Graduate student Centro de Investigación y de Estudios Avanzados del IPN Mexico City, Mexico
Jesús Alberto García-Lerena (Centro de Investigación y de Estudios Avanzados del IPN)| M. Saraí Mendoza-Figueroa (University of Pennsylvania)| Eddy A. Alfonso-Maqueira (Florida International University)| Cristian J.C. Padrón-Manrique (Universidad Nacional Autónoma de México)| Odila Saucedo-Cárdenas (Universidad Autónoma de Nuevo León)| Elisa Azuara-Liceaga (Universidad Autónoma de la Ciudad de México)| C. Selene Zárate-Guerra (Universidad Autónoma de la Ciudad de México)| Nicolás Villegas-Sepúlveda (Centro de Investigación y de Estudios Avanzados del IPN)| Tomoyoshi Nozaki (University of Tokyo)| A. Méndez-Tenorio (Instituto Politécnico Nacional)| Jesús Valdés-Flores (Centro de Investigación y de Estudios Avanzados del IPN)
Entamoeba histolytica is the etiologic agent of amoebiasis which still causes over 55,000 annual deaths worldwide, and 2.2 million disability-adjusted life years. To differentiate E. histolytica from the non-pathogenic commensal E. dispar requires costly and laborious diagnostic tools. Apart from housekeeping ncRNA, the E. histolytica ncRNA inventory includes microRNAs and stress-induced self-circularized 5'-externally transcribed spacer rRNAs, therefore we searched for as many circRNA types as we could detect in this parasite with potential value for diagnostic purposes, as drug targets, and as markers between virulent vs non-virulent strains. CircRNAs are a type of transcripts widely conserved in eukaryotic cells and in E. histolytica we expected to identify potential circRNAs candidates for diagnosis. Using circular RT-PCR with divergent primers, we identified post-splicing derived full-length intronic circles (flicRNAs). In vivo splicing assays and several biochemical approaches revealed that the highly conserved GU-rich intronic 5’ss is required for flicRNA biogenesis, possibly slowing down intron lariat debranching enzyme activity, thus facilitating intron circularization. Similar approaches, and CLIP analyses using an HA-tagged Pol II CTD construct, showed that some flicRNAs interact with the transcription machinery and silence their parental genes in cis. We also found cytoplasmic flicRNAs whose functions remain to be uncovered. To expand our searches, we mined the available Entamoeba transcriptomes with the CIRIFull software, and identified 199 exonic and intergenic circular RNAs from the reptile parasite E. invadens, as well as 310 circRNAs from both virulent (HMI:IMSS) and avirulent (Rahman) E. histolytica strains (inclusion criteria: > 2 Back Splice Junctions, with Reverse Overlaps). A small proportion of the E. histolytica circRNAs were differentially expressed between strains, and some of these were validated by circular RT-PCR. Altogether, our data suggest that E. histolytica expresses a complex repertoire of circRNAs with diverse functions, involved in multiple biochemical pathways.
Support or Funding Information
Funding was provided by CONACYT (Grants 127557-M, 236104 and 864066) and SEP-CINVESTAV (Grant 601410 FIDSC2018/56)