RNA Splicing by the Spliceosome

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.

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Mechanism of 5ʹ splice site transfer for human spliceosome activation

Science ◽

10.1126/science.aax3289 ◽

2019 ◽

Vol 364 (6438) ◽

pp. 362-367 ◽

Cited By ~ 30

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.

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How Is Precursor Messenger RNA Spliced by the Spliceosome?

Annual Review of Biochemistry ◽

10.1146/annurev-biochem-013118-111024 ◽

2020 ◽

Vol 89 (1) ◽

pp. 333-358 ◽

Cited By ~ 4

Author(s):

Ruixue Wan ◽

Rui Bai ◽

Xiechao Zhan ◽

Yigong Shi

Keyword(s):

Active Site ◽

Rna Splicing ◽

Messenger Rna ◽

Small Nuclear Rna ◽

Exon Ligation ◽

Nuclear Rna ◽

Catalytic Metal ◽

Protein Components ◽

Intron Removal ◽

Duplex Formation

Splicing of the precursor messenger RNA, involving intron removal and exon ligation, is mediated by the spliceosome. Together with biochemical and genetic investigations of the past four decades, structural studies of the intact spliceosome at atomic resolution since 2015 have led to mechanistic delineation of RNA splicing with remarkable insights. The spliceosome is proven to be a protein-orchestrated metalloribozyme. Conserved elements of small nuclear RNA (snRNA) constitute the splicing active site with two catalytic metal ions and recognize three conserved intron elements through duplex formation, which are delivered into the splicing active site for branching and exon ligation. The protein components of the spliceosome stabilize the conformation of the snRNA, drive spliceosome remodeling, orchestrate the movement of the RNA elements, and facilitate the splicing reaction. The overall organization of the spliceosome and the configuration of the splicing active site are strictly conserved between human and yeast.

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Predicting the effect of variants on splicing using Convolutional Neural Networks

PeerJ ◽

10.7717/peerj.9470 ◽

2020 ◽

Vol 8 ◽

pp. e9470

Author(s):

Thanyathorn Thanapattheerakul ◽

Worrawat Engchuan ◽

Jonathan H. Chan

Keyword(s):

Splice Site ◽

Predictive Models ◽

Rna Splicing ◽

Messenger Rna ◽

Computational Models ◽

Splice Variants ◽

Splice Sites ◽

Splice Site Prediction ◽

Donor And Acceptor ◽

Rna Splice Sites

Mutations that cause an error in the splicing of a messenger RNA (mRNA) can lead to diseases in humans. Various computational models have been developed to recognize the sequence pattern of the splice sites. In recent studies, Convolutional Neural Network (CNN) architectures were shown to outperform other existing models in predicting the splice sites. However, an insufficient effort has been put into extending the CNN model to predict the effect of the genomic variants on the splicing of mRNAs. This study proposes a framework to elaborate on the utility of CNNs to assess the effect of splice variants on the identification of potential disease-causing variants that disrupt the RNA splicing process. Five models, including three CNN-based and two non-CNN machine learning based, were trained and compared using two existing splice site datasets, Genome Wide Human splice sites (GWH) and a dataset provided at the Deep Learning and Artificial Intelligence winter school 2018 (DLAI). The donor sites were also used to test on the HSplice tool to evaluate the predictive models. To improve the effectiveness of predictive models, two datasets were combined. The CNN model with four convolutional layers showed the best splice site prediction performance with an AUPRC of 93.4% and 88.8% for donor and acceptor sites, respectively. The effects of variants on splicing were estimated by applying the best model on variant data from the ClinVar database. Based on the estimation, the framework could effectively differentiate pathogenic variants from the benign variants (p = 5.9 × 10−7). These promising results support that the proposed framework could be applied in future genetic studies to identify disease causing loci involving the splicing mechanism. The datasets and Python scripts used in this study are available on the GitHub repository at https://github.com/smiile8888/rna-splice-sites-recognition.

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

10.1101/500363 ◽

2018 ◽

Cited By ~ 1

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.

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Yeast pre-messenger RNA splicing efficiency depends on critical spacing requirements between the branch point and 3′ splice site.

The EMBO Journal ◽

10.1002/j.1460-2075.1986.tb04317.x ◽

1986 ◽

Vol 5 (5) ◽

pp. 1023-1030 ◽

Cited By ~ 23

Author(s):

A. Cellini ◽

E. Felder ◽

J.J. Rossi

Keyword(s):

Branch Point ◽

Splice Site ◽

Rna Splicing ◽

Messenger Rna ◽

Critical Spacing ◽

Splicing Efficiency

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A human postcatalytic spliceosome structure reveals essential roles of metazoan factors for exon ligation

Science ◽

10.1126/science.aaw5569 ◽

2019 ◽

Vol 363 (6428) ◽

pp. 710-714 ◽

Cited By ~ 37

Author(s):

Sebastian M. Fica ◽

Chris Oubridge ◽

Max E. Wilkinson ◽

Andrew J. Newman ◽

Kiyoshi Nagai

Keyword(s):

Electron Microscopy ◽

Saccharomyces Cerevisiae ◽

Alternative Splicing ◽

Splice Site ◽

Active Site ◽

Messenger Rna ◽

Functional Assays ◽

Exon Ligation ◽

Cryo Electron Microscopy ◽

Fine Tune

During exon ligation, the Saccharomyces cerevisiae spliceosome recognizes the 3′-splice site (3′SS) of precursor messenger RNA (pre-mRNA) through non–Watson-Crick pairing with the 5′SS and the branch adenosine, in a conformation stabilized by Prp18 and Prp8. Here we present the 3.3-angstrom cryo–electron microscopy structure of a human postcatalytic spliceosome just after exon ligation. The 3′SS docks at the active site through conserved RNA interactions in the absence of Prp18. Unexpectedly, the metazoan-specific FAM32A directly bridges the 5′-exon and intron 3′SS of pre-mRNA and promotes exon ligation, as shown by functional assays. CACTIN, SDE2, and NKAP—factors implicated in alternative splicing—further stabilize the catalytic conformation of the spliceosome during exon ligation. Together these four proteins act as exon ligation factors. Our study reveals how the human spliceosome has co-opted additional proteins to modulate a conserved RNA-based mechanism for 3′SS selection and to potentially fine-tune alternative splicing at the exon ligation stage.

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