Structural investigation of NTC and of the coupling between splicing and polyadenylation

Abstract

This PhD thesis is composed of two parts. The first and foremost part describes the reconstitution, isolation and analysis of human complexes involved in the coupling between splicing and polyadenylation, with a special focus on the definition of terminal exons. Splicing and polyadenylation are essential steps of gene expression, required for the maturation of transcripts by removing non-coding introns and adding a protective polyadenylate tail. Several lines of evidence suggest that splicing and polyadenylation are coupled at the terminal exons. One proposed role of the coupling is the definition of the terminal exons - a process that can occur in alternative ways, generating different messenger RNA molecules from the same gene and therefore enhancing proteome diversity and functional complexity of an organism. Splicing and polyadenylation occur on two task-specific machines in the size of megadaltons named spliceosome, and cleavage and polyadenylation complex (CP), respectively. How the two complexes interact during coupling is unclear. Hence, I aimed to establish the isolation of native human particles that contain spliceosomes in complex with CP in sufficient amounts for compositional, biochemical, and structural analysis. In this way, our understanding of the cross-talk between splicing and polyadenylation can be advanced. To this end, I first established the reconstitution and purification of prespliceosomes (ie. early spliceosomes) in complex with CP from extracts prepared from nuclei of HeLa cells cultivated in a bioreactor. To capture both complexes in a stable interaction, I used substrates pre-mRNA known to support the coupling between splicing and ployadenylation. By this strategy, I assembled two main variants of prespliceosome-CP particles - one across the terminal intron (ie. pre-A-CP) and one across the terminal exon (terminal exon-definition complex, TEDC). The two particles are in the multimegadalton size and contain all expected snRNA and protein subunits expected in U2 and U1 snRNP, the CP and numerous associated proteins. To gain mechanistic insight into the definition of terminal exons, I explored the structure of TEDC by single-particle cryo-electron microscopy (cryo-EM). Despite the moderate resolution, the overall 3D map of TEDC reveals that U2 snRNP occupies the central position and is flanked by the CP and U1 snRNP complexes. By focused classification, I also resolved the U2 snRNP core component at 4.4A resolution. The latter indicates a conserved mechanism of branch sequence recognition within the terminal and internal introns. To extend the structural insights, I carried out protein-protein cross-linking coupled mass spectrometry. The resulting distance constraints are consistent with the four structural modules of TEDC and provides indications about the subunits that connect U2 and U1 snRNP and potentially U2 snRNP and CT. Based on all the findings corroborated with data from the literature, I propose a structure-based model of the coupling between spliceosome and polyadenylation components, with potential implications for defining the terminal exons. In the second part of the thesis, I describe the expression, purification, and biochemical characterisation of the nineteen complex (NTC), which is a building block of the spliceosome that plays a crucial role in the construction of the spliceosome's catalytic centre. Independently of this role, NTC also acts as a multimeric E3 ubiquitin ligase in the DNA damage pathway. To understand how NTC mediates ubiquitination, I first reconstituted the recombinant NTC and identified the E2 conjugating enzyme UbcH5c as an interacting binding partner. By expressing and purifying NTC variants, in the frame of a collaboration, I contributed to elucidating how the autoinhibited Prp19 homotetramer (the E3 ligase and scaffold of the NTC) is derepressed by stepwise binding of three other NTC components.