Promoter-Bound Full-Length Intronic Circular RNAs-RNA Polymerase II Complexes Regulate Gene Expression in the Human Parasite Entamoeba histolytica

Abstract

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.

In Silico Identification and Characterization of circRNAs as Potential Virulence-Related miRNA/siRNA Sponges from Entamoeba histolytica and Encystment-Related circRNAs from Entamoeba invadens

“…Furthermore, whereas human circRNAs originate from longer-than-average exons flanked by large introns [ 41 , 42 ], in both Entamoeba species, we observed no correlation between intron length or the number of introns per locus with the number of circRNAs produced in a given locus ( Table 2 and Table 3 , repository). Altogether, these data support their suggested role in gene or protein expression regulation, as has been demonstrated in the intronic flicRNAs [ 23 , 43 ].…”

“…The drug was added immediately after the denaturing step [ 55 ]. PCR products were amplified as described [ 43 ] and were resolved in 2.5% agarose gels.…”

Multiple types of nuclear localization signals in Entamoeba histolytica

“…As previously described for U1A (EHI_050780) [ 46 ] and RNA Polymerase ll (EHI_121760) [ 34 ], the protein-coding regions of U2AF1 (EHI_192500), and U2AF2 (EHI_098300) genes were amplified by PCR using E. histolytica genomic DNA as a template and their respective primer pairs (U2AF33Nf taacagatctATGACAGAAACAACAAAAAAAGAAGAAAC, and U2AF33Nr taacagatctTTTGTTGTCATAATATCTTCTTCTTGAAG; U2AF84Nf taacggatccATGGCAGGAAGGTATGATAGGTCTCG, and U2AF84Nr taacggatccTTCTTTTATTTCTTCTTCTGTTTTGAC). As previously described, amoeba transfectants were established and analyzed by confocal microscopy [ 34 , 46 ]. Ten to fourteen optical slices were obtained from the nuclei.…”

“…Recently, two monopartite-type NLSs have been identified in the major subunit of RNA Polymerase II towards its amino terminus [ 34 ]. HA-tagged constructs harboring full-length Pol II or the two NLSs fused to the C-terminal domain of Pol II were expressed in amoeba transformants.…”