System-wide analyses of the fission yeast poly(A)+ RNA interactome reveal insights into organisation and function of RNA-protein complexes

Abstract

Abbreviations: gene ontology (GO); messenger RNA (mRNA); mass spectrometry (MS); polyadenylation site (PAS); RNA-binding domain (RBD); RNA-binding protein (RBP);RNA interactome capture (RIC); ribonucleoprotein particle (RNP); ribosomal protein (RP); transcription elongation factor (TEF); wild-type (WT); whole cell extract (WCE); cleavage and polyadenylation factor (CPF); cleavage factors IA and IB (CFIA and CFIB) 2 Abstract Production, function, and turnover of mRNA are orchestrated by multi-subunit machineries that play a central role in gene expression. Within these molecular machines, interactions with the target mRNA are mediated by RNA-binding proteins (RBPs), and the accuracy and dynamics of these RNA-protein interactions are essential for their function. Here, we show that fission yeast whole cell poly(A)+ RNA-protein crosslinking data provides system-wide information on the organisation and function of the RNA-protein complexes. We evaluate relative enrichment of cellular RBPs on poly(A)+ RNA to identify interactors with high RNAbinding activity and provide key information about the RNA-binding properties of large multiprotein complexes, such as the mRNA 3' end processing machinery (cleavage and polyadenylation factor, CPF) and the RNA exosome. We demonstrate that different functional modules within CPF differ in their ability to interact with RNA. Importantly, we reveal that CPF forms additional contacts with RNA via the Fip1 subunit of the polyadenylation module and two subunits of the nuclease module. In addition, our data highlights the central role of the RNA helicase Mtl1 in RNA degradation by the exosome as mutations in Mtl1 lead to disengagement of the exosome from RNA. We examine how routes of substrate access to the complex are affected upon mutation of exosome subunits. Our results provide important insights into how different components of the exosome contribute to engagement of the complex with substrate RNA. Overall, our data uncover how multi-subunit cellular machineries interact with RNA, on a proteome-wide scale.

Discovering the RNA-Binding Proteome of Plant Leaves with an Improved RNA Interactome Capture Method

“…This is not surprising, since it is known that each ribosomal protein assembles with its target RNA sequence via an intimate co-folding process into the ribosome nano-machinery, thus possessing protein-specific modes to interact with rRNA. Ribosomes sit on the translating mRNA and the ribosomal proteins within the mRNA channel are overrepresented in RBPomes, reflecting the fact that those proteins are the most likely to crosslink to polyadenylated RNA during the translation process [52,53]. However, other ribosomal proteins are detected in the RBPome in sub-stoichiometric quantities [53].…”

“…Ribosomes sit on the translating mRNA and the ribosomal proteins within the mRNA channel are overrepresented in RBPomes, reflecting the fact that those proteins are the most likely to crosslink to polyadenylated RNA during the translation process [52,53]. However, other ribosomal proteins are detected in the RBPome in sub-stoichiometric quantities [53]. These can proceed from extra-ribosomal, regulatory interactions with mRNA [54] or can be structural ribosomal proteins isolated through crosslinking to non-polyadenylated rRNA [53].…”

“…However, other ribosomal proteins are detected in the RBPome in sub-stoichiometric quantities [53]. These can proceed from extra-ribosomal, regulatory interactions with mRNA [54] or can be structural ribosomal proteins isolated through crosslinking to non-polyadenylated rRNA [53]. We also identified 10 of the 13 YT521-B homology (YTH)-containing proteins in Arabidopsis [55], which are known to 'read' methyl 6 adenosine modifications within RNA [56].…”

DEAD-box ATPase Dbp2 mediates mRNA release after 3'-end formation

“…Figure 7A) (Lemay et al, 2014). Moreover, in a comparative RNA interactome capture experiment in which we had previously compared occupancies of RNA-binding proteins on polyadenylated RNA between wild type and mtl1-1 or rrp6Δ (mutants of MTREC and the exosome complex, respectively), CPAC components – in particular CFIA – were significantly enriched, suggesting that CPAC release might be impaired in exosome mutants (Birot et al, 2021; Kilchert et al, 2019) (Suppl. Figure 7B and C).…”

From parts lists to functional significance—RNA–protein interactions in gene regulation

“…A very simple means to estimate in vivo RNA-binding activities of RBPs is the normalization of RIC data to cellular protein abundances, which has not been carried out in most of the initial RIC studies where the degree of protein enrichment was instead determined relative to a non-crosslinked control (Baltz et al, 2012;Beckmann et al, 2015;Castello et al, 2012;Conrad et al, 2016;Kwon et al, 2013;Matia-Gonzalez et al, 2015;Mitchell et al, 2013). RNA binders can be discriminated based on their enrichment in the RIC experiment relative to the input proteome (Kilchert et al 2019) (Figure 4). Notably, the behavior of RBPs is by no means uniform, and spans a continuum of RNA binding activity.…”