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.
摘要：
无处不在的真核非编码环状RNA参与许多共同和转录后调控机制。最近,我们报道了溶组织内阿米巴的全长内含子环状RNA（flicRNAs），具有3个ss-5个ss连接点和5个富含ssGU的元件，这些元件对其生物发生及其在转录调节中的作用至关重要。这里,我们探索了flicRNAs如何影响基因表达调控。使用CLIP测定，然后是qRT-PCR，我们确定RabX13对照flicRNA和毒力相关的flicRNA结合到HA标记的RNAPolIIC末端结构域。U2snRNA也存在于这样的复合物中，表明它们属于转录起始/延伸复合物。相应地,使用硼酸抑制剪接的第二步可减少flicRNA的形成并修饰其亲本基因和非相关基因的表达。flicRNAs也从染色质免疫沉淀洗脱液中回收，这表明flicRNA-PolII复合物是在其同源基因的启动子中形成的。最后,发现两个flicRNA是细胞溶质的，其功能有待揭开。这里,我们为flicRNAs在顺式基因表达调控中的作用提供了新的证据，显然是以一种普遍的方式，作为与RNA聚合酶II转录起始复合物结合的元件，在溶组织大肠杆菌中。
