Biology subjects

Pillon, M. C.

Publications and source records attributed to Pillon, M. C..

5 recordsLinked to original sources

bioRxiv2026

Molecular architecture and assembly of the human RNA Degradosome

The PNPase exoribonuclease interacts with the SUV3 helicase to form the human RNA Degradosome responsible for processive mitochondrial RNA turnover and quality control. Although structural data reveals the trimeric PNPase architecture along with the monomeric and dimeric states of SUV3, the molecular basis of their assembly for processive RNA degradation remains poorly understood. Here we present the molecular characterization of the human RNA Degradosome and define critical molecular features that regulate complex formation. We identify the protein regions required for RNA Degradosome formation and establish the minimum components necessary for the SUV3-PNPase interaction. Chemical crosslinking mass spectrometry analysis reveals distance restraints that position the SUV3 N-terminal domain alongside of the PNPase S1 domain, defining an important interface for complex formation. These findings provide mechanistic insight into how PNPase and SUV3 assemble to form the human RNA Degradosome.

biochemistry↗

bioRxiv2026

Accurate Identification of Functional Residues Across the Human Proteome with TAMALE

The central challenge of the post-AlphaFold era is the functional gap. Despite having structure predictions for nearly every human protein, we remain unable to systematically distinguish residues that drive activity from those that merely maintain structural integrity. Here we introduce TAMALE, a machine learning model that calculates graded residue-level functional scores across the human proteome without prior annotation. TAMALE transforms structure models and variant effect predictions to identify residues involved in catalysis, ligand binding, nucleic acid interactions, and regulation. The model also distinguishes pseudo-enzymes from catalytically active homologs. The model was validated across 20 case studies along with experimental characterization of the FASTKD5 ribonuclease, demonstrating its utility for functional discovery. Applied proteome-wide to 19,528 human proteins, TAMALE generates testable hypotheses enabling mechanistic discovery at scale.

bioinformatics↗

bioRxiv2021

Characterization of SARS2 Nsp15 Nuclease Activity Reveals it's Mad About U

Nsp15 is a uridine specific endoribonuclease that coronaviruses employ to cleave viral RNA and evade host immune defense systems. Previous structures of Nsp15 from across Coronaviridae revealed that Nsp15 assembles into a homo-hexamer and has a conserved active site similar to RNase A. Beyond a preference for cleaving RNA 3 of uridines, it is unknown if Nsp15 has any additional substrate preferences. Here we used cryo-EM to capture structures of Nsp15 bound to RNA in pre- and post-cleavage states. The structures along with molecular dynamics and biochemical assays revealed critical residues involved in substrate specificity, nuclease activity, and oligomerization. Moreover, we determined how the sequence of the RNA substrate dictates cleavage and found that outside of polyU tracts, Nsp15 has a strong preference for purines 3 of the cleaved uridine. This work advances our understanding of how Nsp15 recognizes and processes viral RNA and will aid in the development of new anti-viral therapeutics.

biochemistry↗

bioRxiv2020

Cryo-EM Structures of the SARS-CoV-2 Endoribonuclease Nsp15

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 [A] 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.

biochemistry↗

bioRxiv2019

It takes two (Las1 HEPN Endoribonuclease Motifs) to cut the RNA right

Las1 is an essential endoribonuclease that is well-conserved across eukaryotes and a newly established member of the HEPN (higher eukaryotes and prokaryotes nucleotide-binding) nuclease family. HEPN nucleases participate in diverse RNA cleavage pathways and share a short HEPN nuclease motif important for RNA cleavage. While most HEPN nucleases participate in stress activated RNA cleavage pathways, Las1 plays a fundamental role in processing the pre-ribosomal RNA. Underscoring the significance of Las1 function, mutations to the LAS1L gene have been associated with neurological dysfunction. Two juxtaposed Las1 HEPN nuclease motifs create its composite nuclease active site, however the roles of the individual HEPN residues are poorly defined. Here we show through a combination of in vivo and in vitro studies that both Las1 HEPN nuclease motifs are required for nuclease activity and fidelity. Through in-depth sequence analysis and systematic mutagenesis, we define the consensus Las1 HEPN nuclease motif and uncover its canonical and specialized elements. Using reconstituted Las1 HEPN-HEPNchimeras, we define the molecular requirements for RNA cleavage. Intriguingly, both copies of the Las1 HEPN motif are necessary for nuclease specificity revealing that both HEPN motifs participate in coordinating the RNA within the active site. Taken together, our work reveals critical information about HEPN nuclease function and establishes that HEPN nucleases can be re-wired to cleave alternative RNA substrates.

molecular biology↗