Function of the RNA helicase Sub2 from Saccharomyces cerevisiae and its regulation by Tho1

Abstract

In eukaryotes, the transcription of mRNA takes place in the nucleus of each cell. Already during transcription, various proteins bind the mRNA to prepare it for export from the nucleus so that the information of the genome can be translated into a protein. An essential part of this machinery is the TREX complex, which forms an mRNP together with the mRNA and prepares it for export. This is highly conserved in animals, fungi and plants and consists in Saccharomyces cerevisiae of the heteropentameric THO complex and the RNA helicase Sub2 (UAP56/DDX39 in humans). In addition, other RNA binding proteins (RBPs) such as the RNA annealing protein Yra1 (AlyRef in humans) are recruited. In humans, CIP29/SARNP (in Arabidopsis thaliana MOS11) also interacts with the complex. These RBPs play an important role in mRNA export, but their exact functions are not yet known Sub2 is a conserved DEAD-box RNA helicase from S. cerevisiae and is involved in splicing and mRNA export. While deletion of Sub2 leads to cell death, overexpression rescues the mRNA export defect of a non-functioning THO complex. Although Sub2 has an essential role in mRNA export and splicing, its biochemical activities in these processes are largely unclear. In this work, therefore, the biochemical properties of Sub2 were characterised and its regulation by interaction partners was studied in detail in vitro. Suitable biochemical real-time assays were developed for this purpose. In order to be able to follow important conformational changes in the course of the regulation of Sub2 in the future, the prerequisites for Förster resonance energy transfer (FRET) experiments were created. For the necessary site-specific labelling of Sub2, the existing conserved cysteine residues were replaced and additional cysteine residues were introduced at strategic positions on the surface. The function of the conserved cysteines is currently unclear, but the cysteine-free Sub2 variant does not lead to an obvious phenotype in vivo. First labelling experiments with fluorescent dyes show that the prerequisites for FRET experiments have been created. The starting point for the investigations on the regulation of Sub2 by Tho1 was the fact that the human orthologues UAP56 and CIP29/SARNP form an ATP-dependent complex which leads to the stimulation of ATPase and helicase activity. In this work, ATP-dependent ternary complex formation of Sub2 with Tho1 and RNA was demonstrated. However, Tho1 inhibits the helicase activity of Sub2, which is caused by a previously unknown annealing activity of Tho1. This new activity is evolutionarily conserved and could also be demonstrated for the human CIP29/SARNP, as well as the SARNP/Tho1 orthologue from the thermophilic fungus Chaetomium thermophilum. Furthermore, a detailed mutational analysis of Tho1 experimentally identified the conserved RNA-binding domain as an interaction domain for Sub2. This finding is consistent with the recent prediction of the structures of relevant protein complexes in S. cerevisiae. With the help of further biochemical and cell biological assays, first indications for a new function of Tho1 in mRNA export could also be found. While deletion of Tho1 does not show any phenotype, overexpression of Tho1 (similar to Sub2) rescues several phenotypes (hyperrecombination, transcription and mRNA export defect) of a non-functioning THO complex. The phenotype can be manifested by an accumulation of R-loops and indeed overexpression of Tho1 decreases the number of R-loops in vivo. In vitro experiments show that both Sub2 and, surprisingly, Tho1 alone are already capable of resolving R-loop-like structures. While the resolution of R-loops by Sub2 was expected, as the human orthologue UAP56/DDX39B has already been identified as an important cotranscriptional RNA/DNA helicase, the role of Tho1 is surprising and novel. However, to what extent the interaction of Tho1 and Sub2 is relevant for this function and whether this is evolutionarily conserved is yet to be seen.