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

2016 ◽  
Vol 113 (28) ◽  
pp. 7798-7803 ◽  
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.

scholarly journals The Pre-mRNA 5′ Cap Determines Whether U6 Small Nuclear RNA Succeeds U1 Small Nuclear Ribonucleoprotein Particle at 5′ Splice Sites

1998 ◽  
Vol 18 (12) ◽  
pp. 7510-7520 ◽  
Author(s):  
Laura O’Mullane ◽  
Ian C. Eperon
Keyword(s):  
Splice Site ◽  
Major Effect ◽  
Small Nuclear Rna ◽  
Splice Sites ◽  
U6 Snrna ◽  
Small Nuclear Ribonucleoprotein ◽  
U1 Snrna ◽  
U1 Snrnp ◽  
Nuclear Rna ◽  
Nuclear Ribonucleoprotein

ABSTRACT Efficient splicing of the 5′-most intron of pre-mRNA requires a 5′ m7G(5′)ppp(5′)N cap, which has been implicated in U1 snRNP binding to 5′ splice sites. We demonstrate that the cap alters the kinetic profile of U1 snRNP binding, but its major effect is on U6 snRNA binding. With two alternative wild-type splice sites in an adenovirus pre-mRNA, the cap selectively alters U1 snRNA binding at the site to which cap-independent U1 snRNP binding is stronger and that is used predominantly in splicing; with two consensus sites, the cap acts on both, even though one is substantially preferred for splicing. However, the most striking quantitative effect of the 5′ cap is neither on U1 snRNP binding nor on the assembly of large complexes but on the replacement of U1 snRNP by U6 snRNA at the 5′ splice site. Inhibition of splicing by a cap analogue is correlated with the loss of U6 interactions at the 5′ splice site and not with any loss of U1 snRNP binding.

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

1994 ◽  
Vol 14 (3) ◽  
pp. 1689-1697 ◽  
Author(s):  
M I Zavanelli ◽  
J S Britton ◽  
A H Igel ◽  
M Ares
Keyword(s):  
Rna Structure ◽  
Cold Sensitivity ◽  
Small Nuclear Rna ◽  
Stem Loop ◽  
Alternative Structures ◽  
Small Nuclear Ribonucleoprotein ◽  
Alternative Conformation ◽  
Cold Sensitive ◽  
The Stability ◽  
Nuclear Ribonucleoprotein

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.

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

1994 ◽  
Vol 14 (3) ◽  
pp. 1689-1697
Author(s):  
M I Zavanelli ◽  
J S Britton ◽  
A H Igel ◽  
M Ares
Keyword(s):  
Rna Structure ◽  
Cold Sensitivity ◽  
Small Nuclear Rna ◽  
Stem Loop ◽  
Alternative Structures ◽  
Small Nuclear Ribonucleoprotein ◽  
Alternative Conformation ◽  
Cold Sensitive ◽  
The Stability ◽  
Nuclear Ribonucleoprotein

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.

Structures of the fully assembledSaccharomyces cerevisiaespliceosome before activation

Science ◽  
2018 ◽  
Vol 360 (6396) ◽  
pp. 1423-1429 ◽  
Author(s):  
Rui Bai ◽  
Ruixue Wan ◽  
Chuangye Yan ◽  
Jianlin Lei ◽  
Yigong Shi
Keyword(s):  
Messenger Rna ◽  
Small Nuclear Rna ◽  
U6 Snrna ◽  
Small Nuclear Ribonucleoprotein ◽  
Cryo Electron Microscopy ◽  
Branch Point Sequence ◽  
Specific Proteins ◽  
Complex Structural ◽  
Nuclear Ribonucleoprotein ◽  
U5 Snrna

The precatalytic spliceosome (B complex) is preceded by the pre-B complex. Here we report the cryo–electron microscopy structures of theSaccharomyces cerevisiaepre-B and B complexes at average resolutions of 3.3 to 4.6 and 3.9 angstroms, respectively. In the pre-B complex, the duplex between the 5′ splice site (5′SS) and U1 small nuclear RNA (snRNA) is recognized by Yhc1, Luc7, and the Sm ring. In the B complex, U1 small nuclear ribonucleoprotein is dissociated, the 5′-exon–5′SS sequences are translocated near U6 snRNA, and three B-specific proteins may orient the precursor messenger RNA. In both complexes, U6 snRNA is anchored to loop I of U5 snRNA, and the duplex between the branch point sequence and U2 snRNA is recognized by the SF3b complex. Structural analysis reveals the mechanism of assembly and activation for the yeast spliceosome.

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.

scholarly journals 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.

1997 ◽  
Vol 17 (12) ◽  
pp. 7099-7107 ◽  
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.

scholarly journals FUS functions in coupling transcription to splicing by mediating an interaction between RNAP II and U1 snRNP

2015 ◽  
Vol 112 (28) ◽  
pp. 8608-8613 ◽  
Author(s):  
Yong Yu ◽  
Robin Reed
Keyword(s):  
Rna Polymerase Ii ◽  
Coupled System ◽  
Base Pairs ◽  
Fused In Sarcoma ◽  
Small Nuclear Ribonucleoprotein ◽  
U1 Snrnp ◽  
Rnap Ii ◽  
Nuclear Ribonucleoprotein ◽  
Antisense Morpholino ◽  
Lateral Sclerosis

Pre-mRNA splicing is coupled to transcription by RNA polymerase II (RNAP II). We previously showed that U1 small nuclear ribonucleoprotein (snRNP) associates with RNAP II, and both RNAP II and U1 snRNP are also the most abundant factors associated with the protein fused-in-sarcoma (FUS), which is mutated to cause the neurodegenerative disease amyotrophic lateral sclerosis. Here, we show that an antisense morpholino that base-pairs to the 5′ end of U1 snRNA blocks splicing in the coupled system and completely disrupts the association between U1 snRNP and both FUS and RNAP II, but has no effect on the association between FUS and RNAP II. Conversely, we found that U1 snRNP does not interact with RNAP II in FUS knockdown extracts. Moreover, using these extracts, we found that FUS must be present during the transcription reaction in order for splicing to occur. Together, our data lead to a model that FUS functions in coupling transcription to splicing via mediating an interaction between RNAP II and U1 snRNP.

Secondary structure of U6 small nuclear RNA: implications for spliceosome assembly

2010 ◽  
Vol 38 (4) ◽  
pp. 1099-1104 ◽  
Author(s):  
Elizabeth A. Dunn ◽  
Stephen D. Rader
Keyword(s):  
Secondary Structure ◽  
Small Nuclear Rna ◽  
Stem Loop ◽  
New Model ◽  
Rna Molecules ◽  
U6 Snrna ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Rna ◽  
High Level ◽  
And Function

U6 snRNA (small nuclear RNA), one of five RNA molecules that are required for the essential process of pre-mRNA splicing, is notable for its high level of sequence conservation and the important role it is thought to play in the splicing reaction. Nevertheless, the secondary structure of U6 in the free snRNP (small nuclear ribonucleoprotein) form has remained elusive, with predictions changing substantially over the years. In the present review we discuss the evidence for existing models and critically evaluate a fundamental assumption of these models, namely whether the important 3′ ISL (3′ internal stem–loop) is present in the free U6 particle, as well as in the active splicing complex. We compare existing models of free U6 with a newly proposed model lacking the 3′ ISL and evaluate the implications of the new model for the structure and function of U6's base-pairing partner U4 snRNA. Intriguingly, the new model predicts a role for U4 that was unanticipated previously, namely as an activator of U6 for assembly into the splicing machinery.

scholarly journals Loop I of U1 small nuclear RNA is the only essential RNA sequence for binding of specific U1 small nuclear ribonucleoprotein particle proteins.

1988 ◽  
Vol 8 (11) ◽  
pp. 4787-4791 ◽  
Author(s):  
J Hamm ◽  
V L van Santen ◽  
R A Spritz ◽  
I W Mattaj
Keyword(s):  
Protein Interactions ◽  
Protein C ◽  
Small Nuclear Rna ◽  
Structural Element ◽  
Rna Sequence ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Rna ◽  
Egg Extracts ◽  
Specific Proteins ◽  
Nuclear Ribonucleoprotein

The binding of the U1 small nuclear ribonucleoprotein (snRNP)-specific proteins C, A, and 70K to U1 small nuclear RNA (snRNA) was analyzed. Assembly of U1 snRNAs from bean and soybean and a set of mutant Xenopus U1 snRNAs into U1 snRNPs in Xenopus egg extracts was studied. The ability to bind proteins was analyzed by immunoprecipitation with monospecific antibodies and by a protein-sequestering assay. The only sequence essential for binding of the U1-specific proteins was the conserved loop sequence in the 5' hairpin of U1. Further analysis suggested that protein C binds directly to the loop and that the assembly of proteins A and 70K into the RNP requires mainly protein-protein interactions. Protein C apparently recognizes a specific RNA sequence rather than a secondary structural element in the RNA.

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