Quality Control of Gene Expression / RNA Surveillance

The exosome is a large multisubunit complex endowed with exonuclease activity. This nuclease is the major 3'-5' exonuclease of eukaryotic cells. It is constituted of a core containing 10 different proteins to which associate nuclear and cytoplasmic specific subunits. The exosome has been implicated in most decay pathways involved in the removal of aberrant and non-functional RNA molecules. Those include elimination of Cryptic Unstable Transcrips (CUTs), aberrant tRNAs, non-spliced pre-mRNA as well as some snRNAs and rRNAs in the nucleus. In the cytoplasm, the exosome has been show to degrade non-functional mRNAs containing a premature stop codon (NMD), those lacking a stop codon (NSD) or those provoking ribosome stalling (No-Go decay). The exosome thus plays a central and essential role in the control of RNA quality. The exosome has to target and degrade aberrant RNAs without attacking functional molecules. This discrimination results in part from the action of factors that will bind and/or modify biologically relevant substrates and target them to degradation. Such factors include the Trf/Air poly(A) polymerase and Mtr4 DEAD box helicase in the nucleus and the Ski7 GTpase and Ski2/3/8 complex in the cytoplasm. If the enzymes and factors involved in these pathways are known, the mechanisms leading them to selectively degrade aberrant RNA molecules remain unclear. We propose here a multidisciplinary study of the role of the exosome in RNA quality control pathways. This analysis will combine molecular biology and biochemical approaches, native mass spectrometry, structural analyses by electron microscopy and small angle X-ray scattering to provide a structural and functional framework allowing the understanding of the role of the exosome in RNA surveillance. We envision that the methodology, developed in this collaborative project involving experts in complementary fields, will be useful to study other RNA based processes and other molecular mechanisms in the future.

The main enzyme responsible for 3'-5' RNA degradation in eukaryotic cells has been named the exosome (van Hoof and Parker, 1999). This enzyme has been shown to be responsible for the degradation of numerous RNA species including aberrant nuclear RNA species such as CUTs and improperly processed tRNAs, ribosomal RNA and snRNA precursors (Allmang et al., 1999a; Dez et al., 2006; Kadaba et al., 2004; LaCava et al., 2005; Vanacova et al., 2005; Wyers et al., 2005) and unspliced pre-mRNAs in the nucleus (Bousquet-Antonelli et al., 2000). In addition, the exosome is involved in the cytoplasm in degradation of defective mRNA lacking a stop codon (van Hoof et al., 2002), those containing a premature stop codon (Mitchell and Tollervey, 2003; Takahashi et al., 2003) and those provoking ribosome stalling (Doma and Parker, 2006) as well as a fraction of non-defective mRNAs. This rather long list demonstrates not only the importance of the exosome activity in eukaryotic cells but also underline its major contribution to RNA surveillance. Thus, the exosome is involved in the decay of all major types of RNAs in the nucleus (pre-mRNAs, tRNAs, rRNA, snRNAs) once these have been recognized as defective. Thus far, the exosome is the only exonuclease known to contribute to the degradation of all types of aberrant or damaged RNA that eukaryotic cells need to eliminate, even if in some cases, redundant pathways involving other exonucleases have been described. However, despite its ubiquitous involvement in RNA quality control, the structural and mechanistic context allowing the recruitment of exosomes for the selective degradation of defective RNA molecules remains poorly understood. In particular, the function of regulatory factors in targeting and/or activating the exosome to degrade aberrant RNAs remains unclear.