Cassandra K. Hayne

The Target of EGR1 (TOE1) gene encodes the TOE1 deadenylase, which is essential for the maturation of Pol-II transcribed snRNAs in humans. Over a dozen missense mutations in the TOE1 gene have been linked to Pontocerebellar Hypoplasia Type 7 (PCH7), a rare but serious neurodevelopmental and neurodegenerative disease that leads to early mortality. The biochemical mechanisms for why these PCH7-linked mutations alter TOE1’s biochemical characteristics remains vague. Here, we utilized AlphaFold predicted structures of TOE1 and biochemical characterizations to investigate the impact of selected TOE1 variants on TOE1’s biochemical properties. We performed characterization of the thermal stability and activity of eleven PCH-linked TOE1 variants and found that eight variants have significantly reduced protein thermal stability and that all but two variants impair TOE1’s ribonuclease activity, particularly its exonuclease activity. Additionally, we found that the F148Y mutation impacts TOE1’s oligomeric state in vitro and in vivo . Together, these results demonstrate that PCH-linked mutations of TOE1 impact many different aspects of TOE1 biochemistry, providing novel insights which may provide potential therapeutic strategies to treat PCH7 patients. In addition, these mutations provide a library of TOE1 variants that will be useful for future studies of TOE1 function and regulation.

Throughout bacteria, archaea and eukarya, certain tRNA transcripts contain introns. Pre-tRNAs with introns require splicing to form the mature anticodon stem loop. In eukaryotes, tRNA splicing is initiated by the heterotetrameric tRNA splicing endonuclease (TSEN) complex. All TSEN subunits are essential, and mutations within the complex are associated with a family of neurodevelopmental disorders known as pontocerebellar hypoplasia (PCH). Here, we report cryo-electron microscopy structures of the human TSEN–pre-tRNA complex. These structures reveal the overall architecture of the complex and the extensive tRNA binding interfaces. The structures share homology with archaeal TSENs but contain additional features important for pre-tRNA recognition. The TSEN54 subunit functions as a pivotal scaffold for the pre-tRNA and the two endonuclease subunits. Finally, the TSEN structures enable visualization of the molecular environments of PCH-causing missense mutations, providing insight into the mechanism of pre-tRNA splicing and PCH.

The splicing of transfer RNA (tRNA) introns is a critical step of tRNA maturation, for intron‐containing tRNAs. In eukaryotes, tRNA splicing is a multi‐step process that relies on several RNA processing enzymes to facilitate intron removal and exon ligation. Splicing is initiated by the tRNA splicing endonuclease (TSEN) complex which catalyzes the excision of the intron through its two nuclease subunits. Mutations in all four subunits of the TSEN complex are linked to a family of neurodegenerative and neurodevelopmental diseases known as pontocerebellar hypoplasia (PCH). Recent studies provide molecular insights into the structure, function, and regulation of the eukaryotic TSEN complex and are beginning to illuminate how mutations in the TSEN complex lead to neurodegenerative disease. Using new advancements in the prediction of protein structure, we created a three‐dimensional model of the human TSEN complex. We review functions of the TSEN complex beyond tRNA splicing by highlighting recently identified substrates of the eukaryotic TSEN complex and discuss mechanisms for the regulation of tRNA splicing, by enzymes that modify cleaved tRNA exons and introns. Finally, we review recent biochemical and animal models that have worked to address the mechanisms that drive PCH and synthesize these studies with previous studies to try to better understand PCH pathogenesis.This article is categorized under: RNA Processing > tRNA Processing RNA in Disease and Development > RNA in Disease RNA Interactions with Proteins and Other Molecules > Protein–RNA Recognition

Nsp15, a uridine specific endoribonuclease conserved across coronaviruses, processes viral RNA to evade detection by host defense systems. Crystal structures of Nsp15 from different coronaviruses have shown a common hexameric assembly, yet how the enzyme recognizes and processes RNA remains poorly understood. Here we report a series of cryo-EM reconstructions of SARS-CoV-2 Nsp15, in both apo and UTP-bound states. The cryo-EM reconstructions, combined with biochemistry, mass spectrometry, and molecular dynamics, expose molecular details of how critical active site residues recognize uridine and facilitate catalysis of the phosphodiester bond. Mass spectrometry revealed the accumulation of cyclic phosphate cleavage products, while analysis of the apo and UTP-bound datasets revealed conformational dynamics not observed by crystal structures that are likely important to facilitate substrate recognition and regulate nuclease activity. Collectively, these findings advance understanding of how Nsp15 processes viral RNA and provide a structural framework for the development of new therapeutics.

New therapeutics are urgently needed to inhibit SARS-CoV-2, the virus responsible for the on-going Covid-19 pandemic. Nsp15, a uridine-specific endoribonuclease found in all coronaviruses, processes viral RNA to evade detection by RNA-activated host defense systems, making it a promising drug target. Previous work with SARS-CoV-1 established that Nsp15 is active as a hexamer, yet how Nsp15 recognizes and processes viral RNA remains unknown. Here we report a series of cryo-EM reconstructions of SARS-CoV-2 Nsp15. The UTP-bound cryo-EM reconstruction at 3.36 Å resolution provides molecular details into how critical residues within the Nsp15 active site recognize uridine and facilitate catalysis of the phosphodiester bond, whereas the apo-states reveal active site conformational heterogeneity. We further demonstrate the specificity and mechanism of nuclease activity by analyzing Nsp15 products using mass spectrometry. Collectively, these findings advance understanding of how Nsp15 processes viral RNA and provide a structural framework for the development of new therapeutics.

The splicing of tRNA introns is a critical step in pre-tRNA maturation. In archaea and eukaryotes, tRNA intron removal is catalyzed by the tRNA splicing endonuclease (TSEN) complex. Eukaryotic TSEN is comprised of four core subunits (TSEN54, TSEN2, TSEN34 and TSEN15). The human TSEN complex additionally co-purifies with the polynucleotide kinase CLP1; however, CLP1’s role in tRNA splicing remains unclear. Mutations in genes encoding all four TSEN subunits, as well as CLP1, are known to cause neurodegenerative disorders, yet the mechanisms underlying the pathogenesis of these disorders are unknown. Here, we developed a recombinant system that produces active TSEN complex. Co-expression of all four TSEN subunits is required for efficient formation and function of the complex. We show that human CLP1 associates with the active TSEN complex, but is not required for tRNA intron cleavage in vitro. Moreover, RNAi knockdown of the Drosophila CLP1 orthologue, cbc, promotes biogenesis of mature tRNAs and circularized tRNA introns (tricRNAs) in vivo. Collectively, these and other findings suggest that CLP1/cbc plays a regulatory role in tRNA splicing by serving as a negative modulator of the direct tRNA ligation pathway in animal cells.