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 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.

scholarly journals Mechanism of 5ʹ splice site transfer for human spliceosome activation

Science ◽  
2019 ◽  
Vol 364 (6438) ◽  
pp. 362-367 ◽  
Author(s):  
Clément Charenton ◽  
Max E. Wilkinson ◽  
Kiyoshi Nagai
Keyword(s):  
Splice Site ◽  
Messenger Rna ◽  
Catalytic Center ◽  
U6 Snrna ◽  
Dead Box ◽  
Cryo Electron Microscopy ◽  
Branch Point Sequence ◽  
U1 Snrnp ◽  
Spliceosome Activation ◽  
Small Nuclear Ribonucleoproteins

The prespliceosome, comprising U1 and U2 small nuclear ribonucleoproteins (snRNPs) bound to the precursor messenger RNA 5ʹ splice site (5ʹSS) and branch point sequence, associates with the U4/U6.U5 tri-snRNP to form the fully assembled precatalytic pre–B spliceosome. Here, we report cryo–electron microscopy structures of the human pre–B complex captured before U1 snRNP dissociation at 3.3-angstrom core resolution and the human tri-snRNP at 2.9-angstrom resolution. U1 snRNP inserts the 5ʹSS–U1 snRNA helix between the two RecA domains of the Prp28 DEAD-box helicase. Adenosine 5ʹ-triphosphate–dependent closure of the Prp28 RecA domains releases the 5ʹSS to pair with the nearby U6 ACAGAGA-box sequence presented as a mobile loop. The structures suggest that formation of the 5ʹSS-ACAGAGA helix triggers remodeling of an intricate protein-RNA network to induce Brr2 helicase relocation to its loading sequence in U4 snRNA, enabling Brr2 to unwind the U4/U6 snRNA duplex to allow U6 snRNA to form the catalytic center of the spliceosome.

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.

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.

Cryo-EM Studies of Pre-mRNA Splicing: From Sample Preparation to Model Visualization

2018 ◽  
Vol 47 (1) ◽  
pp. 175-199 ◽  
Author(s):  
Max E. Wilkinson ◽  
Pei-Chun Lin ◽  
Clemens Plaschka ◽  
Kiyoshi Nagai
Keyword(s):  
Messenger Rna ◽  
Model Building ◽  
De Novo ◽  
Small Nuclear Ribonucleoprotein ◽  
Cryo Electron Microscopy ◽  
Eukaryotic Gene Expression ◽  
Eukaryotic Gene ◽  
Recent Developments ◽  
Nuclear Ribonucleoprotein ◽  
Model Visualization

The removal of noncoding introns from pre-messenger RNA (pre-mRNA) is an essential step in eukaryotic gene expression and is catalyzed by a dynamic multi-megadalton ribonucleoprotein complex called the spliceosome. The spliceosome assembles on pre-mRNA substrates by the stepwise addition of small nuclear ribonucleoprotein particles and numerous protein factors. Extensive remodeling is required to form the RNA-based active site and to mediate the pre-mRNA branching and ligation reactions. In the past two years, cryo-electron microscopy (cryo-EM) structures of spliceosomes captured in different assembly and catalytic states have greatly advanced our understanding of its mechanism. This was made possible by long-standing efforts in the purification of spliceosome intermediates as well as recent developments in cryo-EM imaging and computational methodology. The resulting high-resolution densities allow for de novo model building in core regions of the complexes. In peripheral and less ordered regions, the combination of cross-linking, bioinformatics, biochemical, and genetic data is essential for accurate modeling. Here, we summarize these achievements and highlight the critical steps in obtaining near-atomic resolution structures of the spliceosome.

scholarly journals Structure of an active human histone pre-mRNA 3′-end processing machinery

Science ◽  
2020 ◽  
Vol 367 (6478) ◽  
pp. 700-703 ◽  
Author(s):  
Yadong Sun ◽  
Yixiao Zhang ◽  
Wei Shen Aik ◽  
Xiao-Cui Yang ◽  
William F. Marzluff ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Messenger Rnas ◽  
Small Nuclear Rna ◽  
Processing Machinery ◽  
Small Nuclear Ribonucleoprotein ◽  
Cryo Electron Microscopy ◽  
Nuclear Rna ◽  
Cleavage And Polyadenylation ◽  
Specificity Factor ◽  
Nuclear Ribonucleoprotein

The 3′-end processing machinery for metazoan replication-dependent histone precursor messenger RNAs (pre-mRNAs) contains the U7 small nuclear ribonucleoprotein and shares the key cleavage module with the canonical cleavage and polyadenylation machinery. We reconstituted an active human histone pre-mRNA processing machinery using 13 recombinant proteins and two RNAs and determined its structure by cryo–electron microscopy. The overall structure is highly asymmetrical and resembles an amphora with one long handle. We captured the pre-mRNA in the active site of the endonuclease, the 73-kilodalton subunit of the cleavage and polyadenylation specificity factor, poised for cleavage. The endonuclease and the entire cleavage module undergo extensive rearrangements for activation, triggered through the recognition of the duplex between the authentic pre-mRNA and U7 small nuclear RNA (snRNA). Our study also has notable implications for understanding canonical and snRNA 3′-end processing.

scholarly journals Structure of the activated human minor spliceosome

Science ◽  
2021 ◽  
pp. eabg0879
Author(s):  
Rui Bai ◽  
Ruixue Wan ◽  
Lin Wang ◽  
Kui Xu ◽  
Qiangfeng Zhang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Splice Site ◽  
Zinc Finger Protein ◽  
Small Nuclear Rna ◽  
Catalytic Center ◽  
Cryo Electron Microscopy ◽  
Finger Protein ◽  
Branch Point Sequence ◽  
Nuclear Rna ◽  
U5 Snrna

The minor spliceosome mediates splicing of the rare but essential U12-type pre-mRNA. Here we report the atomic features of the activated human minor spliceosome determined by cryo-electron microscopy at 2.9-Å resolution. The 5′-splice site and branch point sequence of the U12-type intron are recognized by U6atac and U12 small nuclear RNA (snRNA), respectively. Five newly identified proteins stabilize the conformation of the catalytic center. The zinc finger protein SCNM1 functionally mimics the SF3a complex of the major spliceosome. The RBM48/ARMC7 complex binds the γ-monomethyl phosphate cap at the 5′-end of U6atac snRNA. The U-box protein PPIL2 coordinates loop I of U5 snRNA and stabilizes U5 snRNP. CRIPT stabilizes U12 snRNP. Our study provides a framework for mechanistic understanding of the function of the minor spliceosome.

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

1992 ◽  
Vol 12 (11) ◽  
pp. 5197-5205 ◽  
Author(s):  
D Frank ◽  
B Patterson ◽  
C Guthrie
Keyword(s):  
Mrna Splicing ◽  
Second Step ◽  
Small Nuclear Rna ◽  
Synthetic Lethal ◽  
Lethal Mutations ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Rna ◽  
Nuclear Ribonucleoprotein ◽  
U5 Snrna

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

1992 ◽  
Vol 12 (11) ◽  
pp. 5197-5205
Author(s):  
D Frank ◽  
B Patterson ◽  
C Guthrie
Keyword(s):  
Mrna Splicing ◽  
Second Step ◽  
Small Nuclear Rna ◽  
Synthetic Lethal ◽  
Lethal Mutations ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Rna ◽  
Nuclear Ribonucleoprotein ◽  
U5 Snrna

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.

scholarly journals Conserved Loop I of U5 Small Nuclear RNA Is Dispensable for Both Catalytic Steps of Pre-mRNA Splicing in HeLa Nuclear Extracts

1999 ◽  
Vol 19 (4) ◽  
pp. 2782-2790 ◽  
Author(s):  
Véronique Ségault ◽  
Cindy L. Will ◽  
Maria Polycarpou-Schwarz ◽  
Iain W. Mattaj ◽  
Christiane Branlant ◽  
...  
Keyword(s):  
Mrna Splicing ◽  
Small Nuclear Rna ◽  
Internal Loop ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Extracts ◽  
Nuclear Rna ◽  
Nuclear Ribonucleoprotein ◽  
U5 Snrna ◽  
Loop 2

ABSTRACT The function of conserved regions of the metazoan U5 snRNA was investigated by reconstituting U5 small nuclear ribonucleoprotein particles (snRNPs) from purified snRNP proteins and HeLa orXenopus U5 snRNA mutants and testing their ability to restore splicing to U5-depleted nuclear extracts. Substitution of conserved nucleotides comprising internal loop 2 or deletion of internal loop 1 had no significant effect on the ability of reconstituted U5 snRNPs to complement splicing. However, deletion of internal loop 2 abolished U5 activity in splicing and spliceosome formation. Surprisingly, substitution of the invariant loop 1 nucleotides with a GAGA tetraloop had no effect on U5 activity. Furthermore, U5 snRNPs reconstituted from an RNA formed by annealing the 5′ and 3′ halves of the U5 snRNA, which lacked all loop 1 nucleotides, complemented both steps of splicing. Thus, in contrast to yeast, loop 1 of the human U5 snRNA is dispensable for both steps of splicing in HeLa nuclear extracts. This suggests that its function can be compensated for in vitro by other spliceosomal components: for example, by proteins associated with the U5 snRNP. Consistent with this idea, immunoprecipitation studies indicated that several functionally important U5 proteins associate stably with U5 snRNPs containing a GAGA loop 1 substitution.

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