Biology subjects

Riquelme-Barrios, S.

Publications and source records attributed to Riquelme-Barrios, S..

5 recordsLinked to original sources

bioRxiv2025

Epitranscriptomic control of stress adaptations in Escherichia coli

Bacterial stress responses have been studied at the phenotypic, transcriptional, and translational levels, demonstrating the presence of an "alarm" phase immediately after stress exposure. However, the contributions of RNA modifications during stress adaptation remain largely unexplored. Here, we map the epitranscriptomic changes of Escherichia coli after exposure to oxidative and acid stress using direct RNA sequencing of mRNA, rRNA, and tRNA, combined with mass spectrometry, deletion mutant phenotyping, and single-nucleotide PCR. We identified widespread, dynamic RNA modifications that include central metabolism transcripts and increased levels of rRNA methylations (m4Cm and m5C) under both stresses, with potential consequences for translation. In uncharged tRNAs, stress-specific modifications via the Mnm and Q pathways accumulated at the wobble position; these modifications proved crucial for survival. Together, these findings reveal a multifaceted layer of post-transcriptional regulation, establishing the first comprehensive view of the bacterial epitranscriptome during the alarm phase of stress adaptation.

microbiology↗

bioRxiv2025

Detection of stress-dependent m5C rRNA dynamics in Escherichia coli using m5C-Rol-LAMP

Numerous RNA modifications are known in prokaryotes, but their dynamics and function in regulation remain largely unexplored. In Escherichia coli, three methyltransferases catalyze the 5-methylcytosine (m5C) modification in ribosomal RNA. Here, we introduce m5C-rolling circle loop-mediated isothermal amplification (m5C-Rol-LAMP) as a novel qPCR-based method that offers high sensitivity and site- specific resolution to detect and quantify mC in total RNA. When applying m5C-Rol-LAMP to E. coli under heat stress (45 {degrees}C), we observe a site-specific increase of mC at position 1407 of 16S rRNA from 77% to 89%, while m5C levels at positions 967 (16S) and 1962 (23S) remain unchanged. In recovered cells (at 37{degrees}C), the m5C abundance partially returns to the no stress level. Under oxidative stress, the level of mC1407 also increases, but remains high in recovered cells. These results demonstrate for the first time a reversible, stress-dependent and site-specific change in the rRNA modification level of a bacterium. m5C-Rol-LAMP is a powerful and easy-to-use tool for studying m5C in all RNA species, allowing the quantitative and site-specific detection of this modification.

microbiology↗

bioRxiv2024

Decoding the Escherichia coli epitranscriptome

Modifications of RNA, known as the epitranscriptome, affect gene expression, translation, and splicing in eukaryotes, with implications for developmental processes, cancer, and viral infections. In prokaryotes, regulation at the level of the epitranscriptome is still poorly understood. Here, we used Nanopore direct RNA sequencing of Escherichia coli to study RNA modifications and their changes under heat stress. With a single sequencing reaction, we detected most known modification types in rRNA, tRNA, and mRNA. RNA sequencing was complemented by a multifaceted approach that included mass spectrometry, deletion mutants, single-nucleotide PCR, and in vitro methylation. Known m5C and m6A sites in the rRNA were confirmed, but these types of modifications could not be localized in the mRNA. In response to heat stress, levels of m5C, m6A, and m6,6A increased in the 16S rRNA. Sequencing and mass spectrometry data demonstrated a decrease in tRNA modification abundance in the anticodon loop at 45 {degrees}C. In general, mRNA modifications at 37 {degrees}C were enriched in the coding regions of genes associated with general metabolism and RNA processing, which shifted to genes involved in cell wall synthesis and membrane transport under heat stress. This study provides new insights into the complexity of post-transcriptional regulation in bacteria. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/602490v2_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@11b9bc4org.highwire.dtl.DTLVardef@18f77feorg.highwire.dtl.DTLVardef@efdeaorg.highwire.dtl.DTLVardef@402859_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2019

CBP80/20-dependent translation initiation factor (CTIF) inhibits HIV-1 Gag synthesis by targeting the function of the viral protein Rev

Translation initiation of the human immunodeficiency virus type-1 (HIV-1) unspliced mRNA has been shown to occur through cap-dependent and IRES-driven mechanisms. Previous studies suggested that the nuclear cap-binding complex (CBC) rather than eIF4E drives cap-dependent translation of the unspliced mRNA and we have recently reported that the CBC subunit CBP80 supports the function of the viral protein Rev during nuclear export and translation of this viral transcript. Ribosome recruitment during CBC-dependent translation of cellular mRNAs relies on the activity CBP80/20 translation initiation factor (CTIF), which bridges CBP80 and the 40S ribosomal subunit through interactions with eIF3g. Here, we report that CTIF restricts HIV-1 replication by interfering with Gag synthesis from the unspliced mRNA. Our results indicate that CTIF associates with Rev through its N-terminal domain and is recruited onto the unspliced mRNA ribonucleoprotein complex in order to block translation. We also demonstrate that CTIF induces the cytoplasmic accumulation of Rev impeding the association of the viral protein with CBP80. We finally show that CTIF restricts HIV-2 but not MLV Gag synthesis indicating an inhibitory mechanism conserved in Rev-expressing human lentiviruses.

microbiology↗

bioRxiv2019

An epitranscriptomic switch at the 5′-UTR controls genome selection during HIV-1 genomic RNA packaging

During retroviral replication, the full-length RNA serves both as mRNA and genomic RNA (gRNA). While the simple retrovirus MLV segregates its full-length RNA into two functional populations, the HIV-1 full-length RNA was proposed to exist as a single population used indistinctly for protein synthesis or packaging. However, the mechanisms by which the HIV-1 Gag protein selects the two RNA molecules that will be packaged into nascent virions remain poorly understood. Here, we demonstrate that HIV-1 full-length RNA packaging is regulated through an epitranscriptomic switch requiring demethylation of two conserved adenosine residues present within the 5'-UTR. As such, while m6A deposition by METTL3/METTL14 onto the full-length RNA was associated with increased Gag synthesis and reduced packaging, FTO-mediated demethylation was required for the incorporation of the full-length RNA into viral particles. Interestingly, HIV-1 Gag associates with the RNA demethylase FTO in the nucleus and drives full-length RNA demethylation. Finally, the specific inhibition of the FTO RNA demethylase activity suppressed HIV-1 full-length RNA packaging. Together, our data propose a novel epitranscriptomic mechanism allowing the selection of the full-length RNA molecules that will be used as viral genomes.

molecular biology↗