The Role of the Exonic lncRNA PRKDC-210 in Transcription Regulation

Abstract

In recent years, long noncoding RNAs (lncRNAs) have received increasing attention and have been reported to be associated with various genetic abnormalities. However, the functions of many lncRNAs, including those of long exonic noncoding RNAs (lencRNAs), have not yet been elucidated. Here, we used a novel tethering luciferase assay to analyze the transcriptional regulatory functions of five lencRNAs that are upregulated in cancer. We found that the lencRNA PRKDC-210 interacts with MED12, a component of the CDK8 complex, to regulate the transcription of several genes. The transcriptional activation ability of PRKDC-210 was abolished in siRNA-treated CDK8-depleted cells. We also confirmed the enrichment of PRKDC-210 on RNA polymerase II. RNA-seq analysis of cells in which PRKDC-210 or PRKDC mRNA was knocked down using antisense oligonucleotides revealed that PRKDC-210 can affect the expression levels of genes related to fatty acid metabolism. Finally, we used a ChIRP assay to examine PRKDC-210-enriched sites in the genome. Overall, our findings demonstrate that the lencRNA PRKDC-210 promotes transcription through the CDK8 complex pathway at the transcription initiation site. We propose that PRKDC-210 can affect the transcription of adjacent genes after its transcription and splicing.

Keywords: CDK8; MED12; cancer; directed affect; lncRNA; tethering luciferase assay; transcription regulation.

Figure 1

(a) Schematic illustration of the tethering luciferase assay system. (b,c) Tethering luciferase assay using HEK293T cells, relative fluorescence and mRNA levels were detected. We used the empty MS2 plasmid instead of the MS2 lencRNA plasmid as negative control (neg.ctrl). Value = sample/neg.ctrl. (b) We used the Dual-Luciferase^® Reporter assay to detect the results of the tethering assay; the luciferase gene expression levels of different lencRNAs in the tethering or nontethering were compared. F/R, Firefly luciferase/Renilla luciferase. (c) Total RNA was extracted from cells transfected with tethering luciferase assay plasmid, and the differences of luciferase mRNA transcript between different lencRNAs were compared. Total RNA was reverse-transcribed into cDNA using random primers and quantified by qPCR using specific primer for luciferase gene. (d) The binding of different lencRNAs to MED12 in MCF7 cells was compared by RIP-qPCR assay. Value = sample/input. Data in all panels are represented as the mean  ±  s.e.m. of three independent experiments. * p  <  0.05 by a two-tailed Student’s t-test (compared with nontethering (a) or IgG (d)).

Figure 2

(a) This figure shows the expression of luciferase after tethering of PRKDC-210 in control siRNA-treated HEK293T cells and siCDK8-treated HEK293T cells. F/R, firefly luciferase/Renilla luciferase. (b) RIP-qPCR assays of MCF7 cells using antibodies targeting total RNA pol II, RNA pol II S5P, and RNA pol II S2P. The enrichment of PRKDC mRNA at different sites and the enrichment of PRKDC-210 in different phosphorylation of RNA Pol II are shown. Data in all panels are represented as the mean  ±  s.e.m. of three independent experiments. *** p  <  0.001 by a two-tailed Student’s t-test.

Figure 3

(a,b) Total RNA was extracted from MCF7 cells expressing control (ctrl) ASOs or ASOs targeting the PRKDC mRNA or PRKDC-210, and the RNA transcripts levels were detected via RT-qPCR. Kd means knockdown. Data are represented as the mean  ±  s.e.m. of three independent experiments. (c–g) RNA-seq analyses of PRKDC mRNA knockdown and PRKDC-210 knockdown cells. (c,d) FPKM profiles showing the overall gene expression levels. (e) The numbers of RNAs with altered transcript levels in PRKDC-210 knockdown cells versus control cells. (f) Genes that were significantly differentially expressed (fold change >2 and p < 0.05) in PRKDC-210 knockdown cells versus PRKDC mRNA knockdown cells. (g) Gene ontology biological process enrichment analysis.

Figure 4

MFC7 was used for all ChIRP assays. (a) RT-qPCR analyses of PRKDC-210 and ATP5B (negative control) levels in the RNA precipitated by ChIRP. (b–d) The DNAs associated with the immunoprecipitated RNAs in the ChIRP assay were analyzed by qPCR using specific primers. (b) The distribution of PRKDC-210 at the PRKDC locus. (c) Enrichment of PRKDC-210 at the TSSs of various genes. (d) Enrichment of PRKDC-210 at the TSSs of genes related to fatty acid metabolism. Data in all panels are represented as the mean  ±  s.e.m. of three independent experiments. *** p  <  0.001 by a two-tailed Student’s t-test.