Biology subjects

Chane-Woon-Ming, B.

Publications and source records attributed to Chane-Woon-Ming, B..

6 recordsLinked to original sources

bioRxiv2025

Complete post-transcriptional modification profiles in individual Staphylococcus aureus tRNA species

Transfer RNAs play a critical role in protein synthesis by matching mRNA codons to their corresponding amino acids. Their post-transcriptional modifications, shaping structure, stability, and codon decoding, are now recognized as key regulators of translation and cellular adaptation, including in bacterial pathogens. Here, we provide a comprehensive analysis of tRNA modifications in Staphylococcus aureus using extensive oligonucleotide mass spectrometry and deep-sequencing methods, generating a high-confidence modification map for each individual tRNA species, including the non-proteogenic tRNAGly. While the overall tRNA modification landscape is conserved among Gram-positive bacteria, our data uncovered unexpected S. aureus-specific features. These include the absence of [m2A]37 in tRNAs despite the presence of the methyltransferase RlmN, a single multi-site DusB2 enzyme catalyzing all tRNA dihydrouridylation, and a dedicated pseudouridine synthase responsible for [{Psi}]32 formation. Besides, heterogenous modification patterns were observed in tRNALeu(UAA) and tRNALys(CAA), highlighting a complex interplay in anticodon hypermodification. Integration of ribosome profiling and Nanopore tRNA sequencing offered a global view of the decoding properties of the reduced S. aureus tRNA set, revealing efficient four-way wobble recognition, slower translation of rare codons by low abundant tRNAs, and distinctive decoding dynamics of Gly codons potentially influenced by the unusual modification status of tRNAGly(UCC). This work establishes a framework for future research aimed at dissecting the role of specific tRNA modifications in S. aureus physiology and pathogenesis.

molecular biology↗

bioRxiv2025

Extended Shine-Dalgarno motifs govern translation initiation in Staphylococcus aureus

Regulation of translation initiation is central to bacterial adaptation, but species-specific mechanisms remain poorly understood. We present high-resolution mapping of translation start sites in S. aureus, revealing distinct features of initiation alongside numerous unannotated small ORFs. Our analysis, combined with cryo-EM of a native mRNA-ribosome complex, shows that S. aureus relies on extended, start codon proximal Shine-Dalgarno (SD) interactions, creating specificity against phylogenetically distant bacteria. Several natural S. aureus initiation sites are not correctly decoded by E. coli ribosomes. We identify new and conserved non-canonical start codons, whose regulatory initiation sites contain these characteristic extended SD sequence motifs. Finally, we characterize a striking example of uORF-mediated translational control in S. aureus, demonstrating that translation of a novel small leader peptide modulates expression of a key biofilm regulator. The described mechanism involves codon rarity, ribosome pausing and arginine availability, linking nutrient sensing to biofilm formation in this major human pathogen. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/660503v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1ea5094org.highwire.dtl.DTLVardef@505be5org.highwire.dtl.DTLVardef@1c14ad6org.highwire.dtl.DTLVardef@118549c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

molecular biology↗

bioRxiv2023

Canonical and non-canonical contributions of human Dicer helicase domain in antiviral defense

In mammalian somatic cells, the relative contribution of RNAi and the type I interferon response during viral infection is unclear. The apparent inefficiency of antiviral RNAi might be due to self-limiting properties and mitigating co-factors of the key enzyme Dicer. In particular, the helicase domain of human Dicer appears to be an important restriction factor of its activity. We studied the involvement of several helicase-truncated mutants of human Dicer in the antiviral response. All deletion mutants displayed a PKR-dependent antiviral phenotype against certain viruses and one of them, Dicer N1, acted in a complete RNAi-independent manner. Transcriptomic analyses showed that many genes from the interferon and inflammatory response were upregulated in Dicer N1 expressing cells. We could show that some of these genes appear to be controlled by NF-kB and that blocking this pathway abrogated the antiviral phenotype of Dicer N1. Our findings highlight the crosstalk between Dicer, PKR, and the NF-kB pathway, and suggest that human Dicer may have repurposed its helicase domain to prevent basal activation of antiviral and inflammatory pathways.

molecular biology↗

bioRxiv2021

Determination of dsRNA interactome upon Sindbis virus infection in human cells identifies SFPQ as a critical proviral factor

Viruses are obligate intracellular parasites, which depend on the host cellular machineries to replicate their genome and complete their infectious cycle. Long double stranded (ds)RNA is a common viral by-product originating during RNA virus replication and is universally sensed as a danger signal to trigger the antiviral response. As a result, viruses hide dsRNA intermediates into viral replication factories and have evolved strategies to hijack cellular proteins for their benefit. The characterization of the host factors associated with viral dsRNA and involved in viral replication remains a major challenge to develop new antiviral drugs against RNA viruses. Here, we performed anti-dsRNA immunoprecipitation followed by mass spectrometry analysis to fully characterize the dsRNA interactome in Sindbis virus (SINV) infected human cells. Among the identified proteins, we characterized SFPQ (Splicing factor, proline-glutamine rich) as a new dsRNA-associated proviral factor upon SINV infection. We showed that SFPQ depletion reduces SINV infection in human HCT116 and SK-N-BE(2) cells, suggesting that SFPQ enhances viral production. We demonstrated that the cytoplasmic fraction of SFPQ partially colocalizes with dsRNA upon SINV infection. In agreement, we proved by RNA-IP that SFPQ can bind dsRNA and viral RNA. Furthermore, we showed that overexpression of a wild type, but not an RNA binding mutant SFPQ, increased viral infection, suggesting that RNA binding is essential for its positive effect on the virus. Overall, this study provides the community with a compendium of dsRNA-associated factors during viral infection and identifies SFPQ as a new proviral dsRNA binding protein.

microbiology↗

bioRxiv2020

Human Dicer helicase domain acts as an interaction platform to recruit PKR and dsRNA binding proteins during viral infection

The antiviral innate immune response mainly involves type I interferon (IFN) in mammalian cells. The contribution of the RNA silencing machinery remains to be established, but several recent studies indicate that the ribonuclease DICER can generate viral siRNAs in specific conditions. It has also been proposed that type I IFN and RNA silencing could be mutually exclusive antiviral responses. In order to decipher the implication of DICER during infection of human cells with alphaviruses such as the Sindbis virus and Semliki forest virus, we determined its interactome by proteomics analysis. We show that DICER specifically interacts with several double-stranded RNA binding proteins and RNA helicases during viral infection. In particular, proteins such as DHX9, ADAR-1 and the protein kinase RNA-activated (PKR) are enriched with DICER in virus-infected cells. We demonstrate that the helicase domain of DICER is essential for this interaction and that its deletion confers antiviral properties to DICER in an RNAi-independent, PKR-dependent, manner. Author summaryWhile RNAi has been recognized as an efficient antiviral defense system in organisms such as plants and insects, its physiological importance in mammals remains to be determined. DICER is an enzyme involved in cleaving long double-stranded RNAs and is essential for RNAi induction. Using mass spectrometry analysis, we determined its interactome in human cells and showed that RNA binding proteins such as PKR are specifically enriched upon infection with the Sindbis virus or the Semliki forest virus. We determined that the N terminal helicase domain of the DICER protein acts as a platform to recruit these factors during infection and that its deletion confers an antiviral activity to DICER.

microbiology↗

bioRxiv2019

High-throughput fluorescence-based screen identifies the neuronal microRNA miR-124 as a positive regulator of alphavirus infection

Micro (mi)RNAs are small regulatory RNAs, which act as guide for the RISC complex to modulate the expression of target genes. In addition to their role in maintaining essential physiological functions in the cell, miRNAs can also regulate viral infections. They can do so directly by targeting RNAs of viral origin or indirectly by targeting RNAs from the host and this can result in a positive or negative outcome for the virus. Here, we performed a miRNA genome-wide screen in order to identify cellular miRNAs involved in the regulation of arbovirus infection in human cells. We identified sixteen miRNAs showing a positive effect on the virus, among which a number of neuron-specific ones such as miR-124. We confirmed that overexpression of miR-124 increases Sindbis virus (SINV) viral production and that this effect is mediated by its seed sequence. We further demonstrated that the SINV genome possesses a binding site for miR-124-3p. Both inhibition of miR-124-3p or silent mutations to disrupt this binding site in the viral RNA abolished the positive regulation. We also proved that miR-124 inhibition reduces SINV infection in human differentiated neuronal cells. Finally, we showed that the proviral effect of miR-124 is conserved for other medically relevant alphaviruses. Indeed, inhibition of miR-124 expression reduces chikungunya virus (CHIKV) viral production in human cells. Altogether, our work expands the panel of positive regulation of the viral cycle by direct binding of host miRNAs to the viral RNA and provides new insights into the role of cellular miRNAs as regulators of alphavirus infection.\n\nSIGNIFICANCE STATEMENTArthropod-borne (arbo) viruses are part of a class of pathogens that are transmitted to their final hosts by insects. Because of climate change, the habitat of some of these insects, such as mosquitoes, is shifting, thereby facilitating the emergence of viral epidemics. Among the pathologies associated with arboviruses infection, neurological diseases like meningitis or encephalitis represent a significant health burden. Using a genome-wide miRNA screen, we identified the neuronal miR-124 as a positive regulator of the Sindbis and chikungunya alphaviruses. We also showed that this effect was in part direct, thereby opening novel avenues to treat alphaviruses infection.

molecular biology↗