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 'guide' sequences, and identifies its targets primarily via a 'seed' mediated partial complementarity process. Despite numerous studies, a definitive experimental dataset of AGO2 'guide'-'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 'guide' 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 'guides' 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 'guide' class and experimentally validate several interactions. In conclusion, our comprehensive analysis of the AGO2 targetome broadens our understanding of its 'guide' 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/ .

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 reexpressing fetal contractile proteins via transcriptional and posttranscriptional processes, such as alternative splicing (AS). Here, we demonstrate that the heterogeneous nuclear ribonucleoprotein C (hnRNPC) is up-regulated and relocates to the sarcomeric Z-disc upon ECM pathological remodeling. We show that this is an active site of localized translation, where the ribonucleoprotein associates with the translation machinery. Alterations in hnRNPC expression, phosphorylation, and localization can be mechanically determined and affect the AS of mRNAs involved in mechanotransduction and cardiovascular diseases, including Hippo pathway effector Yes-associated protein 1. We propose that cardiac ECM remodeling serves as a switch in RNA metabolism by affecting an associated regulatory protein of the spliceosome apparatus. These findings offer new insights on the mechanism of mRNA homeostatic mechanoregulation in pathological conditions.

Interplays of different types of epitranscriptomic mRNA modifications.

Eukaryotic mRNAs are modified by several chemical marks which have significant impacts on mRNA biology, gene expression, and cellular metabolism as well as on the survival and development of the whole organism. The most abundant and well-studied mRNA base modifications are m6A and ADAR RNA editing. Recent studies have also identified additional mRNA marks such as m6Am, m5C, m1A and Ψ and studied their roles. Each type of modification is deposited by a specific writer, many types of modification are recognized and interpreted by several different readers and some types of modifications can be removed by eraser enzymes. Several works have addressed the functional relationships between some of the modifications. In this review we provide an overview on the current status of research on the different types of mRNA modifications and about the crosstalk between different marks and its functional consequences.

Plasmodium berghei glycine cleavage system T-protein is non-essential for parasite survival in vertebrate and invertebrate hosts.

T-protein, an aminomethyltransferase, represents one of the four components of glycine cleavage system (GCS) and catalyzes the transfer of methylene group from H-protein intermediate to tetrahydrofolate (THF) forming N(5), N(10)-methylene THF (CH2-THF) with the release of ammonia. The malaria parasite genome encodes T-, H- and L-proteins, but not P-protein which is a glycine decarboxylase generating the aminomethylene group. A putative GCS has been considered to be functional in the parasite mitochondrion despite the absence of a detectable P-protein homologue. In the present study, the mitochondrial localization of T-protein in the malaria parasite was confirmed by immunofluorescence and its essentiality in the entire parasite life cycle was studied by targeting the T-protein locus in Plasmodium berghei (Pb). PbT knock out parasites did not show any growth defect in asexual, sexual and liver stages indicating that the T-protein is dispensable for parasite survival in vertebrate and invertebrate hosts. The absence of P-protein homologue and the non-essentiality of T protein suggest the possible redundancy of GCS activity in the malaria parasite. Nevertheless, the H- and L-proteins of GCS could be essential for malaria parasite because of their involvement in α-ketoacid dehydrogenase reactions.

Purification of a recombinant histidine-tagged lactate dehydrogenase from the malaria parasite, Plasmodium vivax, and characterization of its properties.

Lactate dehydrogenase (LDH) of the malaria parasite, Plasmodium vivax (Pv), serves as a drug target and immunodiagnostic marker. The LDH cDNA generated from total RNA of a clinical isolate of the parasite was cloned into pRSETA plasmid. Recombinant his-tagged PvLDH was over-expressed in E. coli Rosetta2DE3pLysS and purified using Ni(2+)-NTA resin giving a yield of 25-30 mg/litre bacterial culture. The recombinant protein was enzymatically active and its catalytic efficiency for pyruvate was 5.4 × 10(8) min(-1) M(-1), 14.5 fold higher than a low yield preparation reported earlier to obtain PvLDH crystal structure. The enzyme activity was inhibited by gossypol and sodium oxamate. The recombinant PvLDH was reactive in lateral flow immunochromatographic assays detecting pan- and vivax-specific LDH. The soluble recombinant PvLDH purified using heterologous expression system can facilitate the generation of vivax LDH-specific monoclonals and the screening of chemical compound libraries for PvLDH inhibitors.

Malaria parasite-synthesized heme is essential in the mosquito and liver stages and complements host heme in the blood stages of infection.

Heme metabolism is central to malaria parasite biology. The parasite acquires heme from host hemoglobin in the intraerythrocytic stages and stores it as hemozoin to prevent free heme toxicity. The parasite can also synthesize heme de novo, and all the enzymes in the pathway are characterized. To study the role of the dual heme sources in malaria parasite growth and development, we knocked out the first enzyme, δ-aminolevulinate synthase (ALAS), and the last enzyme, ferrochelatase (FC), in the heme-biosynthetic pathway of Plasmodium berghei (Pb). The wild-type and knockout (KO) parasites had similar intraerythrocytic growth patterns in mice. We carried out in vitro radiolabeling of heme in Pb-infected mouse reticulocytes and Plasmodium falciparum-infected human RBCs using [4-(14)C] aminolevulinic acid (ALA). We found that the parasites incorporated both host hemoglobin-heme and parasite-synthesized heme into hemozoin and mitochondrial cytochromes. The similar fates of the two heme sources suggest that they may serve as backup mechanisms to provide heme in the intraerythrocytic stages. Nevertheless, the de novo pathway is absolutely essential for parasite development in the mosquito and liver stages. PbKO parasites formed drastically reduced oocysts and did not form sporozoites in the salivary glands. Oocyst production in PbALASKO parasites recovered when mosquitoes received an ALA supplement. PbALASKO sporozoites could infect mice only when the mice received an ALA supplement. Our results indicate the potential for new therapeutic interventions targeting the heme-biosynthetic pathway in the parasite during the mosquito and liver stages.