MicroRNA-directed transcriptional gene silencing in mammalian cells

Abstract

MicroRNAs (miRNAs) regulate gene expression at the posttranscriptional level in the cytoplasm, but recent findings suggest additional roles for miRNAs in the nucleus. To address whether miRNAs might transcriptionally silence gene expression, we searched for miRNA target sites proximal to known gene transcription start sites in the human genome. One conserved miRNA, miR-320, is encoded within the promoter region of the cell cycle gene POLR3D in the antisense orientation. We provide evidence of a cis-regulatory role for miR-320 in transcriptional silencing of POLR3D expression. miR-320 directs the association of RNA interference (RNAi) protein Argonaute-1 (AGO1), Polycomb group (PcG) component EZH2, and tri-methyl histone H3 lysine 27 (H3K27me3) with the POLR3D promoter. Our results suggest the existence of an epigenetic mechanism of miRNA-directed transcriptional gene silencing (TGS) in mammalian cells.

Fig. 1.

miR-320 levels correlate inversely with POLR3D expression. (A) miR-320 is transcribed from the antisense strand of the POLR3D promoter region and is conserved in human, mouse, rat, and cow. (B–D) Mature miR-320 and POLR3D mRNA expression in human testis and brain tissues (B); bEnd.3, HeLa, and HEK-293 cells (C); and bEnd.3 and HeLa cells transfected with control or anti-miR-320 oligonucleotides (D), as measured by TaqMan miRNA assays and qRT-PCR, respectively, and normalized to GAPDH levels. Error bars indicate SD (n = 3).

Fig. 2.

miR-320 directs transcriptional silencing of POLR3D. (A) Mature miR-320 and POLR3D mRNA levels in HEK-293 cells transfected with control or miR-320 duplexes, as measured by TaqMan miRNA assays and qRT-PCR, respectively, and normalized to GAPDH levels (relative expression for miR-320 is indicated in hundreds). (B) Mature miR-21 and miR-29b miRNA levels in HEK-293 cells transfected with control or miR-320 duplexes, as measured by TaqMan miRNA assays and normalized to GAPDH levels. (C) Nuclear run-on experiments for nascent POLR3D mRNA transcription in HEK-293 cells transfected with control or miR-320 duplexes, as measured by qRT-PCR and normalized to nascent GAPDH mRNA transcription levels. (D) ChIP assays, using antibodies to AGO1, EZH2, or H3K27me3 or no antibody (NA) controls, in HEK-293 cells transfected with control or miR-320 duplexes. Quantification of immunoprecipitated APRT (control) and POLR3D promoter regions determined by real-time PCR and normalized to input DNA. Error bars indicate SD (n = 3).

Fig. 3.

miR-320 regulates POLR3D upon cell cycle arrest. Mature miR-320 and POLR3D mRNA levels in HEK-293 cells grown for 60 h in the presence of serum or under serum starvation conditions, transfected with control or anti-miR-320 oligonucleotides, as measured by TaqMan miRNA assays and qRT-PCR, respectively, and normalized to GAPDH levels. Error bars indicate SD (n = 3).

Fig. 4.

POLR3D promoter RNA transcript expression. Total RNA was prepared from HEK-293 cells grown for 60 h with serum (Asynchronous) or under serum starvation conditions (Quiescent), and levels of primary miR-320 and POLR3D promoter RNA transcript expression were measured using strand-specific qRT-PCR and normalized to GAPDH mRNA levels. Error bars indicate SD (n = 3).

Fig. 5.

Potential trans target genes of miRNAs complementary to promoters. (A) Potential genes with miRNA target sites −1 to −200 bp upstream of UCSC known human gene TSS. Putative miRNA target sites were searched from the sense and antisense strand sequences of gene promoter regions. “mm” refers to the number of mismatches between mature miRNA and target promoter sequences. (B) Most abundantly represented “biological process” gene ontology (GO) categories of annotated genes with putative miRNA target sites containing between 0 and 3 mismatches in gene promoter regions.