Cryo-EM analyses of dimerized spliceosomes provide new insights into the functions of B complex proteins

Zhang, Zhenwei; Kumar, Vinay; Dybkov, Olexandr; Will, Cindy L; Urlaub, Henning; Stark, Holger; Lührmann, Reinhard

doi:10.1038/s44318-024-00052-1

Cited by 5 publications

(11 citation statements)

References 49 publications

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“…We affinity-purified CE B-like complexes (generated as described above) and subsequently determined their cryo-EM structure. Single-particle cryo-EM of the affinity-purified CE B-like complexes revealed dimers comprising two B-like complexes aligned in a parallel manner, similar to the overall organization of human CI B dimers isolated under low salt conditions 23 (Extended Data Fig. 3a–i ).…”

Section: D Structure Of the B-like Complexsupporting

confidence: 56%

“…2f ). Indeed, nearly all of the B-specific proteins localized in the CE B-like complex are spatially organized in an analogous manner to that of the human CI B complex 23 (Extended Data Fig. 4a–c ).…”

Section: D Structure Of the B-like Complexmentioning

confidence: 89%

“…2a ). In the B-like complex, G +1 and U +2 of the 5′ss of the exogenously added RNA oligonucleotide are exclusively recognized by DIM1 and PRP8 in the same manner as the 5′ss of the pre-mRNA in CI B complexes 19 , 22 , 23 (Fig. 2b–d and Extended Data Fig.…”

Section: D Structure Of the B-like Complexmentioning

confidence: 94%

“…This helix is present in an extended form (that is, extended U6/5′ss helix) in CI B complexes. This leads to multiple, major structural rearrangements in the CI pre-B complex 18 , 19 , 22 , 23 and the recruitment of so-called B-specific proteins (including, SMU1, RED, FBP21, SNU23, MFAP1, PRP38A and UBL5) 6 . We previously showed that addition of an excess of a 5′ss-containing RNA oligonucleotide (5′ss oligo) to CE pre-B complexes in the presence of nuclear extract and ATP bypasses the requirement for PRP28 (refs.…”

Section: D Structure Of the B-like Complexmentioning

confidence: 99%

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Structural insights into the cross-exon to cross-intron spliceosome switch

Zhang,

Kumar,

Dybkov

et al. 2024

Nature

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Early spliceosome assembly can occur through an intron-defined pathway, whereby U1 and U2 small nuclear ribonucleoprotein particles (snRNPs) assemble across the intron1. Alternatively, it can occur through an exon-defined pathway2–5, whereby U2 binds the branch site located upstream of the defined exon and U1 snRNP interacts with the 5′ splice site located directly downstream of it. The U4/U6.U5 tri-snRNP subsequently binds to produce a cross-intron (CI) or cross-exon (CE) pre-B complex, which is then converted to the spliceosomal B complex6,7. Exon definition promotes the splicing of upstream introns2,8,9 and plays a key part in alternative splicing regulation10–16. However, the three-dimensional structure of exon-defined spliceosomal complexes and the molecular mechanism of the conversion from a CE-organized to a CI-organized spliceosome, a pre-requisite for splicing catalysis, remain poorly understood. Here cryo-electron microscopy analyses of human CE pre-B complex and B-like complexes reveal extensive structural similarities with their CI counterparts. The results indicate that the CE and CI spliceosome assembly pathways converge already at the pre-B stage. Add-back experiments using purified CE pre-B complexes, coupled with cryo-electron microscopy, elucidate the order of the extensive remodelling events that accompany the formation of B complexes and B-like complexes. The molecular triggers and roles of B-specific proteins in these rearrangements are also identified. We show that CE pre-B complexes can productively bind in trans to a U1 snRNP-bound 5′ splice site. Together, our studies provide new mechanistic insights into the CE to CI switch during spliceosome assembly and its effect on pre-mRNA splice site pairing at this stage.

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Section: D Structure Of the B-like Complexsupporting

confidence: 56%

Section: D Structure Of the B-like Complexmentioning

confidence: 89%

Section: D Structure Of the B-like Complexmentioning

confidence: 94%

Section: D Structure Of the B-like Complexmentioning

confidence: 99%

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Structural insights into the cross-exon to cross-intron spliceosome switch

Zhang,

Kumar,

Dybkov

et al. 2024

Nature

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“…Therefore, this duplex likely appears in most species as long as the intron has the ability to form this motif with U6. Three proteins in the spliceosomal B complex surround this duplex [ 24 ]. Among these proteins, Prp38 and ZMAT2/Snu23 have homologues in E. gracilis [ 15 ].…”

Section: Resultsmentioning

confidence: 99%

Unique features of conventional and nonconventional introns in Euglena gracilis

Gao,

Zhao,

et al. 2024

BMC Genomics

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Background Nuclear introns in Euglenida have been understudied. This study aimed to investigate nuclear introns in Euglenida by identifying a large number of introns in Euglena gracilis (E. gracilis), including cis-spliced conventional and nonconventional introns, as well as trans-spliced outrons. We also examined the sequence characteristics of these introns. Results A total of 28,337 introns and 11,921 outrons were identified. Conventional and nonconventional introns have distinct splice site features; the former harbour canonical GT/C-AG splice sites, whereas the latter are capable of forming structured motifs with their terminal sequences. We observed that short introns had a preference for canonical GT-AG introns. Notably, conventional introns and outrons in E. gracilis exhibited a distinct cytidine-rich polypyrimidine tract, in contrast to the thymidine-rich tracts observed in other organisms. Furthermore, the SL-RNAs in E. gracilis, as well as in other trans-splicing species, can form a recently discovered motif called the extended U6/5’ ss duplex with the respective U6s. We also describe a novel type of alternative splicing pattern in E. gracilis. The tandem repeat sequences of introns in this protist were determined, and their contents were comparable to those in humans. Conclusions Our findings highlight the unique features of E. gracilis introns and provide insights into the splicing mechanism of these introns, as well as the genomics and evolution of Euglenida.

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Phenotypic screens identify SCAF1 as critical activator of RNAPII elongation and global transcription

Bhandare,

Narain,

Hofstetter

et al. 2024

Nucleic Acids Research

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Transcripts produced by RNA polymerase II (RNAPII) are fundamental for cellular responses to environmental changes. It is therefore no surprise that there exist multiple avenues for the regulation of this process. To explore the regulation mediated by RNAPII-interacting proteins, we used a small interfering RNA (siRNA)-based screen to systematically evaluate their influence on RNA synthesis. We identified several proteins that strongly affected RNAPII activity. We evaluated one of the top hits, SCAF1 (SR-related C-terminal domain-associated factor 1), using an auxin-inducible degradation system and sequencing approaches. In agreement with our screen results, acute depletion of SCAF1 decreased RNA synthesis, and showed an increase of Serine-2 phosphorylated-RNAPII (pS2-RNAPII). We found that the accumulation of pS2-RNAPII within the gene body occurred at GC-rich regions and was indicative of stalled RNAPII complexes. The accumulation of stalled RNAPII complexes was accompanied by reduced recruitment of initiating RNAPII, explaining the observed global decrease in transcriptional output. Furthermore, upon SCAF1 depletion, RNAPII complexes showed increased association with components of the proteasomal-degradation machinery. We concluded that in cells lacking SCAF1, RNAPII undergoes a rather interrupted passage, resulting in intervention by the proteasomal-degradation machinery to clear stalled RNAPII. While cells survive the compromised transcription caused by absence of SCAF1, further inhibition of proteasomal-degradation machinery is synthetically lethal.

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Cryo-EM analyses of dimerized spliceosomes provide new insights into the functions of B complex proteins

Cited by 5 publications

References 49 publications

Structural insights into the cross-exon to cross-intron spliceosome switch

Structural insights into the cross-exon to cross-intron spliceosome switch

Unique features of conventional and nonconventional introns in Euglena gracilis

Phenotypic screens identify SCAF1 as critical activator of RNAPII elongation and global transcription

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