Biology subjects

Vanacova, S.

Publications and source records attributed to Vanacova, S..

5 recordsLinked to original sources

bioRxiv2024

Global Analysis by LC-MS/MS of N6-Methyladenosine and Inosine in mRNA Reveals Complex Incidence

The precise and unambiguous detection and quantification of internal RNA modifications represents a critical step for understanding their physiological functions. The methods of direct RNA sequencing are quickly developing allowing for the precise location of internal RNA marks. This detection is however not quantitative and still presents detection limits. One of the biggest remaining challenges in the field is still the detection and quantification of m6A, m6Am and m1A modifications. The second intriguing and timely question remaining to be addressed is the extent to which individual marks are coregulated or potentially can affect each other. Here we present a methodological approach to detect and quantify several key mRNA modifications in human total RNA and in mRNA, which is difficult to purify way from contaminating tRNA. We show that the adenosine demethylase FTO primarily targets m6Am marks in noncoding RNAs in HEK293T cells. Surprisingly, we observe little effect of FTO or ALKBH5 depletion on the m6A mRNA levels. Interestingly, upregulation of ALKBH5 is accompanied by an increase in inosine level in overall mRNA.

molecular biology↗

bioRxiv2023

Beyond microRNAs: Analysis of chimeric reads characterises the diverse targetome of AGO2-mediated regulation.

Argonaute proteins are instrumental in regulating RNA stability and translation. AGO2, the major mammalian Argonaute protein, is known to primarily associate with microRNAs, a family of small RNA driver sequences, and identifies its targets primarily via a seed mediated partial complementarity process Despite numerous studies, a definitive experimental dataset of AGO2 driver-target interactions remains elusive. Our study employs two experimental methods - AGO2 CLASH and AGO2 eCLIP, to generate thousands of AGO2 target sites verified by chimeric reads. These chimeric reads contain both the AGO2 loaded small RNA driver and the target sequence, providing a robust resource for modeling AGO2 binding preferences. Our novel analysis pipeline reveals thousands of AGO2 target sites driven by microRNAs and a significant number of AGO2 drivers derived from fragments of other small RNAs such as tRNAs, YRNAs, snoRNAs, rRNAs, and more. We utilize convolutional neural networks to train machine learning models that accurately predict the binding potential for each driver class and experimentally validate several interactions. In conclusion, our comprehensive analysis of the AGO2 targetome broadens our understanding of its driver repertoire and potential function in development and disease. Moreover, we offer practical bioinformatic tools for future experiments and the prediction of AGO2 targets. All data and code from this study are freely available at https://github.com/ML-Bioinfo-CEITEC/HybriDetector/ Contactpanagiotis.alexiou@um.edu.mt, stepanka.vanacova@ceitec.muni.cz

molecular biology↗

bioRxiv2023

Cryo-electron tomography of enterovirus cell entry and endosome escape

Enveloped viruses deliver their genomes into the cell cytoplasm by membrane fusion; in contrast, membrane penetration by non-enveloped viruses is more diverse and less well understood. Enteroviruses, one of the largest groups of non-enveloped viruses, cause diseases ranging from the common cold to life-threatening encephalitis. To initiate infection, most enteroviruses enter cells by endocytosis. However, how enterovirus particles or RNA genomes cross the endosome membrane into the cytoplasm remains unknown. Here we used cryo-electron tomography of infected cells to show that endosomes containing rhinovirus 2, echovirus 18, echovirus 30, or enterovirus 71 deform, rupture, and release their content into the cytoplasm. Blocking endosome acidification with bafilomycin A1 reduced the number of enterovirus particles that released their genomes in endosomes, but did not prevent them from reaching the cytoplasm. Inhibiting N-WASP-mediated post-endocytic membrane remodeling with wiskostatin promoted abortive enterovirus genome release in endosomes. We show that the rupture of endosomes also occurs in uninfected cells. In summary, our results indicate that cellular membrane remodeling disrupts enterovirus-containing endosomes and thus releases the virus genomes and particles into the cytoplasm. Since the studied enteroviruses employ different receptors for cell entry but are all delivered into the cytoplasm by cell-mediated endosome disruption, it is possible that many other enteroviruses utilize endosome rupture to infect cells.

microbiology↗

bioRxiv2022

Human tRNA methyltransferase ALKBH8 targets wider repertoire of tRNAs and non-coding RNAs

Transfer RNAs acquire a large plethora of chemical modifications. Among those, modifications of the anticodon loop play important roles in translational fidelity and tRNA stability. Four human wobble U containing tRNAs obtain 5-methoxycarbonylmethyluridine (mcm5U34) or 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34), which play a role in decoding. This mark involves a cascade of enzymatic activities. The last step is mediated by Alkylation Repair Homolog 8 (ALKBH8). In this study, we performed a transcriptome-wide analysis of the repertoire of ALKBH8 RNA targets. Using a combination of HITS-CLIP-seq and RIP-seq analyses, we uncover ALKBH8-bound RNAs. It targets an additional wobble U-containing tRNA tRNALys(UUU). More interestingly, the spectrum of bound RNAs includes other types of non-coding RNAs, such as C/D box snoRNAs, 7SK RNA or some miRNAs, respectively.

molecular biology↗

bioRxiv2021

The mechanical regulation of RNA binding protein hnRNPC in the failing heart

Cardiac pathologies are characterized by intense remodeling of the extracellular matrix (ECM) that eventually leads to heart failure. Cardiomyocytes respond to the ensuing biomechanical stress by re-expressing fetal contractile proteins via transcriptional and post-transcriptional processes, like alternative splicing (AS). Here, we demonstrate that the heterogeneous nuclear ribonucleoprotein C (hnRNPC) is upregulated and relocates to the sarcomeric Z-disk upon ECM pathological remodeling. We show that this is an active site of localized translation, where the ribonucleoprotein associates to the translation machinery. Alterations in hnRNPC expression and localization can be mechanically determined and affect the AS of numerous mRNAs involved in mechanotransduction and cardiovascular diseases, like Hippo pathway effector YAP1. We propose that cardiac ECM remodeling serves as a switch in RNA metabolism by impacting an associated regulatory protein of the spliceosome apparatus. These findings offer new insights on the mechanism of mRNAs homeostasis mechanoregulation in pathological conditions.

pathology↗