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.

RNA Splicing by the Spliceosome

2020 ◽  
Vol 89 (1) ◽  
pp. 359-388 ◽  
Author(s):  
Max E. Wilkinson ◽  
Clément Charenton ◽  
Kiyoshi Nagai
Keyword(s):  
Splice Site ◽  
Active Site ◽  
Rna Splicing ◽  
Messenger Rna ◽  
Structural Studies ◽  
U2 Snrna ◽  
U6 Snrna ◽  
Mature Mrna ◽  
Catalytic Metal ◽  
Small Nuclear Ribonucleoproteins

The spliceosome removes introns from messenger RNA precursors (pre-mRNA). Decades of biochemistry and genetics combined with recent structural studies of the spliceosome have produced a detailed view of the mechanism of splicing. In this review, we aim to make this mechanism understandable and provide several videos of the spliceosome in action to illustrate the intricate choreography of splicing. The U1 and U2 small nuclear ribonucleoproteins (snRNPs) mark an intron and recruit the U4/U6.U5 tri-snRNP. Transfer of the 5′ splice site (5′SS) from U1 to U6 snRNA triggers unwinding of U6 snRNA from U4 snRNA. U6 folds with U2 snRNA into an RNA-based active site that positions the 5′SS at two catalytic metal ions. The branch point (BP) adenosine attacks the 5′SS, producing a free 5′ exon. Removal of the BP adenosine from the active site allows the 3′SS to bind, so that the 5′ exon attacks the 3′SS to produce mature mRNA and an excised lariat intron.

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.

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 Phylogenetic comparison and splice site conservation of eukaryotic U1 snRNP-specific U1-70K gene family

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tao Fan ◽  
Yu-Zhen Zhao ◽  
Jing-Fang Yang ◽  
Qin-Lai Liu ◽  
Yuan Tian ◽  
...  
Keyword(s):  
Gene Family ◽  
Splice Site ◽  
Messenger Rna ◽  
Expression Patterns ◽  
Protein Structures ◽  
Single Copy ◽  
Central Component ◽  
Animal Kingdom ◽  
Functional Studies ◽  
U1 Snrnp

AbstractEukaryotic cells can expand their coding ability by using their splicing machinery, spliceosome, to process precursor mRNA (pre-mRNA) into mature messenger RNA. The mega-macromolecular spliceosome contains multiple subcomplexes, referred to as small nuclear ribonucleoproteins (snRNPs). Among these, U1 snRNP and its central component, U1-70K, are crucial for splice site recognition during early spliceosome assembly. The human U1-70K has been linked to several types of human autoimmune and neurodegenerative diseases. However, its phylogenetic relationship has been seldom reported. To this end, we carried out a systemic analysis of 95 animal U1-70K genes and compare these proteins to their yeast and plant counterparts. Analysis of their gene and protein structures, expression patterns and splicing conservation suggest that animal U1-70Ks are conserved in their molecular function, and may play essential role in cancers and juvenile development. In particular, animal U1-70Ks display unique characteristics of single copy number and a splicing isoform with truncated C-terminal, suggesting the specific role of these U1-70Ks in animal kingdom. In summary, our results provide phylogenetic overview of U1-70K gene family in vertebrates. In silico analyses conducted in this work will act as a reference for future functional studies of this crucial U1 splicing factor in animal kingdom.

scholarly journals Structures of the Human Spliceosomes Before and After Release of the Ligated Exon

2018 ◽  
Author(s):  
Xiaofeng Zhang ◽  
Xiechao Zhan ◽  
Chuangye Yan ◽  
Wenyu Zhang ◽  
Dongliang Liu ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Splice Site ◽  
Mrna Splicing ◽  
Small Nuclear Rna ◽  
Exon Ligation ◽  
Cryo Electron Microscopy ◽  
Branch Point Sequence ◽  
Nuclear Rna ◽  
Before And After ◽  
Functional States

Pre-mRNA splicing is executed by the spliceosome, which has eight major functional states each with distinct composition. Five of these eight human spliceosomal complexes, all preceding exon ligation, have been structurally characterized. In this study, we report the cryo-electron microscopy structures of the human post-catalytic spliceosome (P complex) and intron lariat spliceosome (ILS) at average resolutions of 3.0 and 2.9 Å, respectively. In the P complex, the ligated exon remains anchored to loop I of U5 small nuclear RNA, and the 3'-splice site is recognized by the junction between the 5'-splice site and the branch point sequence. The ATPase/helicase Prp22, along with the ligated exon and eight other proteins, are dissociated in the P-to-ILS transition. Intriguingly, the ILS complex exists in two distinct conformations, one with the ATPase/helicase Prp43 and one without. Comparison of these three late-stage human spliceosomes reveals mechanistic insights into exon release and spliceosome disassembly.

scholarly journals Structure of a transcribing RNA polymerase II–U1 snRNP complex

Science ◽  
2021 ◽  
Vol 371 (6526) ◽  
pp. 305-309 ◽  
Author(s):  
Suyang Zhang ◽  
Shintaro Aibara ◽  
Seychelle M. Vos ◽  
Dmitry E. Agafonov ◽  
Reinhard Lührmann ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Splice Site ◽  
Messenger Rna ◽  
Loop Formation ◽  
Mrna Isoforms ◽  
Pol Ii ◽  
Small Nuclear Ribonucleoprotein ◽  
Starting Point ◽  
U1 Snrnp

To initiate cotranscriptional splicing, RNA polymerase II (Pol II) recruits the U1 small nuclear ribonucleoprotein particle (U1 snRNP) to nascent precursor messenger RNA (pre-mRNA). Here, we report the cryo–electron microscopy structure of a mammalian transcribing Pol II–U1 snRNP complex. The structure reveals that Pol II and U1 snRNP interact directly. This interaction positions the pre-mRNA 5′ splice site near the RNA exit site of Pol II. Extension of pre-mRNA retains the 5′ splice site, leading to the formation of a “growing intron loop.” Loop formation may facilitate scanning of nascent pre-mRNA for the 3′ splice site, functional pairing of distant intron ends, and prespliceosome assembly. Our results provide a starting point for a mechanistic analysis of cotranscriptional spliceosome assembly and the biogenesis of mRNA isoforms by alternative 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 Involvement of U1 Small Nuclear Ribonucleoproteins (snRNP) in 5′ Splice Site-U1 snRNP Interaction

1996 ◽  
Vol 271 (39) ◽  
pp. 23985-23991 ◽  
Author(s):  
Ferdinand Rossi ◽  
Thierry Forné ◽  
Etienne Antoine ◽  
Jamal Tazi ◽  
Claude Brunel ◽  
...  
Keyword(s):  
Splice Site ◽  
U1 Snrnp ◽  
Small Nuclear Ribonucleoproteins

Multiple interactions between the splicing substrate and small nuclear ribonucleoproteins in spliceosomes

1987 ◽  
Vol 7 (1) ◽  
pp. 281-293
Author(s):  
B Chabot ◽  
J A Steitz
Keyword(s):  
Splice Site ◽  
Beta Globin ◽  
Branch Site ◽  
U2 Snrnp ◽  
U1 Snrnp ◽  
Branch Formation ◽  
Splice Site Sequence ◽  
Multiple Interactions ◽  
Small Nuclear Ribonucleoproteins

Protection experiments with antibodies against small nuclear ribonucleoproteins (snRNPs) have elucidated the location of and requirements for interactions between snRNPs and human beta-globin transcripts during splicing in vitro. U2 snRNP association with the intron branch site continues after branch formation, requires intact U2 RNA, and is affected by some alterations of the 3' splice site sequence. U2 snRNP binding to the branched intermediate and U1 snRNP protection of an extended 5' splice region are detected exclusively in spliceosome fractions, indicating that both snRNPs are spliceosome components. While each snRNP associates specifically with the pre-mRNA, they also appear to interact with each other. The recovery of fragments mapping upstream of the 5' splice site suggests how the excised exon is held in the spliceosome.

scholarly journals Multiple interactions between the splicing substrate and small nuclear ribonucleoproteins in spliceosomes.

1987 ◽  
Vol 7 (1) ◽  
pp. 281-293 ◽  
Author(s):  
B Chabot ◽  
J A Steitz
Keyword(s):  
Splice Site ◽  
Beta Globin ◽  
Branch Site ◽  
U2 Snrnp ◽  
U1 Snrnp ◽  
Branch Formation ◽  
Splice Site Sequence ◽  
Multiple Interactions ◽  
Small Nuclear Ribonucleoproteins

Protection experiments with antibodies against small nuclear ribonucleoproteins (snRNPs) have elucidated the location of and requirements for interactions between snRNPs and human beta-globin transcripts during splicing in vitro. U2 snRNP association with the intron branch site continues after branch formation, requires intact U2 RNA, and is affected by some alterations of the 3' splice site sequence. U2 snRNP binding to the branched intermediate and U1 snRNP protection of an extended 5' splice region are detected exclusively in spliceosome fractions, indicating that both snRNPs are spliceosome components. While each snRNP associates specifically with the pre-mRNA, they also appear to interact with each other. The recovery of fragments mapping upstream of the 5' splice site suggests how the excised exon is held in the spliceosome.

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