Mechanism of protein-guided folding of the active site U2/U6 RNA during spliceosome activation

Townsend, C.; Leelaram, Majety Naga; Agafonov, Dmitry E.; Dybkov, Olexandr; Will, Cindy L.; Bertram, K.; Urlaub, Henning; Kastner, Berthold; Stark, Holger; Lührmann, Reinhard

doi:10.1126/science.abc3753

Cited by 63 publications

(95 citation statements)

References 56 publications

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“…Human GPATCH1 and DHX35 have both been identified in C complex spliceosomes assembled in vitro (Ilagan et al, 2013), while RBM5 and RBM17 have been found in the early A complex that includes U2 snRNP (Hartmuth et al, 2002). Loss of the nuclear exosome factor Rrp6 produced a signature that tended to cluster adjacent to that of strains lacking the ortholog of CTNNBL1 (Figure 3A-C), a core component of active human spliceosomes recently visualized by cryoEM (Townsend et al, 2020), indicating an overlap between RNA species normally degraded by Rrp6 and those that accumulate in cells lacking CTNNBL1. Other mutants also showed some degree of clustering, suggesting functional/biochemical relationships.…”

Section: Resultsmentioning

confidence: 94%

Coupling of spliceosome complexity to intron diversity

Sales-Lee

Perry

Bowser

et al. 2021

Preprint

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We determined that over 40 spliceosomal proteins are conserved between many fungal species and humans but were lost during the evolution of S. cerevisiae, an intron-poor yeast with unusually rigid splicing signals. We analyzed null mutations in a subset of these factors, most of which had not been investigated previously, in the intron-rich yeast Cryptococcus neoformans. We found they govern splicing efficiency of introns with divergent spacing between intron elements. Importantly, most of these factors also suppress usage of weak nearby cryptic/alternative splice sites. Among these, orthologs of GPATCH1 and the helicase DHX35 display correlated functional signatures and copurify with each other as well as components of catalytically active spliceosomes, identifying a conserved G-patch/helicase pair that promotes splicing fidelity. We propose that a significant fraction of spliceosomal proteins in humans and most eukaryotes are involved in limiting splicing errors, potentially through kinetic proofreading mechanisms, thereby enabling greater intron diversity.

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Section: Resultsmentioning

confidence: 94%

Coupling of spliceosome complexity to intron diversity

Sales-Lee

Perry

Bowser

et al. 2021

Preprint

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“…This domain is atypical because the cluster of residues that are typically positively charged and coordinate nucleic acid binding in a winged helix is not charged, leading to the hypothesis that the highly conserved 3 10 helix is involved in protein binding [24] . KIN17 is predicted to have a disordered central region flanked by α-helices [15] , followed by a tandem of SH3-like domains separated by a flexible linker.…”

Section: Resultsmentioning

confidence: 99%

“…From B complex, the spliceosome undergoes a number of rearrangements through pre-Bact1, pre-Bact2, Bact and C complex. CryoEM studies of these complexes from human spliceosomes [2,15] allow for the study of different snapshots of the spliceosome assembly process. In these complexes there is an exchange of different factors that interact with the region of the 5'ss and its interactions with the U6 ACAGAGA box as the 5'ss is loaded into the catalytic core of the splicing machine.…”

Section: Introductionmentioning

confidence: 99%

A Genetic Screen for Suppressors of Cryptic 5’ Splicing inC. elegansReveals Roles for KIN17 and PRCC in Maintaining Both 5’ and 3’ Splice Site Identity

Jm¹,

Osterhoudt²,

Ch³

et al. 2021

Preprint

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Pre-mRNA splicing is an essential step of eukaryotic gene expression carried out by a series of dynamic macromolecular protein/RNA complexes, known collectively and individually as the spliceosome. This series of spliceosomal complexes define, assemble on, and catalyze the removal of introns. Molecular model snapshots of intermediates in the process have been created from cryo-EM data, however, many aspects of the dynamic changes that occur in the spliceosome are not fully understood. Caenorhabditis elegans follow the GU-AG rule of splicing, with almost all introns beginning with 5′ GU and ending with 3′ AG. These splice sites are identified early in the splicing cycle, but as the cycle progresses and ″custody″ of the pre-mRNA splice sites is passed from factor to factor as the catalytic site is built, the mechanism by which splice site identity is maintained or re-established through these dynamic changes is unclear. We performed a genetic screen in C. elegans for factors that are capable of changing 5′ splice site choice. We report that KIN17 and PRCC are involved in splice site choice, the first functional splicing role proposed for either of these proteins. Previously identified suppressors of cryptic 5′ splicing promote distal cryptic GU splice sites, however, mutations in KIN17 and PRCC instead promote usage of an unusual proximal 5′ splice site which defines an intron beginning with UU, separated by 1nt from a GU donor. We performed high-throughput mRNA sequencing analysis and found that mutations in PRCC but not KIN17 changed 5′ splice sites genome-wide, promoting usage of nearby non-consensus sites. We further found that mutations in KIN17 and PRCC changed dozens of 3′ splice sites, promoting non-consensus sites upstream of canonical splice sites. Our work has uncovered both fine and coarse mechanisms by which the spliceosome maintains splice site identity during the complex assembly process.

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“…Apparently, an opposite function of CBs is in place for the telomerase RNP, in which case CBs function to sequester telomerase from its nucleoplasmic substrates, the telomeres (Bizarro et al, 2019) Interestingly, two 2'-O-methyl groups within U2 snRNA are not impacted by Nopp140 KD, Gm12 and Gm25. In the active spliceosome, those 2'-O-methylated residues lie between helix Ia and Ib, and right adjacent to helix II formed between U2 and U6 snRNAs, perhaps pointing to an especially important role in splicing (Townsend et al, 2020;Zhang et al, 2017). In fact, U2-Gm12 was one of four 2'-O-methylated residues within the first 20 nucleotides of HeLa U2-snRNA that was required for in vitro splicing (Dönmez et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

Nopp140-chaperoned 2’-O-methylation of small nuclear RNAs in Cajal bodies ensures splicing fidelity

Bizarro

Deryusheva

Wacheul

et al. 2021

Preprint

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Spliceosomal small nuclear RNAs (snRNAs) are modified by small Cajal body (CB) specific ribonucleoproteins (scaRNPs) to ensure snRNP biogenesis and pre-mRNA splicing. However, the function and subcellular site of snRNA modification are largely unknown. We show that CB localization of the protein Nopp140 is essential for concentration of scaRNPs in that nuclear condensate; and that phosphorylation by casein kinase 2 (CK2) at some 80 serines targets Nopp140 to CBs. Transiting through CBs, snRNAs are apparently modified by scaRNPs. Indeed, Nopp140 knockdown-mediated release of scaRNPs from CBs severely compromises 2′-O-methylation of spliceosomal snRNAs, identifying CBs as the site of scaRNP catalysis. Additionally, alternative splicing patterns change indicating that these modifications in U1, U2, U5, and U12 snRNAs safeguard splicing fidelity. Given the importance of CK2 in this pathway, compromised splicing could underlie the mode of action of small molecule CK2 inhibitors currently considered for therapy in cholangiocarcinoma, hematological malignancies, and COVID-19.

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Mechanism of protein-guided folding of the active site U2/U6 RNA during spliceosome activation

Cited by 63 publications

References 56 publications

Coupling of spliceosome complexity to intron diversity

Coupling of spliceosome complexity to intron diversity

A Genetic Screen for Suppressors of Cryptic 5’ Splicing inC. elegansReveals Roles for KIN17 and PRCC in Maintaining Both 5’ and 3’ Splice Site Identity

Nopp140-chaperoned 2’-O-methylation of small nuclear RNAs in Cajal bodies ensures splicing fidelity

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