title: Subnuclear compartmentalization of transiently expressed polyadenylated pri-microRNAs: processing at transcription sites or accumulation in SC35 foci.
abstract: MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally regulate expression of their target messenger RNAs. We recently demonstrated that primary miRNA transcripts (pri-miRNAs) retained at transcription sites are processed with enhanced efficiency, suggesting that pri-miRNA processing is coupled to transcription in mammalian cells. We also observed that transiently expressed pri-miRNAs accumulate in nuclear foci with splicing factor SC35 and Microprocessor components, Drosha and DGCR8. Here, we show that pri-miRNAs containing a self-cleaving hepatitis delta ribozyme accumulate in the nucleoplasm after release from their transcription sites, but are not efficiently processed. Pri-miRNAs with ribozyme-generated 3' ends do not localize to SC35-containing foci, whereas cleaved and polyadenylated pri-miRNA transcripts with or without the pre-miRNA hairpin do. 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 is coupled to transcription. We discuss the implications of our findings relative to recent insights into miRNA biogenesis, mRNA metabolism, and the nuclear organization of gene expression.
authors.affiliation: Department of Pharmacology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06536, USA.
authors.forename: Jan M
authors.initials: JM
authors.lastname: Pawlicki
authors.forename: Joan A
authors.initials: JA
authors.lastname: Steitz
mesh_terms: D000818:Animals; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D012323:RNA Processing, Post-Transcriptional; D016337:RNA, Catalytic / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*; D000068103:Serine-Arginine Splicing Factors; D015398:Signal Transduction / Q000502:physiology; D014158:Transcription, Genetic
chemical_list: D035683:MicroRNAs; D009687:Nuclear Proteins; D016337:RNA, Catalytic; D012261:Ribonucleoproteins; C073288:SRSF2 protein, human; D000068103:Serine-Arginine Splicing Factors