Mutations in an essential U2 small nuclear RNA structure cause cold-sensitive U2 small nuclear ribonucleoprotein function by favoring competing alternative U2 RNA structures

Mutations in stem-loop IIa of yeast U2 RNA cause cold-sensitive growth and cold-sensitive U2 small nuclear ribonucleoprotein function in vitro. Cold-sensitive U2 small nuclear RNA adopts an alternative conformation that occludes the loop and disrupts the stem but does so at both restrictive and permissive temperatures. To determine whether alternative U2 RNA structure causes the defects, we tested second-site mutations in U2 predicted to disrupt the alternative conformation. We find that such mutations efficiently suppress the cold-sensitive phenotypes and partially restore correct U2 RNA folding. A genetic search for additional suppressors of cold sensitivity revealed two unexpected mutations in the base of an adjacent stem-loop. Direct probing of RNA structure in vivo indicates that the suppressors of cold sensitivity act to improve the stability of the essential stem relative to competing alternative structures by disrupting the alternative structures. We suggest that many of the numerous cold-sensitive mutations in a variety of RNAs and RNA-binding proteins could be a result of changes in the stability of a functional RNA conformation relative to a competing structure. The presence of an evolutionarily conserved U2 sequence positioned to form an alternative structure argues that this region of U2 is dynamic during the assembly or function of the U2 small nuclear ribonucleoprotein.

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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.

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.6337 ◽

1994 ◽

Vol 14 (9) ◽

pp. 6337-6349 ◽

Cited By ~ 27

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.

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Model of the interaction between the UI A small nuclear ribonucleoprotein and UI small nuclear RNA stem/loop II

Biochemical Society Transactions ◽

10.1042/bst0210605 ◽

1993 ◽

Vol 21 (3) ◽

pp. 605-609 ◽

Cited By ~ 1

Author(s):

P. R. Evans ◽

C. Oubridge ◽

T.-H. Jessen ◽

J. Li ◽

C. H. Teo ◽

...

Keyword(s):

Small Nuclear Rna ◽

Stem Loop ◽

Small Nuclear Ribonucleoprotein ◽

Nuclear Rna ◽

Nuclear Ribonucleoprotein

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Identification of Prp40, a novel essential yeast splicing factor associated with the U1 small nuclear ribonucleoprotein particle.

Molecular and Cellular Biology ◽

10.1128/mcb.16.3.960 ◽

1996 ◽

Vol 16 (3) ◽

pp. 960-967 ◽

Cited By ~ 54

Author(s):

H Y Kao ◽

P G Siliciano

Keyword(s):

Essential Gene ◽

Temperature Sensitive ◽

Small Nuclear Rna ◽

Ribonucleoprotein Particle ◽

Direct Role ◽

Small Nuclear Ribonucleoprotein ◽

Cold Sensitive ◽

Nuclear Ribonucleoprotein ◽

Small Nuclear Ribonucleoprotein Particle

We have used suppressor genetics to identify factors that interact with Saccharomyces cerevisiae U1 small nuclear RNA (snRNA). In this way, we isolated PRP40-1, a suppressor that restores growth at 18 degrees C to a strain bearing a cold-sensitive mutation in U1 RNA. A gene disruption experiment shows that PRP40 is an essential gene. To study the role of PRP40 in splicing, we created a pool of temperature-sensitive prp40 strains. Primer extension analysis of intron-containing transcripts in prp40 temperature-sensitive strains reveals a splicing defect, indicating that Prp40 plays a direct role in pre-mRNA splicing. In addition, U1 RNA coimmunoprecipitates with Pro40, indicating that Prp40 is bound to the U1 small nuclear ribonucleoprotein particle in vivo. Therefore, we conclude that PRP40 encodes a novel, essential splicing component that associates with the yeast U1 small nuclear ribonucleoprotein particle.

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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

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.6337-6349.1994 ◽

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.

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Substrate-assisted mechanism of RNP disruption by the spliceosomal Brr2 RNA helicase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1524616113 ◽

2016 ◽

Vol 113 (28) ◽

pp. 7798-7803 ◽

Cited By ~ 13

Author(s):

Matthias Theuser ◽

Claudia Höbartner ◽

Markus C. Wahl ◽

Karine F. Santos

Keyword(s):

Rna Helicase ◽

Small Nuclear Rna ◽

Base Pairs ◽

U6 Snrna ◽

Small Nuclear Ribonucleoprotein ◽

Alternative Conformation ◽

Sequential Addition ◽

Spliceosome Activation ◽

Set Up ◽

Nuclear Ribonucleoprotein

The Brr2 RNA helicase disrupts the U4/U6 di-small nuclear RNA–protein complex (di-snRNP) during spliceosome activation via ATP-driven translocation on the U4 snRNA strand. However, it is unclear how bound proteins influence U4/U6 unwinding, which regions of the U4/U6 duplex the helicase actively unwinds, and whether U4/U6 components are released as individual molecules or as subcomplexes. Here, we set up a recombinant Brr2-mediated U4/U6 di-snRNP disruption system, showing that sequential addition of the U4/U6 proteins small nuclear ribonucleoprotein-associated protein 1 (Snu13), pre-mRNA processing factor 31 (Prp31), and Prp3 to U4/U6 di-snRNA leads to a stepwise decrease of Brr2-mediated U4/U6 unwinding, but that unwinding is largely restored by a Brr2 cofactor, the C-terminal Jab1/MPN domain of the Prp8 protein. Brr2-mediated U4/U6 unwinding was strongly inhibited by mutations in U4/U6 di-snRNAs that diminish the ability of U6 snRNA to adopt an alternative conformation but leave the number and kind of U4/U6 base pairs unchanged. Irrespective of the presence of the cofactor, the helicase segregated a Prp3-Prp31-Snu13-U4/U6 RNP into an intact Prp31-Snu13-U4 snRNA particle, free Prp3, and free U6 snRNA. Together, these observations suggest that Brr2 translocates only a limited distance on the U4 snRNA strand and does not actively release RNA-bound proteins. Unwinding is then completed by the partially displaced U6 snRNA adopting an alternative conformation, which leads to dismantling of the Prp3-binding site on U4/U6 di-snRNA but leaves the Prp31- and Snu13-binding sites on U4 snRNA unaffected. In this fashion, Brr2 can activate the spliceosome by stripping U6 snRNA of all precatalytic binding partners, while minimizing logistic requirements for U4/U6 di-snRNP reassembly after splicing.

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Structure and conformational plasticity of the U6 small nuclear ribonucleoprotein core

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798316018222 ◽

2017 ◽

Vol 73 (1) ◽

pp. 1-8 ◽

Cited By ~ 5

Author(s):

Eric J. Montemayor ◽

Allison L. Didychuk ◽

Honghong Liao ◽

Panzhou Hu ◽

David A. Brow ◽

...

Keyword(s):

Crystal Structure ◽

Messenger Rna ◽

Rna Recognition Motif ◽

Small Nuclear Rna ◽

Wild Type ◽

Stem Loop ◽

Small Nuclear Ribonucleoprotein ◽

Conformational Plasticity ◽

Gel Shift Assay ◽

Nuclear Ribonucleoprotein

U6 small nuclear RNA (snRNA) is a key component of the active site of the spliceosome, a large ribonucleoprotein complex that catalyzes the splicing of precursor messenger RNA. Prior to its incorporation into the spliceosome, U6 is bound by the protein Prp24, which facilitates unwinding of the U6 internal stem-loop (ISL) so that it can pair with U4 snRNA. A previously reported crystal structure of the `core' of the U6 small nuclear ribonucleoprotein (snRNP) contained an ISL-stabilized A62G mutant of U6 bound to all four RNA-recognition motif (RRM) domains of Prp24 [Montemayoret al.(2014),Nature Struct. Mol. Biol.21, 544–551]. The structure revealed a novel topology containing interlocked rings of protein and RNA that was not predicted by prior biochemical and genetic data. Here, the crystal structure of the U6 snRNP core with a wild-type ISL is reported. This complex crystallized in a new space group, apparently owing in part to the presence of an intramolecular cross-link in RRM1 that was not observed in the previously reported U6-A62G structure. The structure exhibits the same protein–RNA interface and maintains the unique interlocked topology. However, the orientation of the wild-type ISL is altered relative to the A62G mutant structure, suggesting inherent structural dynamics that may facilitate its pairing with U4. Consistent with their similar architectures in the crystalline state, the wild-type and A62G variants of U6 exhibit similar Prp24-binding affinities and electrophoretic mobilities when analyzed by gel-shift assay.

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Direct binding of small nuclear ribonucleoprotein G to the Sm site of small nuclear RNA

Journal of Molecular Biology ◽

10.1016/0022-2836(92)90678-d ◽

1992 ◽

Vol 227 (1) ◽

pp. 15-28 ◽

Cited By ~ 17

Author(s):

Volker Heinrichs ◽

Wolfgang Hackl ◽

Reinhard Lührmann

Keyword(s):

Small Nuclear Rna ◽

Small Nuclear Ribonucleoprotein ◽

Nuclear Rna ◽

Direct Binding ◽

Nuclear Ribonucleoprotein

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U1 small nuclear RNA-promoted exon selection requires a minimal distance between the position of U1 binding and the 3' splice site across the exon.

Molecular and Cellular Biology ◽

10.1128/mcb.17.12.7099 ◽

1997 ◽

Vol 17 (12) ◽

pp. 7099-7107 ◽

Cited By ~ 16

Author(s):

D Y Hwang ◽

J B Cohen

Keyword(s):

Splice Site ◽

Minimum Size ◽

Minimal Distance ◽

Small Nuclear Rna ◽

Small Nuclear Ribonucleoprotein ◽

Optimal Distance ◽

Small Nuclear Rnas ◽

Constitutive Splicing ◽

Nuclear Ribonucleoprotein ◽

Lower Size

Both experimental work and surveys of the lengths of internal exons in nature have suggested that vertebrate internal exons require a minimum size of approximately 50 nucleotides for efficient inclusion in mature mRNA. This phenomenon has been ascribed to steric interference between complexes involved in recognition of the splicing signals at the two ends of short internal exons. To determine whether U1 small nuclear ribonucleoprotein, a multicomponent splicing factor that is involved in the first recognition of splice sites, contributes to the lower size limit of vertebrate internal exons, we have taken advantage of our previous observation that U1 small nuclear RNAs (snRNAs) which bind upstream or downstream of the 5' splice site (5'SS) stimulate splicing of the upstream intron. By varying the position of U1 binding relative to the 3'SS, we show that U1-dependent splicing of the upstream intron becomes inefficient when U1 is positioned 48 nucleotides or less downstream of the 3'SS, suggesting a minimal distance between U1 and the 3'SS of approximately 50 nucleotides. This distance corresponds well to the suggested minimum size of internal exons. The results of experiments in which the 3'SS region of the reporter was duplicated suggest an optimal distance of greater than 72 nucleotides. We have also found that inclusion of a 24-nucleotide miniexon is promoted by the binding of U1 to the downstream intron but not by binding to the 5'SS. Our results are discussed in the context of models to explain constitutive splicing of small exons in nature.

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U5 small nuclear ribonucleoprotein: RNA structure analysis and ATP-dependent interaction with U4/U6.

Molecular and Cellular Biology ◽

10.1128/mcb.9.8.3350 ◽

1989 ◽

Vol 9 (8) ◽

pp. 3350-3359 ◽

Cited By ~ 49

Author(s):

D L Black ◽

A L Pinto

Keyword(s):

Rna Structure ◽

Rna Secondary Structure ◽

Rnase H ◽

Rna Sequences ◽

Large Loop ◽

Small Nuclear Ribonucleoprotein ◽

Nuclear Extracts ◽

The Individual ◽

Nuclear Ribonucleoprotein ◽

Dependent Interaction

To understand how the U5 small nuclear ribonucleoprotein (snRNP) interacts with other spliceosome components, its structure and binding to the U4/U6 snRNP were analyzed. The interaction of the U5 snRNP with the U4/U6 snRNP was studied by separating the snRNPs in HeLa cell nuclear extracts on glycerol gradients. A complex running at 25S and containing U4, U5, and U6 but not U1 or U2 snRNAs was identified. In contrast to results with native gel electrophoresis to separate snRNPs, this U4/U5/U6 snRNP complex requires ATP to assemble from the individual snRNPs. The structure of the U5 RNA within the U5 snRNP and the U4/5/6 snRNP complexes was then compared. Oligonucleotide-targeted RNase H digestion identified one RNA sequence in the U5 snRNP capable of base pairing to other nucleic acid sequences. Chemical modification experiments identified this sequence as well as two other U5 RNA sequences as accessible to modification within the U5 RNP. One of these regions is a large loop in the U5 RNA secondary structure whose sequence is conserved from Saccharomyces cerevisiae to humans. Interestingly, no differences in modification of free U5 snRNP as compared to U5 in the U4/U5/U6 snRNP complex were observed, suggesting that recognition of specific RNA sequences in the U5 snRNP is not required for U4/U5/U6 snRNP assembly.

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