Mechanisms and cellular responses to the loss of the intron-binding complex
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Abstract
The intracellular environment is populated by hundreds of protein complexes, which are responsible for carrying out all cellular processes. Functionality of protein complexes is achieved through the tight, yet dynamic, regulation of each step of protein complex biogenesis, from production of stoichiometric amounts of subunits to the degradation of faulty assembly intermediates and unassembled subunits through the activity of protein quality control pathways.
In this thesis, we focused on the intron-binding complex, a known building block in spliceosome biogenesis. Besides its described role in splicing, the intron-binding complex is poorly characterized and the mechanisms behind its biogenesis and regulation are currently unknown. We observed that protein levels of intron-binding complex subunits display co-expression patterns, and that loss of one subunit is sufficient to trigger the collateral loss of the others. Using a combination of proteomic approaches, we investigated the protein quality control mechanisms responsible for the targeted degradation of unassembled intron-binding complex subunits, and identified E3 ubiquitin ligases UBR5 and TRIP12 as potential regulators.
In addition to regulating splicing as part of the intron-binding complex (IBC), individual intron-binding complex subunits are involved in a variety of biological processes. Informed by the knowledge that the levels of the subunits are interconnected as the result of the quality control mechanisms targeting the intron-binding complex, we set out to compare the cellular responses to the loss of three intron-binding complex subunits. By investigating similarities in the global proteomic alterations upon depletion of each subunit, we were able to define a core cellular response to the loss of the intron-binding complex. Features of this core response are irreparable DNA damage, p53 activation and cell cycle arrest. By complementing these phenotypes with specific signatures in our proteomic datasets, we concluded that loss of the intron-binding complex leads to cellular senescence.
To gain insight into the senescence phenotype, we looked into the mechanisms responsible for the observed genomic instability. Investigation of the interplay between double-strand break formation and nascent transcription revealed that loss of intron-binding complex subunit AQR causes transcription-associated genomic instability. In addition, general loss of intron-binding complex subunits causes increased levels of three-stranded nucleic acid structures known as R-loops: as formation of R-loops is a direct function of transcription, and their accumulation is a known source of genomic instability, we propose that R-loop-dependent DNA damage is another feature of the response to intron-binding complex loss.
Ultimately, this work underlies the importance of taking into account aspects of protein complex regulation when interpreting phenotypes arising from the loss of individual subunits.