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Roles of the phosphorylated SF3B1 in the activated spliceosome - experiment 2

ABSTRACT: SF3B1 is essential for assembly of B-complex spliceosome and activation of the Bact complex and undergoes dynamic phosphorylation and dephosphorylation during the splicing cycle. We have recently showed that the N-terminus of SF3B1 is phosphorylated during spliceosome activation by a little studied cyclin-dependent kinase 11 (CDK11) and remains hyperphosphorylated in catalytically active spliceosomes. However, little is known about the function of this N-terminal phosphorylation, about factors that “read” it, or about a spliceosome intermediate generated by CDK11 targeting. Here we demonstrate that CDK11 inhibition blocks spliceosome in a new intermediate during B to Bact transition. We now show that, phosphorylated SF3B1 N-terminus is crucial for binding of proteins during spliceosome activation.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: David Potesil

LAB HEAD: Zbynek Zdrahal

PROVIDER: PXD063162 | Pride | 2026-02-24

REPOSITORIES: Pride

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			Action	DRS
	240214_FASTA-lib.predicted.speclib	Other
	240409_main_search.tsv	Tabular
	5996_TIMS1_RSLC-74m_TFA_DIA_1_GC3_1_35655.d.zip	Other
	5996_TIMS1_RSLC-74m_TFA_DIA_2_GC4_1_35687.d.zip	Other
	5996_TIMS1_RSLC-74m_TFA_DIA_3_GC5_1_35639.d.zip	Other

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Roles of the phosphorylated SF3B1 in the activated spliceosome - experiment 1

Project description:SF3B1 is essential for assembly of B-complex spliceosome and activation of the Bact complex and undergoes dynamic phosphorylation and dephosphorylation during the splicing cycle. We have recently showed that the N-terminus of SF3B1 is phosphorylated during spliceosome activation by a little studied cyclin-dependent kinase 11 (CDK11) and remains hyperphosphorylated in catalytically active spliceosomes. However, little is known about the function of this N-terminal phosphorylation, about factors that “read” it, or about a spliceosome intermediate generated by CDK11 targeting. Here we demonstrate that CDK11 inhibition blocks spliceosome in a new intermediate during B to Bact transition. We now show that, phosphorylated SF3B1 N-terminus is crucial for binding of proteins during spliceosome activation.

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Project description:Cyclin-dependent kinase 11 (CDK11) is essential for the regulation of pre-mRNA splicing via phosphorylation of the core spliceosome component SF3B1. This phosphorylation is a marker of the catalytically active spliceosomes thus it is important to identify the mechanisms that regulate CDK11 itself. Here, we report that a small subset of CDK11 is phosphorylated on the activation T-loop threonine 595 (Thr595) and is associated with the activated spliceosome on chromatin in gene bodies. Mutational analyses revealed that Thr595 is essential for the formation of the active CDK11 complex with cyclin L and SAP30BP. CDK11 transiently associates with CDK7, a transcriptional kinase that also promotes the activation of other CDKs. Inhibition of CDK7 initially decreases transcription, but longer durations of inhibition lead to production of unspliced pre-mRNAs. The onset of the CDK7-mediated splicing defect correlates with the sequential dephosphorylation of CDK11 Thr595 and SF3B1. SILAC-based phosphoproteomics upon brief CDK11 inhibition identified SF3B1, CDC5L and ESS2 as CDK11 substrates, which overlap with the previously identified CDK7 substrates in the spliceosome. In summary, our study suggests that CDK7 likely acts via CDK11 Thr595 phosphorylation to regulate pre-mRNA splicing in cells. The identification of additional CDK11 substrates points to its broader role in spliceosome regulation.

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CDK11 phosphorylates SF3B1 and is essential for constitutive splicing (ChIP-seq)

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Cancer associated SF3B1 hotspot mutations induce cryptic 3' splice site selection through use of a different branch point

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