Telomerase reverse transcriptase regulates microRNAs

Abstract

MicroRNAs are small non-coding RNAs that inhibit the translation of target mRNAs. In humans, most microRNAs are transcribed by RNA polymerase II as long primary transcripts and processed by sequential cleavage of the two RNase III enzymes, DROSHA and DICER, into precursor and mature microRNAs, respectively. Although the fundamental functions of microRNAs in RNA silencing have been gradually uncovered, less is known about the regulatory mechanisms of microRNA expression. Here, we report that telomerase reverse transcriptase (TERT) extensively affects the expression levels of mature microRNAs. Deep sequencing-based screens of short RNA populations revealed that the suppression of TERT resulted in the downregulation of microRNAs expressed in THP-1 cells and HeLa cells. Primary and precursor microRNA levels were also reduced under the suppression of TERT. Similar results were obtained with the suppression of either BRG1 (also called SMARCA4) or nucleostemin, which are proteins interacting with TERT and functioning beyond telomeres. These results suggest that TERT regulates microRNAs at the very early phases in their biogenesis, presumably through non-telomerase mechanism(s).

Figure 1

Mature miRNAs are regulated by TERT. Fold changes in miRNA expression measured by sequencing in HeLa cells (a) and THP-1 cells (b). Bars highlighted with asterisks represent statistically-significant changes. In HeLa cells, the changes were measured using sh-TERT#1 (gray) and sh-TERT#2 (black). In THP-1 cells, the changes were measured using a siRNA targeting TERT.

Figure 2

Mature miRNAs and their precursors are downregulated upon TERT suppression. The expression levels of primary, precursor and mature miRNAs in HeLa cells were analyzed with RT-qPCR under the suppression of TERT by sh-TERT#1 (gray) or sh-TERT#2 (black). The relative amount of the individual miRNAs was normalized to U6, and the mean expression levels of normalized sh-GFP (white) refer to one. (a,b) RT-qPCR of mature (a) and primary (b) miRNAs performed with total RNAs. The mRNA levels of ACTB and GAPDH were also examined with the same total RNAs; (c) RNAs smaller than 200 nt were used for RT-qPCR of mature (left) and precursor (right) miRNAs. Values represent the means ± SD for three independent experiments. Asterisks represent p < 0.05 compared with sh-GFP.

Figure 3

Comparable effects of TERT, BRG1 and NS in miRNA expression. The expression levels of mature miRNAs and miRNA precursors were examined with RT-qPCR upon suppression of TERT (top), BRG1 (middle) or NS (bottom) by gene-specific shRNAs. For miRNA precursors, there are the primers amplified primary form (^†) or both primary and precursor forms (^‡). The relative amount of the individual miRNAs was normalized to U6, and the mean expression levels in sh-GFP refer to one. Values represent the means ± SD for three independent experiments. Asterisks represent p < 0.05 compared with sh-GFP.