Ubiquitous eukaryotic non-coding circular RNAs are involved in numerous co- and post-transcriptional regulatory mechanisms. Recently, we reported full-length intronic circular RNAs (flicRNAs) in Entamoeba histolytica, with 3′ss–5′ss ligation points and 5′ss GU-rich elements essential for their biogenesis and their suggested role in transcription regulation. Here, we explored how flicRNAs impact gene expression regulation. Using CLIP assays, followed by qRT-PCR, we identified that the RabX13 control flicRNA and virulence-associated flicRNAs were bound to the HA-tagged RNA Pol II C-terminus domain in E. histolytica transformants. The U2 snRNA was also present in such complexes, indicating that they belonged to transcription initiation/elongation complexes. Correspondingly, inhibition of the second step of splicing using boric acid reduced flicRNA formation and modified the expression of their parental genes and non-related genes. flicRNAs were also recovered from chromatin immunoprecipitation eluates, indicating that the flicRNA-Pol II complex was formed in the promoter of their cognate genes. Finally, two flicRNAs were found to be cytosolic, whose functions remain to be uncovered. Here, we provide novel evidence of the role of flicRNAs in gene expression regulation in cis, apparently in a widespread fashion, as an element bound to the RNA polymerase II transcription initiation complex, in E. histolytica.

Ubiquitous eukaryotic non-coding circular RNAs regulate transcription and translation. We have reported full-length intronic circular RNAs (flicRNAs) in Entamoeba histolytica with esterified 3′ss and 5′ss. Their 5′ss GU-rich elements are essential for their biogenesis and their suggested role in transcription regulation. Here, we explored whether exonic, exonic-intronic, and intergenic circular RNAs are also part of the E. histolytica and E. invadens ncRNA RNAome and investigated their possible functions. Available RNA-Seq libraries were analyzed with the CIRI-full software in search of circular exonic RNAs (circRNAs). The robustness of the analyses was validated using synthetic decoy sequences with bona fide back splice junctions. Differentially expressed (DE) circRNAs, between the virulent HM1:IMSS and the nonvirulent Rahman E. histolytica strains, were identified, and their miRNA sponging potential was analyzed using the intaRNA software. Respectively, 188 and 605 reverse overlapped circRNAs from E. invadens and E. histolytica were identified. The sequence composition of the circRNAs was mostly exonic although different to human circRNAs in other attributes. 416 circRNAs from E. histolytica were virulent-specific and 267 were nonvirulent-specific. Out of the common circRNAs, 32 were DE between strains. Finally, we predicted that 8 of the DE circRNAs could function as sponges of the bioinformatically reported miRNAs in E. histolytica, whose functions are still unknown. Our results extend the E. histolytica RNAome and allow us to devise a hypothesis to test circRNAs/miRNAs/siRNAs interactions in determining the virulent/nonvirulent phenotypes and to explore other regulatory mechanisms during amoebic encystment.

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 5ʹ‐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.