Molecular mechanisms underlying efficient and regulated microRNA production

MicroRNAs (miRNAs) are noncoding RNAs with important roles in regulating gene expression. In studying the earliest nuclear steps of miRNA biogenesis, I observe that primary miRNA (pri-miRNA) transcripts retained at transcription sites due to deletion of 3'-end processing signals are converted more efficiently into precursor miRNAs (pre-miRNAs) than pri-miRNAs that are cleaved, polyadenylated, and released. Flanking exons, which also increase retention at transcription sites, likewise contribute to increased levels of intronic pri-miRNAs. Consistently, efficiently processed endogenous pri-miRNAs are enriched in chromatin-associated nuclear fractions. In contrast, pri-miRNAs that accumulate to high nuclear levels after release from transcription sites due to presence of a hepatitis delta ribozyme or a viral RNA element---the ENE of the Kaposi's sarcoma-associated herpesvirus polyadenylated nuclear RNA---are not efficiently processed to pre- or mature miRNAs. Together these results suggest that pri-miRNA processing is enhanced by coupling to transcription.;I also observed that exogenous pri-miRNAs unexpectedly localize to nuclear foci containing splicing factor SC35. Pri-miRNA/SC35 foci contain a number of proteins normally associated with SC35 domains, including ASF/SF2, PABII, and the prolyl isomerase, Pin1. In contrast, RNA polymerase II and PM/Scl-100 do not strongly colocalize with pri-miRNAs in SC35-containing foci. These data argue that pri-miRNA/SC35-containing foci are not major sites of pri-miRNA processing and that pri-miRNA processing occurs cotranscriptionally. I discuss the implications of these findings relative to recent insights into miRNA biogenesis, mRNA metabolism, and the nuclear organization of gene expression.