Cellular localization and processing of primary transcripts of exonic microRNAs.
Processing of miRNAs occurs simultaneous with the transcription and splicing of their primary transcripts. For the small subset of exonic miRNAs it is unclear if the unspliced and/or spliced transcripts are used for miRNA biogenesis. We assessed endogenous levels and cellular location of primary transcripts of three exonic miRNAs. The ratio between unspliced and spliced transcripts varied markedly, i.e. >1 for BIC, <1 for pri-miR-146a and variable for pri-miR-22. Endogenous unspliced transcripts were located almost exclusively in the nucleus and thus available for miRNA processing for all three miRNAs. Endogenous spliced pri-miRNA transcripts were present both in the nucleus and in the cytoplasm and thus only partly available for miRNA processing. Overexpression of constructs containing the 5' upstream exonic or intronic sequence flanking pre-miR-155 resulted in strongly enhanced miR-155 levels, indicating that the flanking sequence does not affect processing efficiency. Exogenously overexpressed full-length spliced BIC transcripts were present both in the nucleus and in the cytoplasm, were bound by the Microprocessor complex and resulted in enhanced miR-155 levels. We conclude that both unspliced and spliced transcripts of exonic miRNAs can be used for pre-miRNA cleavage. Splicing and cytoplasmic transport of spliced transcripts may present a mechanism to regulate levels of exonic microRNAs.
Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
Izabella
I
Slezak-Prochazka
Joost
J
Kluiver
Debora
D
de Jong
Gertrud
G
Kortman
Nancy
N
Halsema
Sibrand
S
Poppema
Bart-Jan
BJ
Kroesen
Anke
A
van den Berg
D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism*; D005091:Exons / Q000235:genetics*; D006801:Humans; D047468:Immunoprecipitation; D007438:Introns / Q000235:genetics; D016393:Lymphoma, B-Cell / Q000235:genetics*; D035683:MicroRNAs / Q000235:genetics*; D012322:RNA Precursors / Q000235:genetics*; D012323:RNA Processing, Post-Transcriptional; D012326:RNA Splicing / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013347:Subcellular Fractions; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
C542542:MIRN146 microRNA, human; C524942:MIRN155 microRNA, human; C548375:MIRN22 microRNA, human; D035683:MicroRNAs; D012322:RNA Precursors; D012333:RNA, Messenger