Biology subjects

Guiguettaz, L.

Publications and source records attributed to Guiguettaz, L..

8 recordsLinked to original sources

bioRxiv2026

The rapid delivery of RNase H variants reveals distinct classes of RNA:DNA hybrids

Multiple lines of evidence suggest that RNase H1-sensitive RNA:DNA hybrids (thereafter referred to as hybrids) could disrupt the stability and restart of stalled replication forks. The prolonged over-expression of RNase H1 is the most popular method to manipulate hybrids in situ but it cannot discriminate between direct and indirect effects resulting from their loss. To overcome this limitation, we developed iCoRD (in Cellulo RNase H Delivery) to rapidly deliver comparable amounts of either Escherichia coli RNase H1 (RnhA) or control proteins in live cells, with a negligible impact on the transcriptome. iCoRD-delivered RnhA recognises hybrids in situ, is enriched in the vicinity of active replication forks and rescues replication fork progression in different stress conditions. While iCoRD-delivered RnhA had a clear impact on stress-induced, fork-proximal hybrids and R-ChIP signals, it had no effect on DRIP or S9.6 Cut&Tag signals, even when these were induced by stress. Our results strongly suggest the existence of distinct populations of hybrids that are best mapped by different methods and are differently sensitive to RNase H1 activity in situ.

molecular biology↗

bioRxiv2026

Viral Infections Drive Functional Remodeling of Ribosome-Associated Proteins

Viruses hijack host ribosomes to synthesize their proteins and have evolved various strategies to manipulate translation, including the association of numerous non-ribosomal proteins, known as ribosome-associated proteins (RAPs), with the core ribosome. However, current methods of capturing RAPs that associate transiently and/or in small amounts with ribosomes are suboptimal. To address this issue, we developed an enhanced ribosome affinity purification (eRAP) method that enables the capture of over 500 previously unreported RAPs. Using eRAP, we investigated whether the composition of ribosomes changes during infection by two very different viruses: herpes simplex virus 1 (HSV-1) and Sindbis virus (SINV). Our results revealed that viral infection does not substantially alter the composition of core ribosomes but does drive extensive remodeling of RAPs in a virus-specific manner. Functional analyses with SINV showed that several of these RAPs are critical for viral propagation. Additionally, we discovered that the RAP ASCC3 plays a novel role in promoting the expression of secretory proteins, including viral glycoproteins. This occurs independently of ASCC3s canonical function of rescuing ribosome collisions. Together, our findings establish the ribosome as a dynamic regulatory hub that undergoes remodeling to achieve virus-specific translational output, highlighting the functional significance of ribosomal heterogeneity.

molecular biology↗

bioRxiv2025

Rapid delivery of RNase H1 enables replication fork progression under stress independently of R-loop resolution

The over-expression of RNase H1 enables the progression of replication forks under stress and safeguards genome stability. The most common interpretation of these observations is that RNase H1 over-expression removes R-loops, whose formation would hinder replication fork progression. Here we challenge this model using an innovative strategy to rapidly deliver ready-made Escherichia coli RNase H1 (RnhA) in live cells. We show that RnhA is enriched in the vicinity of active replication forks and that it rescues replication fork progression in different stress conditions. However, RnhA had no impact on R-loops mapped by the DRIP or Cut&Tag methods. Our results reveal the existence of distinct populations of RNA:DNA hybrids that are differently sensitive to RnhA and invites new interpretations of well-established observations. HIGHLIGHTSO_LIRapid delivery of R-loop sensors and regulators in live cells C_LIO_LIRapid delivery of RnhA rescues replication fork progression in stress conditions C_LIO_LIRapid delivery of RnhA does not impact R-loops C_LIO_LIDRIP detects RNA:DNA species insensitive to RNase H1 in cells C_LI

molecular biology↗

bioRxiv2024

Unveiling novel conserved HIV-1 open reading frames encoding T cell antigens using ribosome profiling.

The development of ribosomal profiling (Riboseq) revealed the immense coding capacity of human and viral genomes. Here, we used Riboseq to delineate the first translatome of HIV-1 in infected CD4+ T cells. In addition to canonical viral protein coding sequences (CDSs), we identify 98 alternative open reading frames (ARFs), corresponding to small Open Reading Frames (sORFs) that are distributed across the HIV genome including the UTR regions. Using a database of HIV genomes, we observed that most ARF amino-acid sequences are likely conserved among clade B and C of HIV-1, with 8 ARF-encoded amino-acid sequences being more conserved than the overlapping CDSs. Using T cell-based assays and mass spectrometry-based immunopeptidomics, we demonstrate that ARFs encode viral polypeptides. In the blood of HIV-infected individuals, ARF-derived peptides elicit potent poly-functional T cell responses mediated by both CD4+ and CD8+ T cells. Our discovery expands the list of conserved viral polypeptides that are targets for vaccination strategies and might reveal the existence of viral microproteins or pseudogenes.

immunology↗

bioRxiv2023

Metabolism-dependent secondary effect of anti-MAPK cancer therapy on DNA repair

Amino acid bioavailability impacts mRNA translation in a codon depending manner. Here, we report that the anti-cancer MAPK inhibitors (MAPKi) decrease the intracellular concentration of aspartate and glutamate in melanoma cells. This results in the accumulation of ribosomes on codons corresponding to these amino acids and triggers the translation-dependent degradation of mRNAs encoding aspartate- and glutamate-rich proteins mostly involved in DNA metabolism. Consequently, cells that survive to MAPKi degrade aspartate and glutamate to generate energy, which simultaneously decreases their needs in amino acids owing to the downregulation of aspartate- and glutamate-rich proteins involved in cell proliferation. Concomitantly, the downregulation of aspartate- and glutamate-rich proteins involved in DNA repair increases DNA damage loads. Thus, DNA repair defects, and therefore mutations, are, at least in part, a secondary effect of the metabolic adaptation of cells exposed to MAPKi.

cancer biology↗

bioRxiv2023

The inflammatory and tumor suppressor SAMD9L acts through a Schlafen-like box to restrict HIV and inhibit cell translation in SAAD/ATXPC

Sterile alpha motif domain-containing proteins 9 and 9L (SAMD9/9L) are associated with life-threatening genetic diseases and are restriction factors of poxviruses. Yet, their cellular function and the extent of their antiviral role are poorly known. Here, we found that interferon-stimulated SAMD9L, and not SAMD9, restricts HIV-1 replication at the translation step, with a strong inhibition of Transmitted/Founder HIV-1 patient strains. More broadly, SAMD9L restricts primate lentiviruses, but not another retrovirus (MLV) or two ssRNA viruses (MOPV, VSV). Using structural modeling and mutagenesis of SAMD9L, we identified a Schlafen(SLFN)-like active site necessary for HIV-1 restriction. By testing a germline gain-of-function variant from patients with SAMD9L-associated autoinflammatory disease (SAAD) and ataxia-pancytopenia (ATXPC), we determined that SAMD9L cellular and pathogenic functions also depend on the SLFN-like active site. Finally, we propose a model in which SAMD9L translational repression could be dependent on codon-usage, linking its cellular function and the virus-specific innate immunity. The identification of another Achilles heel of HIV, as well as the inflammatory SAMD9L effector and auto-regulatory determinants, provide novel avenues against infectious and genetic diseases. Significance statementThis study identifies SAMD9L as a potent HIV-1 antiviral factor from the interferon immunity and deciphers the host determinants underlying SAMD9L translational repression. The characterization of SAMD9L activity and determinants is also of medical importance for patients with rare genetic diseases bearing deleterious mutations in SAMD9L or with specific cancers. We demonstrate that a pathogenic SAMD9L patients variant is inactivated by the mutation of an identified active site in a SLFN-like box, resulting in an abolished translational shutdown. Furthermore, we describe SAMD9L, but not SAMD9, as an antiviral factor of HIV and lentiviruses, through a translational repression mediated by the SLFN-like box and potentially dependent on codon usage. These findings may have implications to better fight against HIV/AIDS as well as SAAD/ATXPC. Key findings- SAMD9L, but not SAMD9, restricts HIV-1, including Transmitted/Founder patient strains. - SAMD9L broadly restricts primate lentiviruses, but not the retrovirus MLV, nor two ssRNA viruses, the Rhabdovirus VSV and the Arenavirus MOPV. - SAMD9L inhibits viral and cellular translation through an essential E198/D243 active site in a SLFN-like box. - The SAMD9L-associated autoinflammatory disease (SAAD) F886Lfs*11 variant has enhanced HIV translational repression, unveiling an autoregulatory domain of the anti-lentiviral function.

immunology↗

bioRxiv2022

Extensive uORF translation from HIV-1 transcripts conditions DDX3 dependency for expression of main ORFs and elicits specific T cell immune responses in infected individuals

Human immunodeficiency virus type-1 (HIV-1) is a complex retrovirus which relies on alternative splicing, translational and post-translational mechanisms to produce more than 15 functional proteins from its single [~]10kb transcriptional unit. Here, we have applied ribosome profiling and nascent protein labeling at different time points during infection of CD4+ T lymphocytes to characterize the translational landscape of cellular and viral transcripts during the course of infection. Our results indicate a strong impact of viral infection on host cellular transcript levels but a modest impact on global translation rates. Analysis of ribosome profiling reads from viral transcripts reveals extensive and productive non-AUG translation of small peptides from multiple upstream open reading-frames (uORFs) located in the 5 long terminal repeat. Remarkably, these uORFs derived peptides elicit specific T cell responses in HIV-infected individuals. uORFs are conserved among other retroviruses and, together with the TAR sequence, condition the dependency on DDX3 for efficient translation of the main viral open-reading frames.

microbiology↗

bioRxiv2020

Translation-dependent and independent mRNA decay occur through mutually exclusive pathways that are defined by ribosome density during T Cell activation.

mRNA translation and degradation are strongly interconnected processes that participate in the fine tuning of gene expression. Particularly, targeting mRNAs to translation-dependent degradation (TDD) could attenuate protein expression by making any increase in mRNA translation self-limiting. However, the extent to which TDD is a general mechanism for limiting protein expression is currently unknown. Here we describe a comprehensive analysis of basal and signal-induced TDD in mouse primary CD4 T cells. Our data indicate that most cellular transcripts are decayed to some extent in a translation-dependent manner, both in resting and activated cells. Our analysis further identifies the length of untranslated regions, the density of ribosomes and the GC content of the coding region as major determinants of TDD magnitude. Consistent with this, all transcripts that undergo changes in ribosome density upon T cell activation display a corresponding change in their TDD level. Surprisingly, the amplitude of translation-independent mRNA decay (TID) appears as a mirror image of TDD. Moreover, TID also responds to changes in ribosome density upon T cell activation but in the opposite direction from the one observed for TDD. Our data demonstrate a strong interconnection between mRNA translation and decay in mammalian cells. Furthermore, they indicate that ribosome density is a major determinant of the pathway by which transcripts are degraded within cells.

genomics↗