New insights into RNA processing by the eukaryotic tRNA splicing endonuclease.

Through its role in intron cleavage, tRNA splicing endonuclease (TSEN) plays a critical function in the maturation of intron-containing pre-tRNAs. The catalytic mechanism and core requirement for this process is conserved between archaea and eukaryotes, but for decades, it has been known that eukaryotic TSENs have evolved additional modes of RNA recognition, which have remained poorly understood. Recent research identified new roles for eukaryotic TSEN, including processing or degradation of additional RNA substrates, and determined the first structures of pre-tRNA-bound human TSEN complexes. These recent discoveries have changed our understanding of how the eukaryotic TSEN targets and recognizes substrates. Here, we review these recent discoveries, their implications, and the new questions raised by these findings.

An unknown essential function of tRNA splicing endonuclease is linked to the integrated stress response and intron debranching.

tRNA splicing endonuclease (TSEN) has a well-characterized role in transfer RNA (tRNA) splicing but also other functions. For yeast TSEN, these other functions include degradation of a subset of mRNAs that encode mitochondrial proteins and an unknown essential function. In this study, we use yeast genetics to characterize the unknown tRNA-independent function(s) of TSEN. Using a high-copy suppressor screen, we found that sen2 mutants can be suppressed by overexpression of SEN54. This effect was seen both for tRNA-dependent and tRNA-independent functions indicating that SEN54 is a general suppressor of sen2, likely through structural stabilization. A spontaneous suppressor screen identified mutations in the intron-debranching enzyme, Dbr1, as tRNA splicing-independent suppressors. Transcriptome analysis showed that sen2 mutation activates the Gcn4 stress response. These Gcn4 target transcripts decreased considerably in the sen2 dbr1 double mutant. We propose that Dbr1 and TSEN may compete for a shared substrate, which TSEN normally processes into an essential RNA, while Dbr1 initiates its degradation. These data provide further insight into the essential function(s) of TSEN. Importantly, single amino acid mutations in TSEN cause the generally fatal neuronal disease pontocerebellar hypoplasia (PCH). The mechanism by which defects in TSEN cause this disease is unknown, and our results reveal new possible mechanisms.

Yeast Dxo1 is required for 25S rRNA maturation and acts as a transcriptome-wide distributive exonuclease.

The Dxo1/Rai1/DXO family of decapping and exonuclease enzymes can catalyze the in vitro removal of chemically diverse 5' ends from RNA. Specifically, these enzymes act poorly on RNAs with a canonical 7mGpppN cap, but instead prefer RNAs with a triphosphate, monophosphate, hydroxyl, or nonconventional cap. In each case, these enzymes generate an RNA with a 5' monophosphate, which is then thought to be further degraded by Rat1/Xrn1 5' exoribonucleases. For most Dxo1/Rai1/DXO family members, it is not known which of these activities is most important in vivo. Here we describe the in vivo function of the poorly characterized cytoplasmic family member, yeast Dxo1. Using RNA-seq of 5' monophosphate ends, we show that Dxo1 can act as a distributive exonuclease, removing a few nucleotides from endonuclease or decapping products. We also show that Dxo1 is required for the final 5' end processing of 25S rRNA, and that this is the primary role of Dxo1. While Dxo1/Rai1/DXO members were expected to act upstream of Rat1/Xrn1, this order is reversed in 25S rRNA processing, with Dxo1 acting downstream from Rat1. Such a hand-off from a processive to a distributive exonuclease may be a general phenomenon in the precise maturation of RNA ends.

Identifying Interaction Partners of Yeast Protein Disulfide Isomerases Using a Small Thiol-Reactive Cross-Linker: Implications for Secretory Pathway Proteostasis.

Protein disulfide isomerases (PDIs) function in forming the correct disulfide bonds in client proteins, thereby aiding the folding of proteins that enter the secretory pathway. Recently, several PDIs have been identified as targets of organic electrophiles, yet the client proteins of specific PDIs remain largely undefined. Here, we report that PDIs expressed in Saccharomyces cerevisiae are targets of divinyl sulfone (DVSF) and other thiol-reactive protein cross-linkers. Using DVSF, we identified the interaction partners that were cross-linked to Pdi1 and Eug1, finding that both proteins form cross-linked complexes with other PDIs, as well as vacuolar hydrolases, proteins involved in cell wall biosynthesis and maintenance, and many ER proteostasis factors involved ER stress signaling and ER-associated protein degradation (ERAD). The latter discovery prompted us to examine the effects of DVSF on ER quality control, where we found that DVSF inhibits the degradation of the ERAD substrate CPY*, in addition to covalently modifying Ire1 and blocking the activation of the unfolded protein response. Our results reveal that DVSF targets many proteins within the ER proteostasis network and suggest that these proteins may be suitable targets for covalent therapeutic development in the future.

Comparative parallel analysis of RNA ends identifies mRNA substrates of a tRNA splicing endonuclease-initiated mRNA decay pathway.

Eukaryotes share a conserved messenger RNA (mRNA) decay pathway in which bulk mRNA is degraded by exoribonucleases. In addition, it has become clear that more specialized mRNA decay pathways are initiated by endonucleolytic cleavage at particular sites. The transfer RNA (tRNA) splicing endonuclease (TSEN) has been studied for its ability to remove introns from pre-tRNAs. More recently it has been shown that single amino acid mutations in TSEN cause pontocerebellar hypoplasia. Other recent studies indicate that TSEN has other functions, but the nature of these functions has remained obscure. Here we show that yeast TSEN cleaves a specific subset of mRNAs that encode mitochondrial proteins, and that the cleavage sites are in part determined by their sequence. This provides an explanation for the counterintuitive mitochondrial localization of yeast TSEN. To identify these mRNA target sites, we developed a "comPARE" (comparative parallel analysis of RNA ends) bioinformatic approach that should be easily implemented and widely applicable to the study of endoribonucleases. The similarity of tRNA endonuclease-initiated decay to regulated IRE1-dependent decay of mRNA suggests that mRNA specificity by colocalization may be an important determinant for the degradation of localized mRNAs in a variety of eukaryotic cells.

Origin, conservation, and loss of alternative splicing events that diversify the proteome in Saccharomycotina budding yeasts.

Many eukaryotes use RNA processing, including alternative splicing, to express multiple gene products from the same gene. The budding yeast Saccharomyces cerevisiae has been successfully used to study the mechanism of splicing and the splicing machinery, but alternative splicing in yeast is relatively rare and has not been extensively studied. Alternative splicing of SKI7/HBS1 is widely conserved, but yeast and a few other eukaryotes have replaced this one alternatively spliced gene with a pair of duplicated, unspliced genes as part of a whole genome doubling (WGD). We show that other examples of alternative splicing known to have functional consequences are widely conserved within Saccharomycotina. A common mechanism by which alternative splicing has disappeared is by replacement of an alternatively spliced gene with duplicate unspliced genes. This loss of alternative splicing does not always take place soon after duplication, but can take place after sufficient time has elapsed for speciation. Saccharomycetaceae that diverged before WGD use alternative splicing more frequently than S. cerevisiae, suggesting that WGD is a major reason for infrequent alternative splicing in yeast. We anticipate that WGDs in other lineages may have had the same effect. Having observed that two functionally distinct splice-isoforms are often replaced by duplicated genes allowed us to reverse the reasoning. We thereby identify several splice isoforms that are likely to produce two functionally distinct proteins because we find them replaced by duplicated genes in related species. We also identify some alternative splicing events that are not conserved in closely related species and unlikely to produce functionally distinct proteins.

Biallelic variants in the RNA exosome gene EXOSC5 are associated with developmental delays, short stature, cerebellar hypoplasia and motor weakness.

The RNA exosome is an essential ribonuclease complex required for processing and/or degradation of both coding and non-coding RNAs. We identified five patients with biallelic variants in EXOSC5, which encodes a structural subunit of the RNA exosome. The clinical features of these patients include failure to thrive, short stature, feeding difficulties, developmental delays that affect motor skills, hypotonia and esotropia. Brain MRI revealed cerebellar hypoplasia and ventriculomegaly. While we ascertained five patients, three patients with distinct variants of EXOSC5 were studied in detail. The first patient had a deletion involving exons 5-6 of EXOSC5 and a missense variant, p.Thr114Ile, that were inherited in trans, the second patient was homozygous for p.Leu206His and the third patient had paternal isodisomy for chromosome 19 and was homozygous for p.Met148Thr. The additional two patients ascertained are siblings who had an early frameshift mutation in EXOSC5 and the p.Thr114Ile missense variant that were inherited in trans. We employed three complementary approaches to explore the requirement for EXOSC5 in brain development and assess consequences of pathogenic EXOSC5 variants. Loss of function for exosc5 in zebrafish results in shortened and curved tails/bodies, reduced eye/head size and edema. We modeled pathogenic EXOSC5 variants in both budding yeast and mammalian cells. Some of these variants cause defects in RNA exosome function as well as altered interactions with other RNA exosome subunits. These findings expand the number of genes encoding RNA exosome subunits linked to human disease while also suggesting that disease mechanism varies depending on the specific pathogenic variant.

Aromatic Residues at the Dimer-Dimer Interface in the Peroxiredoxin Tsa1 Facilitate Decamer Formation and Biological Function.

To prevent the accumulation of reactive oxygen species and limit associated damage to biological macromolecules, cells express a variety of oxidant-detoxifying enzymes, including peroxiredoxins. In Saccharomyces cerevisiae, the peroxiredoxin Tsa1 plays a key role in peroxide clearance and maintenance of genome stability. Five homodimers of Tsa1 can assemble into a toroid-shaped decamer, with the active sites in the enzyme being shared between individual dimers in the decamer. Here, we have examined whether two conserved aromatic residues at the decamer-building interface promote Tsa1 oligomerization, enzymatic activity, and biological function. When substituting either or both of these aromatic residues at the decamer-building interface with either alanine or leucine, we found that the Tsa1 decamer is destabilized, favoring dimeric species instead. These proteins exhibit varying abilities to rescue the phenotypes of oxidant sensitivity and genomic instability in yeast lacking Tsa1 and Tsa2, with the individual leucine substitutions at this interface partially complementing the deletion phenotypes. The ability of Tsa1 decamer interface variants to partially rescue peroxidase function in deletion strains is temperature-dependent and correlates with their relative rate of reactivity with hydrogen peroxide and their ability to interact with thioredoxin. Based on the combined results of in vitro and in vivo assays, our findings indicate that multiple steps in the catalytic cycle of Tsa1 may be impaired by introducing substitutions at its decamer-building interface, suggesting a multifaceted biological basis for its assembly into decamers.

Trapping redox partnerships in oxidant-sensitive proteins with a small, thiol-reactive cross-linker.

A broad range of redox-regulated proteins undergo reversible disulfide bond formation on oxidation-prone cysteine residues. Heightened reactivity of the thiol groups in these cysteines also increases susceptibility to modification by organic electrophiles, a property that can be exploited in the study of redox networks. Here, we explored whether divinyl sulfone (DVSF), a thiol-reactive bifunctional electrophile, cross-links oxidant-sensitive proteins to their putative redox partners in cells. To test this idea, previously identified oxidant targets involved in oxidant defense (namely, peroxiredoxins, methionine sulfoxide reductases, sulfiredoxin, and glutathione peroxidases), metabolism, and proteostasis were monitored for cross-link formation following treatment of Saccharomyces cerevisiae with DVSF. Several proteins screened, including multiple oxidant defense proteins, underwent intermolecular and/or intramolecular cross-linking in response to DVSF. Specific redox-active cysteines within a subset of DVSF targets were found to influence cross-linking; in addition, DVSF-mediated cross-linking of its targets was impaired in cells first exposed to oxidants. Since cross-linking appeared to involve redox-active cysteines in these proteins, we examined whether potential redox partners became cross-linked to them upon DVSF treatment. Specifically, we found that several substrates of thioredoxins were cross-linked to the cytosolic thioredoxin Trx2 in cells treated with DVSF. However, other DVSF targets, like the peroxiredoxin Ahp1, principally formed intra-protein cross-links upon DVSF treatment. Moreover, additional protein targets, including several known to undergo S-glutathionylation, were conjugated via DVSF to glutathione. Our results indicate that DVSF is of potential use as a chemical tool for irreversibly trapping and discovering thiol-based redox partnerships within cells.