Insight into the RNA Exosome Complex Through Modeling Pontocerebellar Hypoplasia Type 1b Disease Mutations in Yeast

Abstract

Pontocerebellar hypoplasia type 1b (PCH1b) is an autosomal recessive disorder that causes cerebellar hypoplasia and spinal motor neuron degeneration, leading to mortality in early childhood. PCH1b is caused by mutations in the RNA exosome subunit gene, EXOSC3. The RNA exosome is an evolutionarily conserved complex, consisting of nine different core subunits, and one or two 39-59 exoribonuclease subunits, that mediates several RNA degradation and processing steps. The goal of this study is to assess the functional consequences of the amino acid substitutions that have been identified in EXOSC3 in PCH1b patients. To analyze these EXOSC3 substitutions, we generated the corresponding amino acid substitutions in the Saccharomyces cerevisiae ortholog of EXOSC3, Rrp40. We find that the rrp40 variants corresponding to EXOSC3-G31A and -D132A do not affect yeast function when expressed as the sole copy of the essential Rrp40 protein. In contrast, the rrp40-W195R variant, corresponding to EXOSC3-W238R in PCH1b patients, impacts cell growth and RNA exosome function when expressed as the sole copy of Rrp40. The rrp40-W195R protein is unstable, and does not associate efficiently with the RNA exosome in cells that also express wild-type Rrp40. Consistent with these findings in yeast, the levels of mouse EXOSC3 variants are reduced compared to wild-type EXOSC3 in a neuronal cell line. These data suggest that cells possess a mechanism for optimal assembly of functional RNA exosome complex that can discriminate between wildtype and variant exosome subunits. Budding yeast can therefore serve as a useful tool to understand the molecular defects in the RNA exosome caused by PCH1b-associated amino acid substitutions in EXOSC3, and potentially extending to disease-associated substitutions in other exosome subunits.KEYWORDS pontocerebellar hypoplasia type 1b; RNA exosome; EXOSC3; Rrp40; EXOSC2; RNA processing/degradation T HE RNA exosome is an evolutionarily conserved ribonuclease complex that is responsible for several essential RNA processing and degradation steps (Mitchell et al. 1997;Allmang et al. 1999;Schneider and Tollervey 2013). Major exosome substrates include mRNA, rRNA, and small RNAs. In addition to degrading aberrant and unneeded transcripts, the RNA exosome also trims precursor RNAs, including 5.8S rRNA (Mitchell et al. 1996). The RNA exosome thus plays critical roles in both RNA degradation and maturation.The subunits of the RNA exosome complex are evolutionarily conserved and many were first identified in Saccharomyces cerevisiae in a screen for ribosomal RNA processing (rrp) mutants (Mitchell et al. 1996(Mitchell et al. , 1997Allmang et al. 1999). S. cerevisiae Rrp subunits correspond to human EXOSC subunits. The functions of the RNA exosome have been extensively characterized in budding yeast (Sloan et al. 2012), and many are conserved in humans (Schilders et al. 2005;Staals et al. 2010;Lubas et al. 2011Lubas et al. , 2015 the RNA exosome, six subunits comprise a core ring, and three putative RNA-binding sub...

Mutations of EXOSC3/Rrp40p associated with neurological diseases impact ribosomal RNA processing functions of the exosome in S. cerevisiae

“…One of the major differences between our results and those reported in this study is that the authors did not find any impact of these mutations on pre-rRNA processing, as no accumulation of the ITS-2 containing pre-rRNA species was detected by RT-PCR for the two mutants analyzed, G8A and W195R (Fasken et al 2016). In contrast, our analysis …”

“…A recently published study also analyzed in S. cerevisiae the impact of some of the same mutations analyzed here (Fasken et al 2016). Our results are consistent with most of the results reported in this study, including the effects on growth.…”

“…Our results are consistent with most of the results reported in this study, including the effects on growth. Interestingly, this study showed that the W195R mutation results in an unstable protein, which does not associate well with the exosome complex and is degraded by the proteasome, especially when coexpressed with the wild-type version of Rrp40p (Fasken et al 2016). This result suggests that the growth and RNA processing phenotypes of the W195R mutant are due to destabilization of the Rrp40p/ EXOSC3 protein, leading to a fraction of the exosome complexes lacking this subunit, which might potentially destabilize the complex or lack the ability to associate with RNA (Fig.…”

“…S2) and is therefore not appropriate to amplify the 7S pre-rRNA and shorter processing products (we also note that there is a switch between the forward and reverse primers in Supplemental Table 2). This difference is sufficient to explain the fact that Fasken et al (2016) did not detect an accumulation of the 7S species in their mutants, leading to a similar ITS2 signal for all strains analyzed.…”

“…4). We believe that this discrepancy may have been caused by the primer set used by Fasken et al (2016) in their RT-PCR analysis. The reverse primer used in this study is positioned just downstream from the C2 cleavage site in the ITS2 (Supplemental Fig.…”