Structural investigation of the Intron-Binding Complex in RNA splicing and HIV integration
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Embargo End Date
2026-09-27
ICR Authors
Authors
Goodarzi, N
Document Type
Thesis or Dissertation
Date
2024-09-27
Date Accepted
Abstract
Pre-mRNA splicing is an essential step of gene expression required for the selection and ligation of coding exons into the mature mRNA. Splicing occurs by two chemical steps – splicing and exon ligation - on the spliceosome, a multimegadalton complex, composed of five ribonucleoprotein particles (snRNPs) and numerous non-snRNP proteins. The spliceosome is a dynamic machinery that undergoes profound remodeling as it transitions through various landmark stages, each differing in composition and conformation. This extensive dynamics is powered by RNA helicases—molecular motors that integrate within the spliceosome and remodel RNA-RNA or RNA-protein interactions using the energy from NTP hydrolysis. Aquarius is a splicing helicase that joins the spliceosomes as part of the pentameric intron-binding complex (IBC) and drives the remodeling necessary to achieve a catalytically competent state of the spliceosome. During this process, known as catalytic activation, the branch duplex, which carries a reactive adenosine, must be liberated from the grasp of the SF3B complex and relocated to the catalytic center of the spliceosome for the branching reaction. How Aquarius and, implicitly, the IBC act in and regulate splicing is unclear. My thesis is structured in three parts. The first and most important part involves the reconstitution and cryo-EM analyses of several IBC-RNA complexes in three different catalytic steps: pre-catalytic, transition state, and post-catalytic. Furthermore, I stalled and analyzed two spliceosomes (i.e., B AQR and C complexes) before and after the action of Aquarius. Collectively, these structures allow us to propose a model for how this helicase remodels the spliceosome to enable branching catalysis. We conclude Aquarius translocates with 5’-3’ polarity, pulling the intron and extracting the branch duplex, facilitating its transfer to the catalytic center. In complementation, Aquarius triggers a coordinated dissociation and recruitment of numerous spliceosome subunits. In the second part of my thesis, I examined a different role of the IBC, specifically its interaction with HIV1 integrase. Integrase is a viral protein that forms multimers at the ends of viral DNA, creating intasomes. These intasomes facilitate the integration of viral DNA into the host genome, aided by the host protein LEDGF/p75. By reconstituting recombinant intasomes, LEDGF, and IBC, I could provide evidence for the direct interaction of these purified components in vitro. This development paves the way for future cryo-EM reconstructions of intasomes in complex with IBC and LEDGF, enhancing our understanding of how IBC mediates HIV genome integration. In the third part of my thesis, I explored the connection between splicing and the DNA damage response (DDR). Building on previously reported interactions between splicing and DDR factors, we aimed to evaluate the stability of these interactions and attempt to reconstitute complexes for structural analysis. I developed molecular baits composed of ssDNA, the different types of recombinant RPA complexes, and various nuclear extracts, including those from cells subjected to X-ray-induced DNA damage. Our systematic analysis indicates that the reported interactions are not reproducible. However, this has facilitated the development of valuable and more reliable protocols for future biochemical studies of splicing and DDR.
Citation
2024
DOI
Source Title
Publisher
Institute of Cancer Research (University Of London)
ISSN
eISSN
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Research Team
Mech of pre-mRNA splicing