Background: H19 was one of the earliest identified, and is the most studied, long noncoding RNAs. It is presumed that H19 is essential for regulating development and disease conditions, and it is associated with carcinogenesis for many types. However the biological function and regulatory mechanism of this conserved RNA, particularly with respect to its effect on transcription, remain largely unknown.
Methods: We performed RNA pulldown, RNA immunoprecipitation and deletion mapping to identify the proteins that are associated with H19. In addition, we employed EU (5-ethynyl uridine) incorporation, immunoprecipitation and Western blotting to investigate the functional aspects of H19.
Results: Our research further verifies that H19 is bound to hnRNP U, and this interaction is located within the 5' 882 nt region of H19. Moreover, H19 disrupts the interaction between hnRNP U and actin, which inhibits phosphorylation at Ser5 of the RNA polymerase II (Pol II) C-terminal domain (CTD), consequently preventing RNA Pol II-mediated transcription. We also showed that hnRNP U is essential for H19-mediated transcription repression.
Conclusions: In this study, we demonstrate that H19 inhibits RNA Pol II-mediated transcription by disrupting the hnRNP U-actin complex.
General significance: These data suggest that H19 regulates general transcription and exerts wide-ranging effects in organisms.
Keywords: 5-Ethynyl uridine incorporation; 5-ethynyl uridine; C-terminal domain; C-terminal domain phosphorylation; CTD; EU; H19; HCC; IP; Pol II; RIP; RNA immunoprecipitation; RNA polymerase II subunit B1; Rpb1; Transcription repression; hepatocellular carcinoma; heterogeneous nuclear ribonucleoprotein; hnRNP; hnRNP U; immunoprecipitation; ncRNAs; noncoding RNAs; polymerase II.
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