and slt21/prp8-21 are not. This difference suggests that the latter two factors are unlikely to be involved in interactions with U2-U6 snRNA helix II but rather are specific to interactions with U2 snRNA. Pairwise synthetic lethality was observed among slt11-1 (which affects the first step of splicing) and several second-step factors, includingslt15/prp17-100, slt17/slu7-100, andprp16-1. Mutations in loop 1 of U5 snRNA, a region that is implicated in the alignment of the two exons, are synthetically lethal with slu4/prp17-2 and slu7-1 (D. Frank, B. Patterson, and C. Guthrie, Mol. Cell. Biol. 12:5179–5205, 1992), as well as with slt11-1, slt15/prp17-100,slt17/slu7-100, and slt21/prp8-21. These same U5 snRNA mutations also interact genetically with certain U2 snRNA mutations that lie in the helix I and helix II regions of the U2-U6 snRNA structure. Our results suggest interactions among U2 snRNA, U5 snRNA, and Slt protein factors that may be responsible for coupling and coordination of the two reactions of pre-mRNA splicing.

An mRNA-type intron is present in the Rhodotorula hasegawae U2 small nuclear RNA gene

1993 ◽  
Vol 13 (9) ◽  
pp. 5613-5619
Author(s):  
Y Takahashi ◽  
S Urushiyama ◽  
T Tani ◽  
Y Ohshima
Keyword(s):  
Mrna Splicing ◽  
Small Nuclear Rna ◽  
U2 Snrna ◽  
U6 Snrna ◽  
Branch Site ◽  
Snrna Gene ◽  
Nuclear Rna ◽  
Catalytic Role ◽  
Mrna Precursor ◽  
Site Recognition

Splicing an mRNA precursor requires multiple factors involving five small nuclear RNA (snRNA) species called U1, U2, U4, U5, and U6. The presence of mRNA-type introns in the U6 snRNA genes of some yeasts led to the hypothesis that U6 snRNA may play a catalytic role in pre-mRNA splicing and that the U6 introns occurred through reverse splicing of an intron from an mRNA precursor into a catalytic site of U6 snRNA. We characterized the U2 snRNA gene of the yeast Rhodotorula hasegawae, which has four mRNA-type introns in the U6 snRNA gene, and found an mRNA-type intron of 60 bp. The intron of the U2 snRNA gene is present in the highly conserved region immediately downstream of the branch site recognition domain. Interestingly, we found that this region can form a novel base pairing with U6 snRNA. We discuss the possible implications of these findings for the mechanisms of intron acquisition and for the role of U2 snRNA in pre-mRNA splicing.

scholarly journals An mRNA-type intron is present in the Rhodotorula hasegawae U2 small nuclear RNA gene.

1993 ◽  
Vol 13 (9) ◽  
pp. 5613-5619 ◽  
Author(s):  
Y Takahashi ◽  
S Urushiyama ◽  
T Tani ◽  
Y Ohshima
Keyword(s):  
Mrna Splicing ◽  
Small Nuclear Rna ◽  
U2 Snrna ◽  
U6 Snrna ◽  
Branch Site ◽  
Snrna Gene ◽  
Nuclear Rna ◽  
Catalytic Role ◽  
Mrna Precursor ◽  
Site Recognition

Splicing an mRNA precursor requires multiple factors involving five small nuclear RNA (snRNA) species called U1, U2, U4, U5, and U6. The presence of mRNA-type introns in the U6 snRNA genes of some yeasts led to the hypothesis that U6 snRNA may play a catalytic role in pre-mRNA splicing and that the U6 introns occurred through reverse splicing of an intron from an mRNA precursor into a catalytic site of U6 snRNA. We characterized the U2 snRNA gene of the yeast Rhodotorula hasegawae, which has four mRNA-type introns in the U6 snRNA gene, and found an mRNA-type intron of 60 bp. The intron of the U2 snRNA gene is present in the highly conserved region immediately downstream of the branch site recognition domain. Interestingly, we found that this region can form a novel base pairing with U6 snRNA. We discuss the possible implications of these findings for the mechanisms of intron acquisition and for the role of U2 snRNA in pre-mRNA splicing.

scholarly journals Interactions between highly conserved U2 small nuclear RNA structures and Prp5p, Prp9p, Prp11p, and Prp21p proteins are required to ensure integrity of the U2 small nuclear ribonucleoprotein in Saccharomyces cerevisiae.

1994 ◽  
Vol 14 (9) ◽  
pp. 6337-6349 ◽  
Author(s):  
S E Wells ◽  
M Ares
Keyword(s):  
Synthetic Lethality ◽  
Small Nuclear Rna ◽  
Rna Structures ◽  
Stem Loop ◽  
U2 Snrna ◽  
Small Nuclear Ribonucleoprotein ◽  
U2 Snrnp ◽  
Nuclear Rna ◽  
Nuclear Ribonucleoprotein

Binding of U2 small nuclear ribonucleoprotein (snRNP) to the pre-mRNA is an early and important step in spliceosome assembly. We searched for evidence of cooperative function between yeast U2 small nuclear RNA (snRNA) and several genetically identified splicing (Prp) proteins required for the first chemical step of splicing, using the phenotype of synthetic lethality. We constructed yeast strains with pairwise combinations of 28 different U2 alleles with 10 prp mutations and found lethal double-mutant combinations with prp5, -9, -11, and -21 but not with prp3, -4, -8, or -19. Many U2 mutations in highly conserved or invariant RNA structures show no phenotype in a wild-type PRP background but render mutant prp strains inviable, suggesting that the conserved but dispensable U2 elements are essential for efficient cooperative function with specific Prp proteins. Mutant U2 snRNA fails to accumulate in synthetic lethal strains, demonstrating that interaction between U2 RNA and these four Prp proteins contributes to U2 snRNP assembly or stability. Three of the proteins (Prp9p, Prp11p, and Prp21p) are associated with each other and pre-mRNA in U2-dependent splicing complexes in vitro and bind specifically to synthetic U2 snRNA added to crude splicing extracts depleted of endogenous U2 snRNPs. Taken together, the results suggest that Prp9p, -11p, and -21p are U2 snRNP proteins that interact with a structured region including U2 stem loop IIa and mediate the association of the U2 snRNP with pre-mRNA.

The phylogenetically invariant ACAGAGA and AGC sequences of U6 small nuclear RNA are more tolerant of mutation in human cells than in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (9) ◽  
pp. 5377-5382
Author(s):  
B Datta ◽  
A M Weiner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transient Expression ◽  
Human Cells ◽  
Mrna Splicing ◽  
Small Nuclear Rna ◽  
Transient Expression Assay ◽  
U6 Snrna ◽  
Nuclear Rna

U6 small nuclear RNA (snRNA) is the most highly conserved of the five spliceosomal snRNAs that participate in nuclear mRNA splicing. The proposal that U6 snRNA plays a key catalytic role in splicing [D. Brow and C. Guthrie, Nature (London) 337:14-15, 1989] is supported by the phylogenetic conservation of U6, the sensitivity of U6 to mutation, cross-linking of U6 to the vicinity of the 5' splice site, and genetic evidence for extensive base pairing between U2 and U6 snRNAs. We chose to mutate the phylogenetically invariant 41-ACAGAGA-47 and 53-AGC-55 sequences of human U6 because certain point mutations within the homologous regions of Saccharomyces cerevisiae U6 selectively block the first or second step of mRNA splicing. We found that both sequences are more tolerant to mutation in human cells (assayed by transient expression in vivo) than in S. cerevisiae (assayed by effects on growth or in vitro splicing). These differences may reflect different rate-limiting steps in the particular assays used or differential reliance on redundant RNA-RNA or RNA-protein interactions. The ability of mutations in U6 nucleotides A-45 and A-53 to selectively block step 2 of splicing in S. cerevisiae had previously been construed as evidence that these residues might participate directly in the second chemical step of splicing; an indirect, structural role seems more likely because the equivalent mutations have no obvious phenotype in the human transient expression assay.

scholarly journals Transient Nucleolar Localization Of U6 Small Nuclear RNA InXenopus Laevis Oocytes

2000 ◽  
Vol 11 (7) ◽  
pp. 2419-2428 ◽  
Author(s):  
Thilo Sascha Lange ◽  
Susan A. Gerbi
Keyword(s):  
Nucleolar Localization ◽  
Xenopus Laevis Oocytes ◽  
Small Nuclear Rna ◽  
U2 Snrna ◽  
U6 Snrna ◽  
Nuclear Rna ◽  
Inxenopus Laevis ◽  
Nucleolar Staining ◽  
Over Time

Recent studies on the 2′-O-methylation and pseudouridylation of U6 small nuclear RNA (snRNA) hypothesize that these posttranscriptional modifications might occur in the nucleolus. In this report, we present direct evidence for the nucleolar localization of U6 snRNA and analyze the kinetics of U6 nucleolar localization after injection of in vitro transcribed fluorescein-labeled transcripts into Xenopus laevis oocytes. In contrast to U3 small nucleolar RNA (snoRNA) which developed strong nucleolar labeling over 4 h and maintained strong nucleolar signals through 24 h, U6 snRNA localized to nucleoli immediately after injection, but nucleolar staining decreased after 4 h. By 24 h after injection of U6 snRNA, only weak nucleolar signals were observed. Unlike the time-dependent profile of strong nucleolar localization of U6 snRNA or U3 snoRNA, injection of fluorescein-labeled U2 snRNA gave weak nucleolar staining at all times throughout a 24-h period; U2 snRNA modifications are believed to occur outside of the nucleolus. The notion that the decrease of U6 signals over time was due to its trafficking out of nucleoli and not to transcript degradation was supported by the demonstration of U6 snRNA stability over time. Therefore, in contrast to snoRNAs like U3, U6 snRNA transiently passes through nucleoli.

scholarly journals Structures of the Catalytically Activated Yeast Spliceosome Reveal the Mechanism of Branching

2018 ◽  
Author(s):  
Ruixue Wan ◽  
Rui Bai ◽  
Chuangye Yan ◽  
Jianlin Lei ◽  
Yigong Shi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Active Site ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Compelling Evidence ◽  
U2 Snrna ◽  
Branch Point Sequence ◽  
Catalytic Metal ◽  
Activated Complex

SummaryPre-mRNA splicing is executed by the spliceosome. Structural characterization of the catalytically activated complex (B*) is pivotal for mechanistic understanding of catalysis of the branching reaction by the spliceosome. In this study, we assembled the B* complex on two different pre-mRNAs from Saccharomyces cerevisiae and determined the cryo-EM structures of four distinct B complexes at overall resolutions of 2.9-3.8 Å. The duplex between U2 snRNA and the branch point sequence (BPS) is located 13-20 Å away from the 5’-splice site (5’SS) in the B* complexes that are devoid of the step I splicing factors Yju2 and Cwc25. Recruitment of Yju2 into the active site brings the U2/BPS duplex into the vicinity of 5’SS, ready for branching. In the absence of Cwc25, the nucleophile from BPS is positioned about 4 Å away from, and remains to be activated by, the catalytic metal M2. This analysis reveals the functional mechanism of Yju2 and Cwc25 in branching. These four structures constitute compelling evidence for substrate-specific conformations of the spliceosome in a major functional state.

scholarly journals PRP38 encodes a yeast protein required for pre-mRNA splicing and maintenance of stable U6 small nuclear RNA levels.

1992 ◽  
Vol 12 (9) ◽  
pp. 3939-3947 ◽  
Author(s):  
S Blanton ◽  
A Srinivasan ◽  
B C Rymond
Keyword(s):  
Structural Similarity ◽  
Mrna Splicing ◽  
Heat Inactivation ◽  
Temperature Sensitive ◽  
Small Nuclear Rna ◽  
Virtual Absence ◽  
U6 Snrna ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Rna

An essential pre-mRNA splicing factor, the product of the PRP38 gene, has been genetically identified in a screen of temperature-sensitive mutants of Saccharomyces cerevisiae. Shifting temperature-sensitive prp38 cultures from 23 to 37 degrees C prevents the first cleavage-ligation event in the excision of introns from mRNA precursors. In vitro splicing inactivation and complementation studies suggest that the PRP38-encoded factor functions, at least in part, after stable splicing complex formation. The PRP38 locus contains a 726-bp open reading frame coding for an acidic 28-kDa polypeptide (PRP38). While PRP38 lacks obvious structural similarity to previously defined splicing factors, heat inactivation of PRP38, PRP19, or any of the known U6 (or U4/U6) small nuclear ribonucleoprotein-associating proteins (i.e., PRP3, PRP4, PRP6, and PRP24) leads to a common, unexpected consequence: intracellular U6 small nuclear RNA (snRNA) levels decrease as splicing activity is lost. Curiously, U4 snRNA, normally extensively base paired with U6 snRNA, persists in the virtual absence of U6 snRNA.

scholarly journals Invariant U2 RNA sequences bordering the branchpoint recognition region are essential for interaction with yeast SF3a and SF3b subunits.

1996 ◽  
Vol 16 (3) ◽  
pp. 818-828 ◽  
Author(s):  
D Yan ◽  
M Ares
Keyword(s):  
Yeast Cells ◽  
Small Nuclear Rna ◽  
Recognition Sequence ◽  
Rna Sequences ◽  
Synthetic Lethal ◽  
U2 Snrna ◽  
U6 Snrna ◽  
Synthetic Lethal Interactions ◽  
Protein Components ◽  
And Function

U2 small nuclear RNA (snRNA) contains a sequence (GUAGUA) that pairs with the intron branchpoint during splicing. This sequence is contained within a longer invariant sequence of unknown secondary structure and function that extends between U2 and I and stem IIa. A part of this region has been proposed to pair with U6 in a structure called helix III. We made mutations to test the function of these nucleotides in yeast U2 snRNA. Most single base changes cause no obvious growth defects; however, several single and double mutations are lethal or conditional lethal and cause a block before the first step of splicing. We used U6 compensatory mutations to assess the contribution of helix III and found that if it forms, helix III is dispensable for splicing in Saccharomyces cerevisiae. On the other hand, mutations in known protein components of the splicing apparatus suppress or enhance the phenotypes of mutations within the invariant sequence that connect the branchpoint recognition sequence to stem IIa. Lethal mutations in the region are suppressed by Cus1-54p, a mutant yeast splicing factor homologous to a mammalian SF3b subunit. Synthetic lethal interactions show that this region collaborates with the DEAD-box protein Prp5p and the yeast SF3a subunits Prp9p, Prp11p, and Prp21p. Together, the data show that the highly conserved RNA element downstream of the branchpoint recognition sequence of U2 snRNA in yeast cells functions primarily with the proteins that make up SF3 rather than with U6 snRNA.

scholarly journals Mutation in the U2 snRNA influences exon interactions of U5 snRNA loop 1 during pre-mRNA splicing

2006 ◽  
Vol 25 (16) ◽  
pp. 3813-3822 ◽  
Author(s):  
Joanne C McGrail ◽  
Elaine M Tatum ◽  
Raymond T O'Keefe
Keyword(s):  
Mrna Splicing ◽  
U2 Snrna ◽  
U5 Snrna

scholarly journals Genetic and Physical Interactions Between Factors Involved in Both Cell Cycle Progression and Pre-mRNA Splicing inSaccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (4) ◽  
pp. 1503-1517 ◽  
Author(s):  
Sigal Ben-Yehuda ◽  
Ian Dix ◽  
Caroline S Russell ◽  
Margaret McGarvey ◽  
Jean D Beggs ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Synthetic Lethality ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Restrictive Temperature ◽  
Cycle Progression ◽  
Cellular Processes ◽  
Physical Interactions ◽  
Mutant Cells

AbstractThe PRP17/CDC40 gene of Saccharomyces cerevisiae functions in two different cellular processes: pre-mRNA splicing and cell cycle progression. The Prp17/Cdc40 protein participates in the second step of the splicing reaction and, in addition, prp17/cdc40 mutant cells held at the restrictive temperature arrest in the G2 phase of the cell cycle. Here we describe the identification of nine genes that, when mutated, show synthetic lethality with the prp17/cdc40Δ allele. Six of these encode known splicing factors: Prp8p, Slu7p, Prp16p, Prp22p, Slt11p, and U2 snRNA. The other three, SYF1, SYF2, and SYF3, represent genes also involved in cell cycle progression and in pre-mRNA splicing. Syf1p and Syf3p are highly conserved proteins containing several copies of a repeated motif, which we term RTPR. This newly defined motif is shared by proteins involved in RNA processing and represents a subfamily of the known TPR (tetratricopeptide repeat) motif. Using two-hybrid interaction screens and biochemical analysis, we show that the SYF gene products interact with each other and with four other proteins: Isy1p, Cef1p, Prp22p, and Ntc20p. We discuss the role played by these proteins in splicing and cell cycle progression.

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