Identification of potent inhibitors of arenavirus and SARS-CoV-2 exoribonucleases by fluorescence polarization assay.

Viral exoribonucleases are uncommon in the world of RNA viruses. To date, they have only been identified in the Arenaviridae and the Coronaviridae families. The exoribonucleases of these viruses play a crucial role in the pathogenicity and interplay with host innate immune response. Moreover, coronaviruses exoribonuclease is also involved in a proofreading mechanism ensuring the genetic stability of the viral genome. Because of their key roles in virus life cycle, they constitute attractive target for drug design. Here we developed a sensitive, robust and reliable fluorescence polarization assay to measure the exoribonuclease activity and its inhibition in vitro. The effectiveness of the method was validated on three different viral exoribonucleases, including SARS-CoV-2, Lymphocytic Choriomeningitis and Machupo viruses. We performed a screening of a focused library consisting of 113 metal chelators. Hit compounds were recovered with an IC50 at micromolar level. We confirmed 3 hits in SARS-CoV-2 infected Vero-E6 cells.

Potent Inhibition of SARS-CoV-2 nsp14 N7-Methyltransferase by Sulfonamide-Based Bisubstrate Analogues.

Enzymes involved in RNA capping of SARS-CoV-2 are essential for the stability of viral RNA, translation of mRNAs, and virus evasion from innate immunity, making them attractive targets for antiviral agents. In this work, we focused on the design and synthesis of nucleoside-derived inhibitors against the SARS-CoV-2 nsp14 (N7-guanine)-methyltransferase (N7-MTase) that catalyzes the transfer of the methyl group from the S-adenosyl-l-methionine (SAM) cofactor to the N7-guanosine cap. Seven compounds out of 39 SAM analogues showed remarkable double-digit nanomolar inhibitory activity against the N7-MTase nsp14. Molecular docking supported the structure-activity relationships of these inhibitors and a bisubstrate-based mechanism of action. The three most potent inhibitors significantly stabilized nsp14 (ΔTm ≈ 11 °C), and the best inhibitor demonstrated high selectivity for nsp14 over human RNA N7-MTase.

Multi-stage structure-based virtual screening approach towards identification of potential SARS-CoV-2 NSP13 helicase inhibitors.

On account of its crucial role in the virus life cycle, SARS-COV-2 NSP13 helicase enzyme was exploited as a promising target to identify a novel potential inhibitor using multi-stage structure-based drug discovery approaches. Firstly, a 3D pharmacophore was generated based on the collected data from a protein-ligand interaction fingerprint (PLIF) study using key interactions between co-crystallised fragments and the NSP13 helicase active site. The ZINC database was screened through the generated 3D-pharmacophore retrieving 13 potential hits. All the retrieved hits exceeded the benchmark score of the co-crystallised fragments at the molecular docking step and the best five-hit compounds were selected for further analysis. Finally, a combination between molecular dynamics simulations and MM-PBSA based binding free energy calculations was conducted on the best hit (compound FWM-1) bound to NSP13 helicase enzyme, which identified FWM-1 as a potential potent NSP13 helicase inhibitor with binding free energy equals -328.6 ± 9.2 kcal/mol.

Characterization of the SARS-CoV-2 ExoN (nsp14ExoN-nsp10) complex: implications for its role in viral genome stability and inhibitor identification.

The SARS-CoV-2 coronavirus is the causal agent of the current global pandemic. SARS-CoV-2 belongs to an order, Nidovirales, with very large RNA genomes. It is proposed that the fidelity of coronavirus (CoV) genome replication is aided by an RNA nuclease complex, comprising the non-structural proteins 14 and 10 (nsp14-nsp10), an attractive target for antiviral inhibition. Our results validate reports that the SARS-CoV-2 nsp14-nsp10 complex has RNase activity. Detailed functional characterization reveals nsp14-nsp10 is a versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3'-terminus. Consistent with a role in maintaining viral genome integrity during replication, we find that nsp14-nsp10 activity is enhanced by the viral RNA-dependent RNA polymerase complex (RdRp) consisting of nsp12-nsp7-nsp8 (nsp12-7-8) and demonstrate that this stimulation is mediated by nsp8. We propose that the role of nsp14-nsp10 in maintaining replication fidelity goes beyond classical proofreading by purging the nascent replicating RNA strand of a range of potentially replication-terminating aberrations. Using our developed assays, we identify drug and drug-like molecules that inhibit nsp14-nsp10, including the known SARS-CoV-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for multifunctional inhibitors in COVID-19 treatment.

Genome-scale CRISPR screens identify host factors that promote human coronavirus infection.

BACKGROUND: The COVID-19 pandemic has resulted in 275 million infections and 5.4 million deaths as of December 2021. While effective vaccines are being administered globally, there is still a great need for antiviral therapies as antigenically novel SARS-CoV-2 variants continue to emerge across the globe. Viruses require host factors at every step in their life cycle, representing a rich pool of candidate targets for antiviral drug design. METHODS: To identify host factors that promote SARS-CoV-2 infection with potential for broad-spectrum activity across the coronavirus family, we performed genome-scale CRISPR knockout screens in two cell lines (Vero E6 and HEK293T ectopically expressing ACE2) with SARS-CoV-2 and the common cold-causing human coronavirus OC43. Gene knockdown, CRISPR knockout, and small molecule testing in Vero, HEK293, and human small airway epithelial cells were used to verify our findings. RESULTS: While we identified multiple genes and functional pathways that have been previously reported to promote human coronavirus replication, we also identified a substantial number of novel genes and pathways. The website https://sarscrisprscreens.epi.ufl.edu/ was created to allow visualization and comparison of SARS-CoV2 CRISPR screens in a uniformly analyzed way. Of note, host factors involved in cell cycle regulation were enriched in our screens as were several key components of the programmed mRNA decay pathway. The role of EDC4 and XRN1 in coronavirus replication in human small airway epithelial cells was verified. Finally, we identified novel candidate antiviral compounds targeting a number of factors revealed by our screens. CONCLUSIONS: Overall, our studies substantiate and expand the growing body of literature focused on understanding key human coronavirus-host cell interactions and exploit that knowledge for rational antiviral drug development.

Hibernation-Promoting Factor Sequesters Staphylococcus aureus Ribosomes to Antagonize RNase R-Mediated Nucleolytic Degradation.

Bacterial and eukaryotic hibernation factors prevent translation by physically blocking the decoding center of ribosomes, a phenomenon called ribosome hibernation that often occurs in response to nutrient deprivation. The human pathogen Staphylococcus aureus lacking the sole hibernation factor HPF undergoes massive ribosome degradation via an unknown pathway. Using genetic and biochemical approaches, we find that inactivating the 3'-to-5' exonuclease RNase R suppresses ribosome degradation in the Δhpf mutant. In vitro cell-free degradation assays confirm that 30S and 70S ribosomes isolated from the Δhpf mutant are extremely susceptible to RNase R, in stark contrast to nucleolytic resistance of the HPF-bound 70S and 100S complexes isolated from the wild type. In the absence of HPF, specific S. aureus 16S rRNA helices are sensitive to nucleolytic cleavage. These RNase hot spots are distinct from that found in the Escherichia coli ribosomes. S. aureus RNase R is associated with ribosomes, but unlike the E. coli counterpart, it is not regulated by general stressors and acetylation. The results not only highlight key differences between the evolutionarily conserved RNase R homologs but also provide direct evidence that HPF preserves ribosome integrity beyond its role in translational avoidance, thereby poising the hibernating ribosomes for rapid resumption of translation. IMPORTANCE Ribosome hibernation is pivotal for the rapid recovery of translation after quiescence in both bacteria and eukaryotes. Ribosome hibernation factors sterically occlude the entry of mRNA and tRNA and are thought to primarily maintain ribosomes in a translation-repressive state, thereby providing a pool of readily recyclable 70S or 80S complexes upon dissociation of the hibernation factors. Ribosomes in Staphylococcus aureus cells lacking the sole hibernation factor HPF are extremely unstable. Here, we show that HPF binding inhibits ribosome degradation by the evolutionarily conserved exoribonuclease RNase R. The data not only uncover a direct protective role of HPF in ribosome stability but also reinforce the versatility of RNase R in RNA processing, decay, and ribosome quality control.

The Antiviral Effect of the Chemical Compounds Targeting DED/EDh Motifs of the Viral Proteins on Lymphocytic Choriomeningitis Virus and SARS-CoV-2.

Arenaviruses and coronaviruses include several human pathogenic viruses, such as Lassa virus, Lymphocytic choriomeningitis virus (LCMV), SARS-CoV, MERS-CoV, and SARS-CoV-2. Although these viruses belong to different virus families, they possess a common motif, the DED/EDh motif, known as an exonuclease (ExoN) motif. In this study, proof-of-concept studies, in which the DED/EDh motif in these viral proteins, NP for arenaviruses, and nsp14 for coronaviruses, could be a drug target, were performed. Docking simulation studies between two structurally different chemical compounds, ATA and PV6R, and the DED/EDh motifs in these viral proteins indicated that these compounds target DED/EDh motifs. The concentration which exhibited modest cell toxicity was used with these compounds to treat LCMV and SARS-CoV-2 infections in two different cell lines, A549 and Vero 76 cells. Both ATA and PV6R inhibited the post-entry step of LCMV and SARS-CoV-2 infection. These studies strongly suggest that DED/EDh motifs in these viral proteins could be a drug target to combat two distinct viral families, arenaviruses and coronaviruses.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp14/nsp10 exoribonuclease.

SARS-CoV-2 is a coronavirus that emerged in 2019 and rapidly spread across the world causing a deadly pandemic with tremendous social and economic costs. Healthcare systems worldwide are under great pressure, and there is an urgent need for effective antiviral treatments. The only currently approved antiviral treatment for COVID-19 is remdesivir, an inhibitor of viral genome replication. SARS-CoV-2 proliferation relies on the enzymatic activities of the non-structural proteins (nsp), which makes them interesting targets for the development of new antiviral treatments. With the aim to identify novel SARS-CoV-2 antivirals, we have purified the exoribonuclease/methyltransferase (nsp14) and its cofactor (nsp10) and developed biochemical assays compatible with high-throughput approaches to screen for exoribonuclease inhibitors. We have screened a library of over 5000 commercial compounds and identified patulin and aurintricarboxylic acid (ATA) as inhibitors of nsp14 exoribonuclease in vitro. We found that patulin and ATA inhibit replication of SARS-CoV-2 in a VERO E6 cell-culture model. These two new antiviral compounds will be valuable tools for further coronavirus research as well as potentially contributing to new therapeutic opportunities for COVID-19.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp14 RNA cap methyltransferase.

The COVID-19 pandemic has presented itself as one of the most critical public health challenges of the century, with SARS-CoV-2 being the third member of the Coronaviridae family to cause a fatal disease in humans. There is currently only one antiviral compound, remdesivir, that can be used for the treatment of COVID-19. To identify additional potential therapeutics, we investigated the enzymatic proteins encoded in the SARS-CoV-2 genome. In this study, we focussed on the viral RNA cap methyltransferases, which play key roles in enabling viral protein translation and facilitating viral escape from the immune system. We expressed and purified both the guanine-N7 methyltransferase nsp14, and the nsp16 2'-O-methyltransferase with its activating cofactor, nsp10. We performed an in vitro high-throughput screen for inhibitors of nsp14 using a custom compound library of over 5000 pharmaceutical compounds that have previously been characterised in either clinical or basic research. We identified four compounds as potential inhibitors of nsp14, all of which also showed antiviral capacity in a cell-based model of SARS-CoV-2 infection. Three of the four compounds also exhibited synergistic effects on viral replication with remdesivir.

Identification and evaluation of potential SARS-CoV-2 antiviral agents targeting mRNA cap guanine N7-Methyltransferase.

SARS-CoV-2, the cause of the currently ongoing COVID-19 pandemic, encodes its own mRNA capping machinery. Insights into this capping system may provide new ideas for therapeutic interventions and drug discovery. In this work, we employ a previously developed Py-FLINT screening approach to study the inhibitory effects of compounds against the cap guanine N7-methyltransferase enzyme, which is involved in SARS-CoV-2 mRNA capping. We screened five commercially available libraries (7039 compounds in total) to identify 83 inhibitors with IC50 < 50 µM, which were further validated using RP HPLC and dot blot assays. Novel fluorescence anisotropy binding assays were developed to examine the targeted binding site. The inhibitor structures were analyzed for structure-activity relationships in order to define common structural patterns. Finally, the most potent inhibitors were tested for antiviral activity on SARS-CoV-2 in a cell based assay.

A cell-based assay to discover inhibitors of SARS-CoV-2 RNA dependent RNA polymerase.

Antiviral therapeutics is one effective avenue to control and end this devastating COVID-19 pandemic. The viral RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 has been recognized as a valuable target of antivirals. However, the cell-free SARS-CoV-2 RdRp biochemical assay requires the conversion of nucleotide prodrugs into the active triphosphate forms, which regularly occurs in cells yet is a complicated multiple-step chemical process in vitro, and thus hinders the utility of this cell-free assay in the rapid discovery of RdRp inhibitors. In addition, SARS-CoV-2 exoribonuclease provides the proof-reading capacity to viral RdRp, thus creates relatively high resistance threshold of viral RdRp to nucleotide analog inhibitors, which must be examined and evaluated in the development of this class of antivirals. Here, we report a cell-based assay to evaluate the efficacy of nucleotide analog compounds against SARS-CoV-2 RdRp and assess their tolerance to viral exoribonuclease-mediated proof-reading. By testing seven commonly used nucleotide analog viral polymerase inhibitors, Remdesivir, Molnupiravir, Ribavirin, Favipiravir, Penciclovir, Entecavir and Tenofovir, we found that both Molnupiravir and Remdesivir showed the strong inhibition of SARS-CoV-2 RdRp, with EC50 value of 0.22 µM and 0.67 µM, respectively. Moreover, our results suggested that exoribonuclease nsp14 increases resistance of SARS-CoV-2 RdRp to nucleotide analog inhibitors. We also determined that Remdesivir presented the highest resistance to viral exoribonuclease activity in cells. Therefore, we have developed a cell-based SARS-CoV-2 RdRp assay which can be deployed to discover SARS-CoV-2 RdRp inhibitors that are urgently needed to treat COVID-19 patients.

Label-Free Screening of SARS-CoV-2 NSP14 Exonuclease Activity Using SAMDI Mass Spectrometry.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the global COVID-19 pandemic. Nonstructural protein 14 (NSP14), which features exonuclease (ExoN) and guanine N7 methyltransferase activity, is a critical player in SARS-CoV-2 replication and fidelity and represents an attractive antiviral target. Initiating drug discovery efforts for nucleases such as NSP14 remains a challenge due to a lack of suitable high-throughput assay methodologies. This report describes the combination of self-assembled monolayers and matrix-assisted laser desorption ionization mass spectrometry to enable the first label-free and high-throughput assay for NSP14 ExoN activity. The assay was used to measure NSP14 activity and gain insight into substrate specificity and the reaction mechanism. Next, the assay was optimized for kinetically balanced conditions and miniaturized, while achieving a robust assay (Z factor > 0.8) and a significant assay window (signal-to-background ratio > 200). Screening 10,240 small molecules from a diverse library revealed candidate inhibitors, which were counterscreened for NSP14 selectivity and RNA intercalation. The assay methodology described here will enable, for the first time, a label-free and high-throughput assay for NSP14 ExoN activity to accelerate drug discovery efforts and, due to the assay flexibility, can be more broadly applicable for measuring other enzyme activities from other viruses or implicated in various pathologies.

New targets for drug design: importance of nsp14/nsp10 complex formation for the 3'-5' exoribonucleolytic activity on SARS-CoV-2.

SARS-CoV-2 virus has triggered a global pandemic with devastating consequences. The understanding of fundamental aspects of this virus is of extreme importance. In this work, we studied the viral ribonuclease nsp14, one of the most interferon antagonists from SARS-CoV-2. Nsp14 is a multifunctional protein with two distinct activities, an N-terminal 3'-to-5' exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), both critical for coronaviruses life cycle, indicating nsp14 as a prominent target for the development of antiviral drugs. In coronaviruses, nsp14 ExoN activity is stimulated through the interaction with the nsp10 protein. We have performed a biochemical characterization of nsp14-nsp10 complex from SARS-CoV-2. We confirm the 3'-5' exoribonuclease and MTase activities of nsp14 and the critical role of nsp10 in upregulating the nsp14 ExoN activity. Furthermore, we demonstrate that SARS-CoV-2 nsp14 N7-MTase activity is functionally independent of the ExoN activity and nsp10. A model from SARS-CoV-2 nsp14-nsp10 complex allowed mapping key nsp10 residues involved in this interaction. Our results show that a stable interaction between nsp10 and nsp14 is required for the nsp14-mediated ExoN activity of SARS-CoV-2. We studied the role of conserved DEDD catalytic residues of SARS-CoV-2 nsp14 ExoN. Our results show that motif I of ExoN domain is essential for the nsp14 function, contrasting to the functionality of these residues in other coronaviruses, which can have important implications regarding the specific pathogenesis of SARS-CoV-2. This work unraveled a basis for discovering inhibitors targeting specific amino acids in order to disrupt the assembly of this complex and interfere with coronaviruses replication.

Development of a High-Throughput Screening Assay to Identify Inhibitors of the SARS-CoV-2 Guanine-N7-Methyltransferase Using RapidFire Mass Spectrometry.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) represents a significant threat to human health. Despite its similarity to related coronaviruses, there are currently no specific treatments for COVID-19 infection, and therefore there is an urgent need to develop therapies for this and future coronavirus outbreaks. Formation of the cap at the 5' end of viral RNA has been shown to help coronaviruses evade host defenses. Nonstructural protein 14 (nsp14) is responsible for N7-methylation of the cap guanosine in coronaviruses. This enzyme is highly conserved among coronaviruses and is a bifunctional protein with both N7-methyltransferase and 3'-5' exonuclease activities that distinguish nsp14 from its human equivalent. Mutational analysis of SARS-CoV nsp14 highlighted its role in viral replication and translation efficiency of the viral genome. In this paper, we describe the characterization and development of a high-throughput assay for nsp14 utilizing RapidFire technology. The assay has been used to screen a library of 1771 Food and Drug Administration (FDA)-approved drugs. From this, we have validated nitazoxanide as a selective inhibitor of the methyltransferase activity of nsp14. Although modestly active, this compound could serve as a starting point for further optimization.

Ritonavir may inhibit exoribonuclease activity of nsp14 from the SARS-CoV-2 virus and potentiate the activity of chain terminating drugs.

SARS-CoV-2is the causative agent for the ongoing COVID19 pandemic, and this virus belongs to the Coronaviridae family. The nsp14 protein of SARS-CoV-2 houses a 3' to 5' exoribonuclease activity responsible for removing mismatches that arise during genome duplication. A homology model of nsp10-nsp14 complex was used to carry out in silico screening to identify molecules among natural products, or FDA approved drugs that can potentially inhibit the activity of nsp14. This exercise showed that ritonavir might bind to the exoribonuclease active site of the nsp14 protein. A model of the SARS-CoV-2-nsp10-nsp14 complex bound to substrate RNA showed that the ritonavir binding site overlaps with that of the 3' nucleotide of substrate RNA. A comparison of the calculated energies of binding for RNA and ritonavir suggested that the drug may bind to the active site of nsp14 with significant affinity. It is, therefore, possible that ritonavir may prevent association with substrate RNA and thus inhibit the exoribonuclease activity of nsp14. Overall, our computational studies suggest that ritonavir may serve as an effective inhibitor of the nsp14 protein. nsp14 is known to attenuate the inhibitory effect of drugs that function through premature termination of viral genome replication. Hence, ritonavir may potentiate the therapeutic properties of drugs such as remdesivir, favipiravir and ribavirin.

Synthesis of adenine dinucleosides SAM analogs as specific inhibitors of SARS-CoV nsp14 RNA cap guanine-N7-methyltransferase.

The spreading of new viruses is known to provoke global human health threat. The current COVID-19 pandemic caused by the recently emerged coronavirus SARS-CoV-2 is one significant and unfortunate example of what the world will have to face in the future with emerging viruses in absence of appropriate treatment. The discovery of potent and specific antiviral inhibitors and/or vaccines to fight these massive outbreaks is an urgent research priority. Enzymes involved in the capping pathway of viruses and more specifically RNA N7- or 2'O-methyltransferases (MTases) are now admitted as potential targets for antiviral chemotherapy. We designed bisubstrate inhibitors by mimicking the transition state of the 2'-O-methylation of the cap RNA in order to block viral 2'-O MTases. This work resulted in the synthesis of 16 adenine dinucleosides with both adenosines connected by various nitrogen-containing linkers. Unexpectedly, all the bisubstrate compounds were barely active against 2'-O MTases of several flaviviruses or SARS-CoV but surprisingly, seven of them showed efficient and specific inhibition against SARS-CoV N7-MTase (nsp14) in the micromolar to submicromolar range. The most active nsp14 inhibitor identified is as potent as but particularly more specific than the broad-spectrum MTase inhibitor, sinefungin. Molecular docking suggests that the inhibitor binds to a pocket formed by the S-adenosyl methionine (SAM) and cap RNA binding sites, conserved among SARS-CoV nsp14. These dinucleoside SAM analogs will serve as starting points for the development of next inhibitors for SARS-CoV-2 nsp14 N7-MTase.

Identification of Phosphate-Containing Compounds as New Inhibitors of 14-3-3/c-Abl Protein-Protein Interaction.

The 14-3-3/c-Abl protein-protein interaction (PPI) is related to carcinogenesis and in particular to pathogenesis of chronic myeloid leukemia (CML). Previous studies have demonstrated that molecules able to disrupt this interaction improve the nuclear translocation of c-Abl, inducing apoptosis in leukemia cells. Through an X-ray crystallography screening program, we have identified two phosphate-containing compounds, inosine monophosphate (IMP) and pyridoxal phosphate (PLP), as binders of human 14-3-3σ, by targeting the protein amphipathic groove. Interestingly, they also act as weak inhibitors of the 14-3-3/c-Abl PPI, demonstrated by NMR, SPR, and FP data. A 37-compound library of PLP and IMP analogues was investigated using a FP assay, leading to the identification of three further molecules acting as weak inhibitors of the 14-3-3/c-Abl complex formation. The antiproliferative activity of IMP, PLP, and the three derivatives was tested against K-562 cells, showing that the parent compounds had the most pronounced effect on tumor cells. PLP and IMP were also effective in promoting the c-Abl nuclear translocation in c-Abl overexpressing cells. Further, these compounds demonstrated low cytotoxicity on human Hs27 fibroblasts. In conclusion, our data suggest that 14-3-3σ targeting compounds represent promising hits for further development of drugs against c-Abl-dependent cancers.

A solid-phase method for synthesis of dimeric and trimeric ligands: Identification of potent bivalent ligands of 14-3-3σ.

Multivalent protein-protein interactions including bivalent and trivalent interactions play a critical role in mediating a wide range of biological processes. Hence, there is a significant interest in developing molecules that can modulate those signaling pathways mediated by multivalent interactions. For example, multimeric molecules capable of binding to a receptor protein through a multivalent interaction could serve as modulators of such interactions. However, it is challenging to efficiently generate such multimeric ligands. Here, we have developed a facile solid-phase method that allows for the rapid generation of (homo- and hetero-) dimeric and trimeric protein ligands. The feasibility of this strategy was demonstrated by efficiently synthesizing fluorescently-labeled dimeric peptide ligands, which led to dramatically increased binding affinities (~400-fold improvement) relative to a monomeric 14-3-3σ protein ligand.

PNPT1 Release from Mitochondria during Apoptosis Triggers Decay of Poly(A) RNAs.

Widespread mRNA decay, an unappreciated feature of apoptosis, enhances cell death and depends on mitochondrial outer membrane permeabilization (MOMP), TUTases, and DIS3L2. Which RNAs are decayed and the decay-initiating event are unknown. Here, we show extensive decay of mRNAs and poly(A) noncoding (nc)RNAs at the 3' end, triggered by the mitochondrial intermembrane space 3'-to-5' exoribonuclease PNPT1, released during MOMP. PNPT1 knockdown inhibits apoptotic RNA decay and reduces apoptosis, while ectopic expression of PNPT1, but not an RNase-deficient mutant, increases RNA decay and cell death. The 3' end of PNPT1 substrates thread through a narrow channel. Many non-poly(A) ncRNAs contain 3'-secondary structures or bind proteins that may block PNPT1 activity. Indeed, mutations that disrupt the 3'-stem-loop of a decay-resistant ncRNA render the transcript susceptible, while adding a 3'-stem-loop to an mRNA prevents its decay. Thus, PNPT1 release from mitochondria during MOMP initiates apoptotic decay of RNAs lacking 3'-structures.

PARN and TOE1 Constitute a 3' End Maturation Module for Nuclear Non-coding RNAs.

Poly(A)-specific ribonuclease (PARN) and target of EGR1 protein 1 (TOE1) are nuclear granule-associated deadenylases, whose mutations are linked to multiple human diseases. Here, we applied mTAIL-seq and RNA sequencing (RNA-seq) to systematically identify the substrates of PARN and TOE1 and elucidate their molecular functions. We found that PARN and TOE1 do not modulate the length of mRNA poly(A) tails. Rather, they promote the maturation of nuclear small non-coding RNAs (ncRNAs). PARN and TOE1 act redundantly on some ncRNAs, most prominently small Cajal body-specific RNAs (scaRNAs). scaRNAs are strongly downregulated when PARN and TOE1 are compromised together, leading to defects in small nuclear RNA (snRNA) pseudouridylation. They also function redundantly in the biogenesis of telomerase RNA component (TERC), which shares sequence motifs found in H/ACA box scaRNAs. Our findings extend the knowledge of nuclear ncRNA biogenesis, and they provide insights into the pathology of PARN/TOE1-associated genetic disorders whose therapeutic treatments are currently unavailable.