Comparative analyses of disease-linked missense mutations in the RNA exosome modeled in budding yeast reveal distinct functional consequences in translation

Abstract

The RNA exosome is an evolutionarily conserved exoribonuclease complex that consists of a 3-subunit cap, a 6-subunit barrel-shaped core, and a catalytic base subunit. Missense mutations in genes encoding structural subunits of the RNA exosome cause a growing family of diseases with diverse pathologies, collectively termed RNA exosomopathies. The disease symptoms vary and can manifest as neurological defects or developmental disorders. The diversity of the RNA exosomopathy pathologies suggests that the different missense mutations in structural genes result in distinct in vivo consequences. To investigate these functional consequences and distinguish whether they are unique to each RNA exosomopathy mutation, we generated a collection of in vivo models using budding yeast by introducing pathogenic missense mutations in orthologous S. cerevisiae genes. We then performed a comparative RNA-seq analysis to assess broad transcriptomic changes in each mutant model. Three of the mutant models rrp4-G226D, rrp40-W195R and rrp46-L191H, which model mutations in the genes encoding structural subunits of the RNA exosome, EXOSC2, EXOSC3 and EXOSC5 showed the largest transcriptomic differences. Further analyses revealed shared increased transcripts enriched in translation or ribosomal RNA modification/processing pathways across the three mutant models. Studies of the impact of the mutations on translation revealed shared defects in ribosome biogenesis but distinct impacts on translation. Collectively, our results provide the first comparative analysis of several RNA exosomopathy mutant models and suggest that different RNA exosomopathy mutations result in in vivo consequences that are both unique and shared across each variant, providing more insight into the biology underlying each distinct pathology.

A Biallelic Variant of the RNA Exosome GeneEXOSC4Causes Translational Defects Associated with a Neurodevelopmental Disorder

“…As 3’-end trimming of the 7S pre-rRNA to the mature 5.8S required to assemble functional ribosomes by the RNA exosome is one of the best defined function of this complex, defects in this process would be predicted to alter translation. Indeed, a recent study demonstrated that several different budding yeast models of exosomopathies impair translation albeit, there appear to be differences in how translation is affected in different yeast models of exosomopathies (29). The analysis of the core subunit here is consistent with the previous finding (29), suggesting that mutations that affect the core subunits vs the cap subunits of the RNA exosome cause distinct translational defects with defects in core subunits such as Rrp41 causing an overall reduction of ribosomes while defects in cap subunits leading to formation of halfmers indicating a potential problem in 60S maturation or subunit joining.…”

“…Indeed, a recent study demonstrated that several different budding yeast models of exosomopathies impair translation albeit, there appear to be differences in how translation is affected in different yeast models of exosomopathies (29). The analysis of the core subunit here is consistent with the previous finding (29), suggesting that mutations that affect the core subunits vs the cap subunits of the RNA exosome cause distinct translational defects with defects in core subunits such as Rrp41 causing an overall reduction of ribosomes while defects in cap subunits leading to formation of halfmers indicating a potential problem in 60S maturation or subunit joining. Further studies will be required to determine whether these changes in translation are confirmed in patient cells or mammalian disease models as well as how these molecular changes alter the proteome.…”

“…The pcDNA3-Myc- Exosc4-L187P (pAC4231) variant plasmid was generated by site-directed mutagenesis of pcDNA3-Myc- Exosc4 (pAC3517) plasmid using oligonucleotides encoding the L187P amino acid change (AC9505 & AC9506; Integrated DNA Technologies) and QuickChange II SDM Kit (Agilent). The pRS316-TEF1p-Cas9-CYC1t-SNR52p (pAC3846) pCas9 plasmid ( URA3 , CEN6 ), which derives from p414- TEF1p-Cas9-CYC1t plasmid [Addgene #43802; (36)] and p426-SNR52p-gRNA.CAN1.Y-SUP4t plasmid [Addgene #43803; (36)] has been described previously (29). The pRS316- TEF1p-Cas9-CYC1t-SNR52p-RRP41_565.gRNA-SUP4t (pAC4339) pCas9 plasmid containing the gRNA for targeting RRP41 was constructed by PCR amplification of RRP41_565.gRNA with oligonucleotides AC9891 and AC6809 using p426-SNR52p-gRNA.CAN1.Y-SUP4t plasmid template (Addgene #43803) and cloning of SphI/KpnI-digested gRNA product into pAC3846 digested with SphI/KpnI.…”