Functional characterization of Saccharomyces cerevisiae protein Sub2 in nuclear mRNP formation

Abstract

RNA-protein interactions play a critical role in numerous key biological processes. These interactions form ribonucleoprotein complexes (RNPs) that regulate gene expression and are involved in all steps of RNA biogenesis, including transcription, RNA processing, nuclear export, localization, stability, and translation. Owing to their importance, failure to appropriately assemble protein-RNA complexes triggers various genetic diseases and cancers. RNA-binding proteins (RBPs) represent a large class of proteins that interact with and regulate transcripts. RBPs can mediate single RNA-protein interactions or be part of multi-protein complexes. An example of such a multi-protein complex is the TREX complex, that couples transcription with nuclear mRNA export. It is composed of the pentameric THO complex (Tho2, Hpr1, Mft1, Thp1 and Tex1), the SR-like proteins Gbp2 and Hrb1, the RNA helicase Sub2 and the export adaptor Yra1. Yra1 links the TREX-packaged mRNA with the export receptor Mex67-Mtr2, which transports the mRNP to the cytoplasm. The RNA helicase Sub2, is highly conserved and functions in splicing, polyadenylation, nuclear mRNA export and removing RNA-DNA hybrids (R-loop resolution). It is recruited to active chromatin by the THO complex and interacts with RNA using amino acid residues in its RNA-binding motifs. In this study, we assessed the role of novel RNA-binding sites of Sub2 in mRNP formation. To achieve this, we mutated amino acids of Sub2 that were identified to interact with RNA in vivo and characterized them functionally. Using this approach, we identified amino acid residues that are vital for Sub2 function, as these mutations affected the viability of our cells. Of the viable sub2 mutants, we characterized three into detail. We identified sub2-T62D, which had mild growth, but strong export defects, implicating it as an essential residue for mRNA export. It also demonstrated slightly reduced binding to RNA and could not resolve R-loops in vivo. We also identified sub2-K70D which was not essential for growth, as it had no growth defects, but had mRNA export defects, and reduced RNA-binding and helicase activities. Additionally, we identified the essential sub2-K202E-Y203E that resulted in growth and mRNA export defects, and reduced RNA-binding and helicase activities. This mutant also mislocalized Sub2. Analysis revealed the essential amino acid in this mutant to be K202. By further characterizing this mutant, we could show that, the ability of Sub2 to support growth depends on its nuclear localization. Altogether, mutating the putative RNA-binding sites of Sub2 affects growth, mRNA export, dsRNA helicase activity, R-loop resolution and impairs interaction with other mRNP components. Moreover, not all residues of Sub2 mediate similar function. Using this mutagenic approach, we identified residues that are not essential for growth, residues vital for mRNA export and residues that are crucial for binding to RNA and protein localization.