Abstract
RNA exosomopathies, a growing family of diseases, are linked to missense mutations in genes encoding structural subunits of the evolutionarily conserved, 10-subunit exoribonuclease complex, the RNA exosome. This complex consists of a three-subunit cap, a six-subunit, barrel-shaped core, and a catalytic base subunit. While a number of mutations in RNA exosome genes cause pontocerebellar hypoplasia, mutations in the cap subunit gene EXOSC2 cause an apparently distinct clinical presentation that has been defined as a novel syndrome SHRF (short stature, hearing loss, retinitis pigmentosa, and distinctive facies). We generated the first in vivo model of the SHRF pathogenic amino acid substitutions using budding yeast by modeling pathogenic EXOSC2 missense mutations (p.Gly30Val and p.Gly198Asp) in the orthologous S. cerevisiae gene RRP4. The resulting rrp4 mutant cells show defects in cell growth and RNA exosome function. Consistent with altered RNA exosome function, we detect significant transcriptomic changes in both coding and noncoding RNAs in rrp4-G226D cells that model EXOSC2 p.Gly198Asp, suggesting defects in nuclear surveillance. Biochemical and genetic analyses suggest that the Rrp4 G226D variant subunit shows impaired interactions with key RNA exosome cofactors that modulate the function of the complex. These results provide the first in vivo evidence that pathogenic missense mutations present in EXOSC2 impair the function of the RNA exosome. This study also sets the stage to compare exosomopathy models to understand how defects in RNA exosome function underlie distinct pathologies.
Highlights
The RNA exosome is an evolutionarily conserved, multisubunit riboexonuclease complex that plays multiple roles in RNA processing and decay
To explore how EXOSC2 G30V and EXOSC2 G198D variants could alter the structure of the EXOSC2 protein or the RNA exosome complex (Fig. 1C), we modeled these EXOSC2 amino acid substitutions using recent structures www.rnajournal.org 1049
We modeled and analyzed pathogenic amino acid substitutions in the S. cerevisiae EXOSC2 ortholog, Rrp4
Summary
The RNA exosome is an evolutionarily conserved, multisubunit riboexonuclease complex that plays multiple roles in RNA processing and decay. First identified in Saccharomyces cerevisiae in a screen for ribosomal RNA processing (rrp) mutants (Mitchell et al 1996, 1997), the RNA exosome is essential in all systems studied far (Mitchell et al 1997; Lorentzen et al 2007; Hou et al 2012; Lim et al 2013; Pefanis et al 2014). RNA (2021) 27:1046–1067; Published by Cold Spring Harbor Laboratory Press for the RNA Society. This essential RNA processing/degradation machine is composed of nine structural subunits associated with a catalytic 3′–5′ exo/endoribonuclease subunit (DIS3/DIS3L [human]; Dis3/Rrp44 [budding yeast]) (Mitchell et al 1997; Makino et al 2013). Structural studies revealed conservation in the structural organization of the RNA exosome (Fig. 1B; Liu et al 2006; Bonneau et al 2009; Makino et al 2013; Wasmuth et al 2014; Zinder et al 2016), suggesting evolutionary conservation not just of subunit sequence but of overall complex structure and organization
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