Cellular Localization and Processing of Primary Transcripts of Exonic MicroRNAs

Abstract

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.

Human natural killer cell microRNA: differential expression of MIR181A1B1 and MIR181A2B2 genes encoding identical mature microRNAs

“…For both MIR181A1B1 and MIR181A2B2 , pri-miRNA levels were relatively constant over many kb. This strongly suggests that the dominant forms of primiR-181ab1 and pri-miR-181ab2 are unspliced transcripts, as has been reported for other primiRNAs 25 . We confirmed and precisely mapped the MIR181A1B1 and MIR181A2B2 TSS using 5’RACE on freshly isolated NK cells with normal intact miRNA processing.…”

“…Variations in RT-qPCR signal strength may be due to unequal RNA degradation rates, differences in priming efficiency, or RNA secondary structure formation affecting reverse transcription efficiency. Some unspliced pri-miRNAs are present at higher levels than their spliced counterparts 25 and the relative constancy of pri-miR-181 levels across the transcriptional units suggests that the major forms are unspliced transcripts. Levels of pri-miR-181ab-1 gradually fell 67-fold in the interval 2-49 kb downstream of the mature miRNA sequences ( Fig.…”

“…This avoided artifacts that might be associated with manipulation of miRNA processing. miRNA can be the products of spliced or unspliced primary transcripts 6 , 25 . For both MIR181A1B1 and MIR181A2B2 , pri-miRNA levels were relatively constant over many kb.…”

Long noncoding RNAs as a novel component of the Myc transcriptional network

“…RNA concentration was measured with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), RNA integrity was assessed by 1% agarose electrophoresis. Nuclear and cytoplasmic fractions were separated from P493‐6 MycOFF cells by adding 200 μL lysis buffer (140 mM NaCl, 1.5 mM MgCl 2 , 10 mM Tris‐HCl pH8.0, 1 mM DTT, 0.5% Nonidet P‐40) to pellets of ~8 million cells, followed by 5 minutes incubation on ice and centrifugation for 3 minutes at 4°C and 100 g (19, 20). The supernatant was collected as the cytoplasmic fraction.…”

Role of bioinformatics in establishing microRNAs as modulators of abiotic stress responses: the new revolution

“…Two miRs, miR436, and miR444, were mapped to the exonic regions of the protein-coding genes J023035E19 ( AK120922 ) and J033125N22 ( AK103332 ), respectively (Sunkar et al, 2005 ). It is hypothesized that the miRs control the host gene expression via a negative feedback loop mechanism that affects alternative splicing and cytoplasmic movement of transcripts (Slezak-Prochazka et al, 2013 ). Recently, CDC5 was identified as a MYB-related DNA binding protein that positively regulates miR production (Zhang et al, 2013a ) by binding to their promoters and through interaction with the RNase III enzyme DCL1 (Dicer-Like 1).…”