Subnuclear compartmentalization of transiently expressed polyadenylated pri-microRNAs: processing at transcription sites or accumulation in SC35 foci.
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.
Pawlicki
Jan M
JM
Department of Pharmacology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06536, USA.
Steitz
Joan A
JA
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
D035683:MicroRNAs; D009687:Nuclear Proteins; D016337:RNA, Catalytic; D012261:Ribonucleoproteins; C073288:SRSF2 protein, human; D000068103:Serine-Arginine Splicing Factors