Structure of the human activated spliceosome in three conformational states

Zhang, Xiaofeng; Yan, Chuangye; Zhan, Xiechao; Li, Lijia; Lei, Jianlin; Shi, Yigong

doi:10.1038/cr.2018.14

Cited by 182 publications

(252 citation statements)

References 70 publications

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“…A recently reported structure of human B act spliceosomes suggests that RNF113A joins the complex after U4 dissociation and is important for the complex rearrangements and activation. 35 All together, our data indicate that RNF113A joins the spliceosome either on B or B act intermediate complexes (Figure 4). Second, our results indicated that the addition of excess recombinant RNF113A led to splicing inhibition.…”

Section: Discussionsupporting

confidence: 52%

Human RNF113A participates of pre‐mRNA splicing in vitro

Silva

Jurica

Cunha

et al. 2018

J of Cellular Biochemistry

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Pre‐messenger RNA (mRNA) splicing is an essential step in the control of eukaryotic gene expression. During splicing, the introns are removed from the gene transcripts as the exons are ligated to create mature mRNA sequences. Splicing is performed by the spliceosome, which is a macromolecular complex composed of five small nuclear RNAs (snRNAs) and more than 100 proteins. Except for the core snRNP proteins, most spliceosome proteins are transiently associated and presumably involved with the regulation of spliceosome activity. In this study, we explored the association and participation of the human protein RNF113A in splicing. The addition of excess recombinant RNF113A to in vitro splicing reactions results in splicing inhibition. In whole‐cell lysates, RNF113A co‐immunoprecipitated with U2, U4, and U6 snRNAs, which are components of the tri‐snRNP, and with proteins PRP19 and BRR2. When HeLa cells were CRISPR‐edited to reduce the RNF113A levels, the in vitro splicing efficiency was severely affected. Consistently, the splicing activity was partially restored after the addition of the recombinant GST‐RNF113A. On the basis on these results, we propose a model in which RNF113A associates with the spliceosome by interacting with PRP19, promoting essential rearrangements that lead to splicing.

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Section: Discussionsupporting

confidence: 52%

Human RNF113A participates of pre‐mRNA splicing in vitro

Silva

Jurica

Cunha

et al. 2018

J of Cellular Biochemistry

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“…These changes lead to release of some proteins and recruitment of others to the spliceosome, ensuring the fidelity and directionality of spliceosome assembly and catalysis (Koodathingal and Staley 2013). Recent cryo-electron microscopy (cryo-EM) models of spliceosomes at distinct steps (tri-snRNP, the A, B, B act , C, and P complexes) have brilliantly illuminated the composition and conformation of the spliceosome before and after each transition Yan et al 2015;Galej et al 2016;Rauhut et al 2016;Wan et al 2016;Yan et al 2016;Bai et al 2017;Bertram et al 2017a;Bertram et al 2017b;Liu et al 2017;Plaschka et al 2017;Wan et al 2017;Wilkinson et al 2017;Yan et al 2017;Zhang et al 2017;Plaschka et al 2018;Zhan et al 2018b;Zhan et al 2018a;Zhang et al 2018). A challenge now is to understand the distinct mechanism of action of each DExD/H protein in catalyzing its specific transition.…”

Section: Introductionmentioning

confidence: 99%

Cus2 enforces the first ATP-dependent step of splicing by binding to yeast SF3b1 through a UHM-ULM interaction

Talkish

Igel

Hunter

et al. 2019

Preprint

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Stable recognition of the intron branchpoint by the U2 snRNP to form the prespliceosome is the first ATP-dependent step of splicing. Genetic and biochemical data from yeast indicate that Cus2 aids U2 snRNA folding into the stem IIa conformation prior to pre-spliceosome formation. Cus2 must then be removed by an ATP-dependent function of Prp5 before assembly can progress. However, the location from which Cus2 is displaced and the nature of its binding to the U2 snRNP are unknown. Here, we show that Cus2 contains a conserved UHM (U2AF homology motif) that binds Hsh155, the yeast homolog of human SF3b1, through a conserved ULM (U2AF ligand motif).Mutations in either motif block binding and allow pre-spliceosome formation without ATP. A 2.0 Å resolution structure of the Hsh155 ULM in complex with the UHM of Tat-SF1, the human homolog of Cus2, and complementary binding assays show that the interaction is highly similar between yeast and humans. Furthermore, we show that Tat-SF1 can replace Cus2 function by enforcing ATP-dependence of pre-spliceosome formation in yeast extracts. Cus2 is removed before pre-spliceosome formation, and both Cus2 and its Hsh155 ULM binding site are absent from available cryo-EM structure models. However, our data are consistent with the apparent location of the disordered Hsh155 ULM between the U2 stem-loop IIa and the HEAT-repeats of Hsh155 that interact with Prp5. We propose a model in which Prp5 uses ATP to remove Cus2 from Hsh155 such that extended base pairing between U2 snRNA and the intron branchpoint can occur.

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“…RNA splicing relies on the spliceosome and is a conserved and very large complex consisting of 5 small nuclear RNP (snRNP) complexes (U1, U2, U4, U5, and U6) and ;150 proteins (31). Several studies have demonstrated the resolution of the spliceosome complex by cryogenic electron microscopy structural analysis (32)(33)(34)(35)(36). The structural analysis revealed a number of striking features that advance our mechanistic understanding of exon ligation.…”

Section: Discussionmentioning

confidence: 99%

Small ribonucleoprotein particle protein SmD3 governs the homeostasis of germline stem cells and the crosstalk between the spliceosome and ribosome signals in Drosophila

Luan

Yan

et al. 2019

FASEB j.

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The ribonucleoprotein (RNP) spliceosome machinery triggers the precursor RNA splicing process in eukaryotes. Major spliceosome defects are implicated in male infertility; however, the underlying mechanistic links between the spliceosome and the ribosome in Drosophila testes remains largely unresolved. Small ribonucleoprotein particle protein SmD3 (SmD3) is a novel germline stem cell (GSC) regulatory gene identified in our previous screen of Drosophila testes. In the present study, using genetic manipulation in a Drosophila model, we demonstrated that SmD3 is required for the GSC niche and controls the self‐renewal and differentiation of GSCs in the testis. Using in vitro assays in Schneider 2 cells, we showed that SmD3 also regulates the homeostasis of proliferation and apoptosis in Drosophila. Furthermore, using liquid chromatography–tandem mass spectrometry methods, SmD3 was identified as binding with ribosomal protein (Rp)L18, which is a key regulator of the large subunit in the ribosome. Moreover, SmD3 was observed to regulate spliceosome and ribosome subunit expression levels and controlled spliceosome and ribosome function via RpL18. Significantly, our findings revealed the genetic causes and molecular mechanisms underlying the stem cell niche and the crosstalk between the spliceosome and the ribosome.—Yu, J., Luan, X., Yan, Y., Qiao, C, Liu, Y., Zhao, D., Xie, B., Zheng, Q., Wang, M., Chen, W., Shen, C, He, Z., Hu, X., Huang, X., Li, H., Chen, B., Zheng, B., Chen, X., Fang, J. Small ribonucleoprotein particle protein SmD3 governs the homeostasis of germline stem cells and the crosstalk between the spliceosome and ribosome signals in Drosophila. FASEB J. 33, 8125–8137 (2019). http://www.fasebj.org

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Structure of the human activated spliceosome in three conformational states

Cited by 182 publications

References 70 publications

Human RNF113A participates of pre‐mRNA splicing in vitro

Human RNF113A participates of pre‐mRNA splicing in vitro

Cus2 enforces the first ATP-dependent step of splicing by binding to yeast SF3b1 through a UHM-ULM interaction

Small ribonucleoprotein particle protein SmD3 governs the homeostasis of germline stem cells and the crosstalk between the spliceosome and ribosome signals in Drosophila

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