microRNAs (miRNAs), a class of non-coding small RNAs, play important roles in gene regulation and impact a myriad of biological processes and diseases.1
Most miRNAs are generated by the canonical biogenesis pathway2
(). miRNA genes are transcribed by RNA polymerase II (RNAP II) into primary miRNA transcripts (pri-miRNAs), which are further processed into miRNA precursors (pre-miRNAs) in the nucleus by the microprocessor complex Drosha/Dgcr8. Pre-miRNAs are then exported to the cytoplasm by Exportin 5 and converted into ~22-nt mature miRNAs by Dicer. Based on the thermodynamic properties, one of the strands is preferentially incorporated into the Argonaute (Ago) protein, a key component of the RNA-induced silencing complex (RISC) complex, and guides it to target(s). miRNAs are conventionally regarded as negative regulators of gene expression, mostly through post-transcriptional events taking place in the cytoplasm. They are known to target complementary sequence on the mRNA at different sites or on many different mRNAs through base-pairing between the miRNA seed region and the 3′ untranslated region (UTR) in the target mRNA. It has been reported that miRNAs can also regulate gene expression by targeting the 5′ UTR,3
and gene termini.9
In addition, miRNAs are predicted by several genome-wide computational analyses to target gene promoters because potential targets for miRNAs are commonly found based on sequence homology in promoter sequences as in 3′ UTRs,10,11
with some targets highly complementary to miRNA sequences.8
Intriguingly, functional RISC activity and RNAi components were detected in the nucleus12-14
and the mitochondria,15
suggesting that miRNAs also regulate gene expression in cellular compartments other than the cytoplasm. A well-illustrated example of small-RNA dependent transcription gene silencing through heterochromatin formation came from fission yeast and plants.16
Deep sequencing analysis revealed a subset of miRNAs predominantly localized in the nucleus of human cells and most miRNAs are imported into the nucleus.17
It has also been shown that nuclear-cytoplasmic shuttling of miRNAs and RNAi components involve CRM1/Exportin 1 and Importin 818,19
(). Therefore, cytoplasmically processed miRNAs can be imported into the nucleus to regulate gene expression. In support, a number of miRNAs have been recently identified to regulate gene expression in the nucleus by binding to the promoter of targeted genes.5-8
In our recent work, by using a combination of computational prediction and experimental validation, we identified miRNAs highly complementary to promoter sequences (also known as promoter-targeting miRNAs) which can activate gene expression in both human and mouse cells.6,7
Figure 1. Actions of miRNAs in the nucleus. Canonical miRNAs are transcribed by RNA polymerase II (RNAP II) into primary miRNA transcripts (pri-miRNAs), which are further processed into miRNA precursors (pre-miRNAs) in the nucleus by Drosha/Dgcr8. (more ...)
Promoter targeting miRNAs
We and others showed that synthetic double-stranded RNA (dsRNA) targeting gene promoters, also known as small activating RNA (saRNA), activate gene expression via a process known as RNA activation (RNAa) (see recent review by Portnoy et al.11
). Mutation analysis showed that dsRNAs with mismatches between dsRNA and targeted promoter sequences retained the ability to induce gene expression,20,21
indicating that RNAa does not require perfect complementarity between the guide RNA and target sequences, a feature reminiscent of miRNA targeting mRNA sequence. This observation led to the hypothesis that endogenously expressed miRNAs might also trigger RNAa. We supported this notion by identifying miR-373 as the first example of a promoter-targeting miRNA in human cells7
(). We showed that miR-373 can readily activate E-Cadherin (CDH1) and cold-shock domain-containing protein C2 (CSDC2), which contain putative miR-373 target sites with at least 80% sequence complementarity in their promoters. Furthermore, gene activation by miR-373 is Dicer dependent and involves recruitment of RNAP II at the target promoter.
Table 1. Promoter-targeting miRNAs
Kim et al.8
subsequently reported another promoter-targeting miRNA, miR-320. Computational analysis revealed that miR-320 is among one of the 9 mature miRNAs which exhibited perfect sequence complementarity with gene promoters and is transcribed from the promoter of POLR3D gene. miR-320 levels correlated inversely with POLR3D expression in different tissues examined and transfection of miR-320 mimics induced gene silencing of POLR3D, implying that miR-320 targets the promoter of POLR3D and directs transcriptional gene silencing (TGS) of POLR3D in cis
(). Following transfection of miR-320, enrichment of Ago1 and H3K27me3 was observed at the POLR3D promoter. mR-320 also induced enrichment of EZH2, a histone methyltransferase, suggesting miR-320 mediated TGS of POLR3D associated with epigenetic changes.
Very recently, Younger et al.5
identified multiple exogenous miRNA mimics (miR-423-5p, miR-372, miR-373, miR-520c-3p) that inhibit the expression of progesterone receptor (PR), a locus well-characterized for small RNA mediated gene regulation (). Consistent with their previous reports using perfectly matched dsRNAs, they showed that TGS at the PR promoter mediated by miR-423-5p in trans is associated with recruitment of Ago2 to a non-coding RNA (ncRNA) transcript transcribed from the PR promoter. Similar to miR-373 which targets multiple promoters for transcriptional regulation,7
miR-423-5p can target additional genes which bear its targets within their promoters (). An increase in H3K9me2 was detected at the PR promoter, again, suggesting that epigenetic changes were associated with miRNA-induced TGS ().
The authors also evaluated the endogenous functions of miR-423-5p in PR regulation. However, they were unable to detect changes in PR gene expression following the expression of antisense RNAs against miR-423-5p in the two cell lines used in this study. The use of exogenous miRNA mimics allowed for proof-of-principle demonstration for small RNA-mediated gene regulation studies at the well-characterized PR locus. However, due to the lack of functional evidence of miR-423-5p, the endogenous functions of this miRNA in mediating TGS still needs to be further examined in other cell types and/or other physiological conditions.