Structure and Conformational Dynamics of the Human Spliceosomal Bact Complex

Haselbach, David; Komarov, I.; Agafonov, Dmitry E.; Hartmuth, Klaus; Graf, B.; Dybkov, Olexandr; Urlaub, Henning; Kastner, Berthold; Lührmann, Reinhard; Stark, Holger

doi:10.1016/j.cell.2018.01.010

Cited by 199 publications

(285 citation statements)

References 55 publications

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“…This interaction sequesters the hydroxyl group of the bulged adenosine, the first-step nucleophile, from the 5'SS and prevents formation of an active catalytic core. Release of SF3B1 from the Bact complex allows U2 snRNA to undergo a large rotation that collapses the U2:BS duplex into the active site and positions the reactants for first-step catalysis (Galej et al, 2016;Haselbach et al, 2018;Yan et al, 2016;Zhan et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Transcriptional analysis supports the expression of human snRNA variants and reveals U2 snRNA homeostasis by an abundant U2 variant

Kosmyna

Gupta²,

Query³

2020

Preprint

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Although expansion of snRNA genes in the human genome and sequence variation in expressed transcripts were both identified long ago, no study has comprehensively analyzed which genes are transcriptionally active. Here, we use comprehensive bioinformatic analysis to differentiate between similar or identical genomic loci to determine that 49 snRNA genes are actively transcribed. This greatly expands on previous observation of sequence variation within snRNA transcripts. Further analysis of U2 snRNA variants reveals sequence variation maintains conserved secondary structures, yet sensitizes these U2 snRNAs to modulation of assembly factors. Homeostasis of total U2 snRNA level is maintained by altering the ratio of canonical and an abundant U2 snRNA variant. Both canonical and variant snRNA promoters respond to MYC and appear differentially sensitive to increased MYC levels. Thus, we identify transcribed snRNA variants and the sequence variation within, and propose mechanisms of transcriptional and posttranscriptional regulation of snRNA levels and pre-mRNA splicing. (Summary: 150/150 words) KEYWORDS: U2 snRNA, snRNA pseudogene, spliceosome, Sm binding site HIGHLIGHTS • ChIP-seq of active promoters identifies uncharacterized snRNA genes • Transcribed repetitive snRNA genes are distinguished from falsely-mapped snRNA loci • U2 snRNA variants are sensitive to modulations in snRNP assembly • Widely expressed U2 snRNA variants provide homeostasis for total U2 snRNP levels

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Section: Introductionmentioning

confidence: 99%

Transcriptional analysis supports the expression of human snRNA variants and reveals U2 snRNA homeostasis by an abundant U2 variant

Kosmyna

Gupta²,

Query³

2020

Preprint

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“…Many splicing regulatory factors and general splicing factors purify with SWI/SNF subunits (Zhao et al, 1998;Dellaire et al, 2002) and purifications of the snRNP U2 spliceosome component also include several SWI/SNF subunits (Makarov et al, 2012;Allemand et al, 2016). We performed an analysis of BRG1 and BRM interacting proteins in the RNP fraction and it revealed that BRG1, in particular, interacted with U2 snRNP and U5-U6 snRNP factors that assemble early in the splicing cycle (Lardelli et al, 2010;Hegele et al, 2012;Agafonov et al, 2016;Haselbach et al, 2018). In addition to general splicing factors, we also found that BRG1 interacted with many regulatory RNA binding factors, such as hnRNPs and RNA helicases.…”

Section: Discussionmentioning

confidence: 91%

“…In addition to general splicing factors, we also found that BRG1 interacted with many regulatory RNA binding factors, such as hnRNPs and RNA helicases. Recent structural determinations of the spliceosome at different steps show that many rearrangements and compositional changes that occur during the splicing cycle require snRNA, splicing factors and regulatory factors (Bertram et al, 2017a;Bertram et al, 2017b;;Haselbach et al, 2018;Zhang et al, 2018). BRG1 recruits splicing factors and RNA binding proteins to affected exons, and we propose that it affects the interactions of RNA binding factors with their sites in RNA, which in turn influences the composition and the activity of the spliceosome and promote changes in splicing outcome.…”

Section: Discussionmentioning

confidence: 99%

The SWI/SNF subunits BRG1 affects alternative splicing by changing RNA binding factor interactions with RNA

Mackowiak

Guo

et al. 2019

Preprint

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BRG1 and BRM are ATPase core subunits of the human SWI/SNF chromatin remodelling complexes. The function of the SWI/SNF complexes in transcriptional initiation has been well studied, while a function in alternative splicing has only been studied for a few cases for BRM-containing SWI/SNF complexes. Here, we have expressed BRG1 in C33A cells, a BRG1 and BRM-deficient cell line, and we have analysed the effects on the transcriptome by RNA sequencing. We have shown that BRG1 expression affects the splicing of a subset of genes. For some, BRG1 expression favours exon inclusion and for others, exon skipping. Some of the changes in alternative splicing induced by BRG1 expression do not require the ATPase activity of BRG1. Among the exons regulated through an ATPase-independent mechanism, the included exons had signatures of high GC-content and lacked a positioned nucleosome at the exon. By investigating three genes in which the expression of either wild-type BRG1 or a BRG1-ATPase-deficient variant favoured exon inclusion, we showed that expression of the ATPases promotes the local recruitment of RNA binding factors to chromatin and RNA in a differential manner. The hnRNPL, hnRNPU and SAM68 proteins associated to chromatin in C33A cells expressing BRG1 or BRM, but their association with RNA varied. We propose that SWI/SNF can regulate alternative splicing by interacting with splicing-RNA binding factor and altering their binding to the nascent pre-mRNA, which changes RNP structure. Author summarySplicing, in particular alternative splicing, is a combinatorial process which involves splicing factor complexes and many RNA binding splicing regulatory proteins in different constellations. Most splicing events occur during transcription, which also makes the DNA sequence, the chromatin state and the transcription rate at the exons important components that influence the splicing outcome. We show here that the ATP-dependent chromatin remodelling complex SWI/SNF influences the interactions of splicing regulatory factors with RNA during transcription on certain exons that have a high GC-content. The splicing on this type of exon rely on the ATPase BRG1 and favour inclusion of alternative exons in an ATP-independent manner. SWI/SNF complexes are known to alter the chromatin structure at promoters in transcription initiation, and have been previously shown to alter the transcription rate or nucleosome position in splicing. Our results suggests a further mechanism for chromatin remodelling proteins in splicing: to change the interaction patterns of RNA binding splicing regulatory factors at alternative exons to alter the splicing outcome. Biegel JA, Busse TM, Weissman BE. SWI/SNF chromatin remodeling complexes and cancer. Am Lynch KW. Global analysis of physical and functional RNA targets of hnRNP L reveals distinct sequence and epigenetic features of repressed and enhanced exons. :52. Custódio N, Carmo-Fonseca M. Co-transcriptional splicing and the CTD code. Crit Rev Biochem Mol Biol. 2016 Sep;51(5):395-411. De Conti L, Baralle M...

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“…In addition to mog-2 and Y111B2A.25, we identified three different RNAi clones targeting the emb-4 locus as strong enhancers of the sacy-1(tn1385) sterility and gamete degeneration phenotypes (Table 1; Figure S1). emb-4 encodes a nuclear protein orthologous to human Aquarius/IBP160/KIAA0560/fSAP164, an intron-binding spliceosomal protein with a helicase-like domain (Sam et al 1998;Jurica et al 2002;Bessonov et al 2008;Herold et al 2009;De et al 2015;Haselbach et al 2018). To extend these RNAi results, we examined genetic interactions between sacy-1 and emb-4, employing the emb-4(sa44) reduction-of-function allele.…”

Section: Synthetic Lethal Interactions Between a Sacy-1 Reduction-of-mentioning

confidence: 99%

Insights into the involvement of spliceosomal mutations in myelodysplastic disorders from an analysis of SACY-1/DDX41 inCaenorhabditis elegans

Greenstein

Tsukamoto

Gearhart

et al. 2019

Preprint

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Mutations affecting spliceosomal proteins are frequently found in hematological malignancies. DDX41/Abstrakt is a metazoan-specific spliceosomal DEAD-box RNA helicase recurrently mutated in inherited and relapsing myelodysplastic syndromes and acute myeloid leukemia. The genetic properties and genomic impacts of disease-causing mutations in spliceosomal proteins have been uncertain. Here we conduct a comprehensive molecular genetic analysis of the C. elegans DDX41 ortholog, SACY-1. Our results reveal that multiple sacy-1/DDX41 missense mutations, including the R525H human oncogenic variant, exhibit antimorphic activity that likely compromises the function of the spliceosome. The genomic consequences of SACY-1 depletion include splicing-splicingindependent and splicing-dependent alterations in the transcriptome. ABSTRACTMutations affecting spliceosomal proteins are frequently found in hematological malignancies, including myelodysplastic syndromes and acute myeloid leukemia. DDX41/Abstrakt is a metazoanspecific spliceosomal DEAD-box RNA helicase found to be recurrently mutated in inherited myelodysplastic syndromes and in relapsing cases of acute myeloid leukemia. The genetic properties and genomic impacts of disease-causing missense mutations in DDX41 and other spliceosomal proteins have been uncertain. Here we conduct a comprehensive molecular genetic analysis of the C. elegans DDX41 ortholog, SACY-1. Our results reveal general essential functions for SACY-1 in both the germline and the soma, as well as specific functions affecting germline sex determination and cell cycle control. Certain sacy-1/DDX41 mutations, including the R525H human oncogenic variant, confer antimorphic activity, suggesting that they compromise the function of the spliceosome. Consistent with these findings, sacy-1 exhibits synthetic lethal interactions with several spliceosomal components, and biochemical analyses suggest that SACY-1 is a component of the C. elegans spliceosome. We used the auxin-inducible degradation system to analyze the impact of SACY-1 on the transcriptome using RNA sequencing. SACY-1 depletion impacts the transcriptome through splicing-independent and splicingdependent mechanisms. The observed transcriptome changes suggest that disruption of spliceosomal function induces a stress response. Altered 3' splice site usage represents the predominant splicing defect observed upon SACY-1 depletion, consistent with a role for SACY-1 as a second-step splicing factor. Missplicing events appear more prevalent in the soma than the germline, suggesting that surveillance mechanisms protect the germline from aberrant splicing.

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Structure and Conformational Dynamics of the Human Spliceosomal Bact Complex

Cited by 199 publications

References 55 publications

Transcriptional analysis supports the expression of human snRNA variants and reveals U2 snRNA homeostasis by an abundant U2 variant

Transcriptional analysis supports the expression of human snRNA variants and reveals U2 snRNA homeostasis by an abundant U2 variant

The SWI/SNF subunits BRG1 affects alternative splicing by changing RNA binding factor interactions with RNA

Insights into the involvement of spliceosomal mutations in myelodysplastic disorders from an analysis of SACY-1/DDX41 inCaenorhabditis elegans

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