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

Abstract

Cornelia Kilchert, Tea Kecman, Emily Priest, et al. protein complexes − interactome reveal insights into organization and function of RNA RNA + System-wide analyses of the fission yeast poly(A)

RNA-binding protein Mub1 and the nuclear RNA exosome act to fine-tune environmental stress response

“…Poly(A)+ S. pombe RNA interactome capture (RIC) was obtained from (54, 72). Briefly, two sets of triplicate experiments (Wild-type 1 (WT1) + mtl1-1 = First eXperiment (FX); WT2 + rrp6Δ + dis3-54 = Second eXperiment (SX)) were performed.…”

“…Following UV crosslinking, poly(A)+ RNA was enriched by oligo-d(T) selection and RNA-associated proteins were identified by mass spectrometry ( Figure 1B). The abundance of individual proteins in the whole cell extract (WCE) was also determined by proteomics and used to normalise the RIC data (see Material and Methods) as in (49,50). The RIC/WCE ratio was used to determine the enrichment profile reflected the association of each individual protein with RNA in the mutants relative to the WT (Supplementary Table 1).…”

“…As proof of principle, we assessed if crosslinking of the RNA-binding protein Mmi1, a known exosome regulatory RNA-binding factor involved in substrate recognition, that was found to be enriched in the WT poly(A)+ RNA interactome (49), has been affected in the exosome mutant. This analysis has demonstrated further 1.5, 2 and 4 times increase of Mmi1 association with poly(A)+ RNA (p-value=0.768, p-value=0.039 and p-value=0.001) in the exosome mutants dis3-54, mtl1-1 and rrp6 RIC respectively, compared to WT cells ( Figure 1C).…”

“…1/33 of the oligo-d(T) pull-down total volume was used for RNA sequencing analysis. The rest was subjected to RNase-A and RNase T1 treatment and subjected to mass spectrometry analyses as described in (49).…”

The increasing diversity and complexity of the RNA-binding protein repertoire in plants

“…(3) Characterize at the functional level the biological significance of RBPs, especially the novel cand-AtRBPs and their relevance in stress responses. (4) Dissect the RNP complex remodelling events under various developmental and stress stimuli including abiotic and biotic stresses (as described in [73] for example). ( 5) Unveil the RBPs regulatory network impacting the fate of targeted RNAs.…”