Investigation of spliceosome assembly and the cross-talk with miRNA biogenesis
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Embargo End Date
2026-11-08
ICR Authors
Authors
Maruntel, I
Document Type
Thesis or Dissertation
Date
2024-11-08
Date Accepted
Abstract
Pre-mRNA splicing is a critical RNA processing event that occurs on the spliceosome, a large and dynamic protein-RNA complex comprising over 100 proteins. The spliceosome initially forms in an autoinhibited state and becomes catalytically active through several remodelling events driven by RNA helicases, as well as other enzymes that include kinases. Although the function of helicases in splicing is well-documented, the specific role of kinases remains poorly understood.
The inactivation of the kinase CDK11 using a specific inhibitor was recently shown to block the phosphorylation of SF3B1—a key component of the spliceosome essential for its assembly and activation. The kinase inhibition results in the spliceosome being arrested at an unclear stage and via an unknown mechanism. To investigate this, I developed a method to purify spliceosomes stalled by the CDK11 inhibitor. Biochemical and biophysical analyses, combined with mass spectrometric proteomics and electron microscopy, reveal that CDK11 inhibition arrests the spliceosome in a precatalytic B-like complex state. This finding aligns with prior studies and paves the way for future cryo-EM structural analysis of the B-like spliceosome halted by CDK11 inactivation.
Next to splicing, microRNA biogenesis is another essential RNA processing event. While splicing is executed by spliceosomes that gradually form on the introns, microRNA biogenesis is initiated by the microprocessor complex (MPC), often on the same introns. Several lines of evidence indicate that splicing and microRNA biogenesis can influence each other, suggesting a cross-talk between spliceosomes and the MPC. To explore this interaction, we aimed to isolate the complexes containing both the microprocessor and spliceosomes on the same RNA substrate in preparative amounts. Utilizing human nuclear extracts and in vitro-transcribed RNA substrates, we isolated and characterized the proteome of spliceosomal A, pre-A, and B complexes associated with the MPC and MPC-associated proteins (MAP). This study provides a good foundation for future structural analysis of the interaction between spliceosomes and the MPC.
Citation
2024
DOI
Source Title
Publisher
Institute of Cancer Research (University Of London)
ISSN
eISSN
Collections
Research Team
Mech of pre-mRNA splicing