Regulation of 3′ splice site selection after step 1 of splicing by spliceosomal C* proteins

Dybkov, Olexandr; Preußner, Marco; Ayoubi, Leyla El; Feng, Vivi-Yun; Harnisch, Caroline; Merz, Kilian; Leupold, Paula; Yudichev, Peter; Agafonov, Dmitry E.; Will, Cindy L.; Girard, Cyrille; Dienemann, Christian; Urlaub, Henning; Kastner, Berthold; Lührmann, Reinhard

doi:10.1126/sciadv.adf1785

Cited by 13 publications

(12 citation statements)

References 67 publications

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“…Based on the weak, but yet statistically significant effects of RBM41 knockout in human HEK293 cells specifically on 3’ss selection of U12-type introns (Figure 6B-D), we hypothesize that while RBM41 is dispensable at the cellular level, it may nevertheless be able to exert a weak kinetic effect on splicing in addition to later participating in the disassembly process. The effect on 3’ss choice is similar to that observed after major spliceosome catalytic step II factor knockdowns, which similarly influence the 3’ss choice, particularly with NAGNAG introns (Dybkov et al ., 2023), further suggesting that the exchange from the U11/U12-65K to RBM41 may take place prior to step II. However, given that our BioID analysis did not provide supporting evidence for this possibility, it is also possible that the exchange from U11/U12-65K to RBM41 takes place at a later step and the effects on minor intron splicing are secondary effects of downstream processes being disturbed.…”

Section: Discussionsupporting

confidence: 60%

Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome

Norppa,

Chowdhury,

van Rooijen

et al. 2023

Preprint

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In this work, we identify RBM41 as a novel unique protein component of the minor spliceosome. RBM41 has no previously recognized cellular function but has been identified as a paralog of the U11/U12-65K protein, a known unique component of the minor spliceosome that functions during the early steps of minor intron recognition as a component of the U11/U12 di-snRNP. We show that both proteins use their highly similar C-terminal RRMs to bind to 3’-terminal stem-loops in U12 and U6atac snRNAs with comparable affinity. Our BioID data indicate that the unique N-terminal domain of RBM41 is necessary for its association with complexes containing DHX8, an RNA helicase, which in the major spliceosome drives the release of mature mRNA from the spliceosome. Consistently, we show that RBM41 associates with excised U12-type intron lariats, is present in the U12 mono-snRNP, and is enriched in Cajal bodies, together suggesting that RBM41 functions in the post-splicing steps of the minor spliceosome assembly/disassembly cycle. This contrasts with the U11/U12-65K protein, which uses the N-terminal region to interact with U11 snRNP during the intron recognition step. Finally, we show that while RBM41 knockout cells are viable, they show alterations in the splicing of U12-type introns, particularly differential U12-type 3’ splice site usage. Together, our results highlight the role 3’-terminal stem-loop of U12 snRNA as a dynamic binding platform for the paralogous U11/U12-65K and RBM41 proteins, which function at distinct stages of minor spliceosome assembly/disassembly cycle.

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Section: Discussionsupporting

confidence: 60%

Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome

Norppa,

Chowdhury,

van Rooijen

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…Based on the weak, yet statistically significant effects of RBM41 knockout in human HEK293 cells specifically on 3′ss selection of U12-type introns (Figure 6B – D ), we hypothesize that while RBM41 is dispensable at the cellular level, it may nevertheless be able to exert a weak kinetic effect on splicing in addition to later participating in the disassembly process. The effect on 3′ss choice is similar to that observed after major spliceosome catalytic step II factor knockdowns, which similarly influence the 3′ss choice, particularly with NAGNAG introns ( 73 ), further suggesting that the exchange from the U11/U12-65K to RBM41 may take place prior to step II. However, given that our BioID analysis did not provide supporting evidence for this possibility, it is also possible that the exchange from U11/U12-65K to RBM41 takes place at a later step and the effects on minor intron splicing are secondary effects of downstream processes being disturbed.…”

Section: Discussionsupporting

confidence: 55%

Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome

Norppa,

Chowdhury,

van Rooijen

et al. 2024

Nucleic Acids Research

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Here, we identify RBM41 as a novel unique protein component of the minor spliceosome. RBM41 has no previously recognized cellular function but has been identified as a paralog of U11/U12-65K, a known unique component of the U11/U12 di-snRNP. Both proteins use their highly similar C-terminal RRMs to bind to 3′-terminal stem-loops in U12 and U6atac snRNAs with comparable affinity. Our BioID data indicate that the unique N-terminal domain of RBM41 is necessary for its association with complexes containing DHX8, an RNA helicase, which in the major spliceosome drives the release of mature mRNA from the spliceosome. Consistently, we show that RBM41 associates with excised U12-type intron lariats, is present in the U12 mono-snRNP, and is enriched in Cajal bodies, together suggesting that RBM41 functions in the post-splicing steps of the minor spliceosome assembly/disassembly cycle. This contrasts with U11/U12-65K, which uses its N-terminal region to interact with U11 snRNP during intron recognition. Finally, while RBM41 knockout cells are viable, they show alterations in U12-type 3′ splice site usage. Together, our results highlight the role of the 3′-terminal stem-loop of U12 snRNA as a dynamic binding platform for the U11/U12-65K and RBM41 proteins, which function at distinct stages of the assembly/disassembly cycle.

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“…U6 snRNA interacts with both the 5′SS and 3′SS during the C to C* transition of the spliceosome (78), as was discussed for the A. thaliana alternative 3′SS usage (21), and this interaction can explain why U6 snRNA m6A43 is important for 3′SS selection. Indeed, many spliceosomal proteins associated with the C* complex affect 3′SS usage (79). However, SNRNP27K is no longer present in human C and C* complexes.…”

Section: Discussionmentioning

confidence: 99%

U6 snRNA m6A modification is required for accurate and efficient cis- and trans-splicing ofC. elegansmRNAs

Shen,

Hencel,

Parker

et al. 2023

Preprint

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pre-mRNA splicing is a critical feature of eukaryotic gene expression. Many eukaryotes use cis-splicing to remove intronic sequences from pre-mRNAs. In addition to cis-splicing, many organisms use trans-splicing to replace the 5′ ends of mRNAs with a non-coding spliced-leader RNA. Both cis- and trans-splicing rely on accurately recognising splice site sequences by spliceosomal U snRNAs and associated proteins. Spliceosomal snRNAs carry multiple RNA modifications with the potential to affect different stages of pre-mRNA splicing. Here, we show that m6A modification of U6 snRNA A43 by the RNA methyltransferase METT-10 is required for accurate and efficient cis- and trans-splicing of C. elegans pre-mRNAs. The absence of U6 snRNA m6A modification primarily leads to alternative splicing at 5′ splice sites. Furthermore, weaker 5′ splice site recognition by the unmodified U6 snRNA A43 affects splicing at 3′ splice sites. U6 snRNA m6A43 and the splicing factor SNRNP27K function to recognise an overlapping set of 5′ splice sites with an adenosine at +4 position. Finally, we show that U6 snRNA m6A43 is required for efficient SL trans-splicing at weak 3′ trans-splice sites. We conclude that the U6 snRNA m6A modification is important for accurate and efficient cis- and trans-splicing in C. elegans.

show abstract

Regulation of 3′ splice site selection after step 1 of splicing by spliceosomal C* proteins

Cited by 13 publications

References 67 publications

Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome

Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome

Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome

U6 snRNA m6A modification is required for accurate and efficient cis- and trans-splicing ofC. elegansmRNAs

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