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FEBS Open Bio. 2014 May 2;4:441-9. doi: 10.1016/j.fob.2014.04.010. eCollection 2014.

Small RNA expression and deep sequencing analyses of the nucleolus reveal the presence of nucleolus-associated microRNAs.

Author information

1
Department of Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins University School of Medicine, 1550 Orleans Street, Baltimore, MD 21287, USA.
2
Department of Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins University School of Medicine, 1550 Orleans Street, Baltimore, MD 21287, USA ; Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, 1550 Orleans Street, Baltimore, MD 21287, USA.

Abstract

Micro RNAs (miRNA) are non-coding RNAs expressed in the cytoplasm as their mature, 21-22-nucleotide short forms. More recently, mature miRNAs have also been detected in the nucleus, raising the possibility that their spatial distribution may be more complex than anticipated. Here we undertook comprehensive systematic analyses of miRNA distribution in several subcellular compartments of human cancer cells. In particular, we focused on the potential presence of miRNAs in the nucleolus, which contains an abundance of small non-coding RNAs. We employed two miRNA expression array platforms and small RNA deep sequencing of small RNAs isolated from cells, nuclei, cytoplasm and the nucleoli. We developed an assay to compare RNAs of isolated nucleoli before and after denaturation and used Northern hybridization to verify the presence of miRNAs in the subcellular compartments. Consistently, we found more than 10 miRNAs associated with the nucleolar preparations. Several miRNAs had greater relative abundance in the nucleolus compared to the other compartments. The nucleolar presence of miRNAs was independent of Dicer and the main activity of the nucleolus, RNA polymerase I transcription, but was dependent on CRM1 previously associated with nucleolar trafficking of small nucleolar RNAs. These results highlight the complexity of miRNA spatial arrangement and regulation.

KEYWORDS:

ActD, Actinomycin D; CRM1, chromosomal maintenance 1; Deep sequencing; LNA, locked nucleic acid; Nucleolus; Pol I, RNA polymerase I; Small RNA; miRNA expression profiling; pre, precursor; qPCR, quantitative real-time PCR; rRNA, ribosomal RNA; siRNA, short interfering RNA; snoRNA, small nucleolar RNA

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