The Exosome Associates Cotranscriptionally with the Nascent Pre-mRNP through Interactions with Heterogeneous Nuclear Ribonucleoproteins

Abstract

Eukaryotic cells have evolved quality control mechanisms to degrade aberrant mRNA molecules and prevent the synthesis of defective proteins that could be deleterious for the cell. The exosome, a protein complex with ribonuclease activity, is a key player in quality control. An early quality checkpoint takes place cotranscriptionally but little is known about the molecular mechanisms by which the exosome is recruited to the transcribed genes. Here we study the core exosome subunit Rrp4 in two insect model systems, Chironomus and Drosophila. We show that a significant fraction of Rrp4 is associated with the nascent pre-mRNPs and that a specific mRNA-binding protein, Hrp59/hnRNP M, interacts in vivo with multiple exosome subunits. Depletion of Hrp59 by RNA interference reduces the levels of Rrp4 at transcription sites, which suggests that Hrp59 is needed for the exosome to stably interact with nascent pre-mRNPs. Our results lead to a revised mechanistic model for cotranscriptional quality control in which the exosome is constantly recruited to newly synthesized RNAs through direct interactions with specific hnRNP proteins. INTRODUCTIONExpression of protein-coding genes is a complex multistep process. Precursor messenger RNAs (pre-mRNAs) are synthesized by RNA polymerase II (Pol-II) and assembled into ribonucleoprotein (RNP) complexes during transcription. The pre-mRNPs must be processed to become mature mRNPs, which are exported to the cytoplasm and translated into protein. Mutations in the DNA or errors in the transcription and processing reactions can lead to the synthesis of aberrant mRNPs. Eukaryotic cells have evolved quality control mechanisms that identify aberrant mRNPs and prevent their expression into protein. In the yeast Saccharomyces cerevisiae there are at least three nuclear steps of mRNP biogenesis that are under surveillance: the cleavage and polyadenylation of the 3Ј end (Hilleren et al., 2001), the assembly of the mRNA-protein complexes (Zenklusen et al., 2002;Luna et al., 2005), and the removal of introns (Galy et al., 2004). In all cases, mRNPs with assembly and/or processing defects are detected by nuclear surveillance mechanisms, retained in the nucleus, and degraded (reviewed by Vinciguerra and Stutz, 2004;Sommer and Nehrbass, 2005;Saguez et al., 2005). Genetic studies in S. cerevisiae have identified the nuclear exosome as a key player in the recognition and retention of defective transcripts (reviewed by Jensen et al., 2003;Houseley et al., 2006;Vanacova and Stefl, 2007;Schmid and Jensen, 2008).The exosome is a protein complex with ribonuclease activity (Mitchell et al., 1997;Allmang et al., 1999;Mitchell and Tollervey, 2000). The core of the exosome has a barrel-like architecture (reviewed by Lorentzen et al., 2008). The barrel is made of nine protein subunits organized into two rings, a hexameric ring and a trimeric ring, and the structure of the barrel is conserved throughout evolution (Lorentzen et al., 2005;Liu et al., 2006;Wang et al., 2007). Two additional proteins, Dis3/Rrp44 and R...

Rrp6 is recruited to transcribed genes and accompanies the spliced mRNA to the nuclear pore

“…S2). The nucleoplasm was strongly labeled, and the polytene chromosomes displayed a banded pattern similar to that observed with antibodies against the core exosome subunit Rrp4 (Hessle et al 2009). Furthermore, a small fraction of Rrp6 was concentrated in discrete nuclear bodies (Fig.…”

The Incorporation of 5-Fluorouracil into RNA Affects the Ribonucleolytic Activity of the Exosome Subunit Rrp6

“…1B), as described for the endogenous Rrp6 (20). And thirdly, we showed in a previous study that Rrp6-V5 associates in vivo with proteins such as Spt5 and Spt6, which are known to interact with the endogenous exosome (16,19).…”

“…Cells stably transfected with a plasmid that encodes V5-tagged Rrp6 have been described by Hessle and colleagues (19). The mock cells used in this study are S2 cells stably transfected with the hygromycin selection plasmid but without the Rrp6-V5 expression plasmid.…”

“…To address this question, we carried out a first series of experiments in which we analyzed whether the assembly of Rrp6 into multimolecular complexes in vivo was affected by 5FU. We used Drosophila S2 cells that expressed a V5-tagged version of Rrp6 under the control of a metallothionein promoter (19). The expression of the tagged Rrp6 was induced with copper sulfate, as shown in Figure 1A.…”

The Role of the Carcinoembryonic Antigen Receptor in Colorectal Cancer Progression

“…[10]. HnRNPM is a multifunctional protein that is involved in mRNA processing [17], splicing [18], stress response [19], mRNA transport and stability of exosomes [20]. In human cells, hnRNPM plays a role in regulating FGFR2 alternative splicing and can affect the splicing of several other genes [21].…”