Biology subjects

De Matos, M.

Publications and source records attributed to De Matos, M..

4 recordsLinked to original sources

bioRxiv2025

Active site assembly by SMG5 as a mechanism for SMG6 endonuclease licensing in nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic surveillance pathway that eliminates transcripts containing premature termination codons (PTCs). While the recognition phase of NMD is well characterised, the effector step - how targeted mRNAs are nucleolytically degraded - remains incompletely understood. In metazoans, NMD employs an endonucleolytic route mediated by SMG6, a PIN-domain nuclease, alongside SMG5 and SMG7, which act downstream of PTC recognition. SMG5 has recently been proposed to license SMG6 activity, yet the molecular basis of this licensing has remained elusive. Here, we combine AlphaFold structural predictions with biochemical assays to investigate interactions among human SMG5, SMG6, and SMG7. Structural models predict a high-confidence interface between SMG5 and SMG6 PIN domains that forms a composite active site: a conserved SMG5 aspartate (D893) complements the SMG6 acidic triad to reinstate the canonical tetrad required for PIN-domain catalysis. In vitro, SMG6 alone exhibits weak endonucleolytic activity, which is enhanced [~]10-fold by SMG5 PIN. Mutational analyses confirm that conserved residues from both proteins are essential for this composite configuration. Our findings reveal that the SMG5 PIN domain, previously considered catalytically inert, plays a critical role in activating SMG6 by completing its active site. This work provides mechanistic insight into the SMG5-dependent licensing step and uncovers a composite PIN nuclease architecture at the heart of the metazoan NMD effector phase.

molecular biology↗

bioRxiv2024

An in vitro assay of MCTS1-DENR-dependent re-initiation and ribosome profiling uncover the activity of MCTS2 and distinct function of eIF2D

Ribosomes scanning from the mRNA 5' cap to the start codon may initiate at upstream open reading frames (uORFs), decreasing protein biosynthesis. Termination at a uORF can lead to re-initiation, where the 40S subunit resumes scanning and initiates another translation event downstream. In mammals, the noncanonical translation factors MCTS1-DENR participate in re-initiation at specific uORFs, but knowledge of other trans-acting factors and uORF features influencing re-initiation is limited. Here, we describe a cell-free re-initiation assay using HeLa cell lysates. Comparing in vivo and in vitro re-initiation activities on uORF-containing model reporters, we validate that MCTS1-DENR-dependent re-initiation is accurately recapitulated in vitro. Using this system and ribosome profiling in cultured cells, we found that knockdown of the homolog eIF2D causes widespread gene expression deregulation unrelated to uORF translation, suggesting distinct functions from MCTS1-DENR. Additionally, we identified MCTS2, encoded by a retrogene copy of Mcts1, as an alternative DENR partner that promotes re-initiation in vitro, providing a plausible explanation for the striking clinical differences associated with Denr vs. Mcts1 mutations in humans. Our findings on re-initiation and the new assay provide valuable insights and a powerful tool for future research on uORF features and trans-acting factors.

molecular biology↗

bioRxiv2019

Transcriptome-wide sites of collided ribosomes reveal principles of translational pausing

Translation initiation is the major regulatory step defining the rate of protein production from an mRNA. Meanwhile, the impact of non-uniform ribosomal elongation rates is largely unknown. Using a modified ribosome profiling protocol based on footprints from two closely packed ribosomes (disomes), we have mapped ribosomal collisions transcriptome-wide in mouse liver. We uncover that the stacking of an elongating onto a paused ribosome occurs frequently and scales with translation rate, trapping [~]10% of translating ribosomes in the disome state. A distinct class of pause sites, independent of translation rate, is indicative of deterministic pausing signals. Pause site association with specific amino acids, peptide motifs and nascent polypeptide structure, is suggestive of programmed pausing as a widespread mechanism associated with protein folding. Evolutionary conservation at disome sites indicates functional relevance of translational pausing. Collectively, our disome profiling approach allows unique insights into gene regulation occurring at the step of translation elongation.

cell biology↗

bioRxiv2018

Charting DENR-dependent translation reinitiation uncovers predictive uORF features and links to circadian timekeeping via Clock

The non-canonical initiation factor DENR promotes translation reinitiation on mRNAs harbouring upstream open reading frames (uORFs). Moreover, DENR depletion shortens circadian period in mouse fibroblasts, suggesting involvement of uORF usage and reinitiation in clock regulation. To identify DENR-regulated translation events transcriptome-wide and, in particular, specific core clock transcripts affected by this mechanism, we have used ribosome profiling in DENR-deficient NIH3T3 cells. We uncovered 240 transcripts with altered translation rate, and used linear regression analysis to extract 5 UTR features predictive of DENR dependence. Among core clock genes, we identified Clock as a DENR target. Using Clock 5 UTR mutants, we mapped the specific uORF through which DENR acts to regulate CLOCK protein biosynthesis. Notably, these experiments revealed an alternative downstream start codon, likely representing the bona fide CLOCK N-terminus. Our findings provide insights into uORF-mediated translational regulation that can regulate the mammalian circadian clock and gene expression at large.

genomics↗