POINT-OF-VIEW RNA Biology 9:3, 1–5; March 2012; G 2012 Landes Bioscience miRNA goes nuclear Vera Huang and Long-Cheng Li Department of Urology and Helen-Diller Comprehensive Cancer Center, University of California—San Francisco; San Francisco, CA USA icroRNAs (miRNAs), defined as to target(s). miRNAs are convention- m 21–24 nucleotide non-coding ally regarded as negative regulators of RNAs, are important regulators of gene gene expression, mostly through post- expression. Initially, the functions of transcriptional events taking place in the miRNAs were recognized as post- cytoplasm. They are known to target transcriptional regulators on mRNAs complementary sequence on the mRNA that result in mRNA degradation and/or at different sites or on many different translational repression. It is becoming mRNAs through base-pairing between evident that miRNAs are not only the miRNA seed region and the 3' restricted to function in the cytoplasm, untranslated region (UTR) in the target they can also regulate gene expression in mRNA. It has been reported that other cellular compartments by a spec- miRNAs can also regulate gene expres- trum of targeting mechanisms via coding sion by targeting the 5' UTR,3 coding © 2012 Landesregions, 5' and 3'untransalated Bioscience. regions regions,4 promoters,5-8 and gene termini.9 (UTRs), promoters, and gene termini. In addition, miRNAs are predicted by In this point-of-view, we will speci- several genome-wide computational ana- fically focus on the nuclear functions lyses to target gene promoters because of miRNAs and discuss examples of potential targets for miRNAs are com- miRNA-directed transcriptional gene monly found based on sequence homo- regulation identified in recent years. logy in promoter sequences as in 3' Do not distribute.10,11 UTRs, with some targets highly complementary to miRNA sequences.8 Introduction Intriguingly, functional RISC activity and RNAi components were detected in microRNAs (miRNAs), a class of non- the nucleus12-14 and the mitochondria,15 coding small RNAs, play important roles suggesting that miRNAs also regulate in gene regulation and impact a myriad gene expression in cellular compartments of biological processes and diseases.1 Most other than the cytoplasm. A well-illustrated miRNAs are generated by the canonical example of small-RNA dependent trans- biogenesis pathway2 (Fig. 1). miRNA cription gene silencing through hetero- genes are transcribed by RNA polymerase chromatin formation came from fission II (RNAP II) into primary miRNA trans- yeast and plants.16 Deep sequencing Keywords: promoter-targeting miRNAs, cripts (pri-miRNAs), which are further analysis revealed a subset of miRNAs gene regulation, RNA activation, processed into miRNA precursors (pre- predominantly localized in the nucleus transcriptional gene silencing, miRNAs) in the nucleus by the micropro- of human cells and most miRNAs are Argonaute proteins, chromatin remodeling cessor complex Drosha/Dgcr8. Pre-miRNAs imported into the nucleus.17 It has also Submitted: 12/13/11 are then exported to the cytoplasm by been shown that nuclear-cytoplasmic Exportin 5 and converted into ~22-nt shuttling of miRNAs and RNAi com- Revised: 01/11/12 mature miRNAs by Dicer. Based on the ponents involve CRM1/Exportin 1 and Accepted: 01/13/12 thermodynamic properties, one of the Importin 818,19 (Fig. 1). Therefore, cyto- http://dx.doi.org/10.4161/rna.9.3.19354 strands is preferentially incorporated into plasmically processed miRNAs can be *Correspondence to: V Huang and L-C Li; the Argonaute (Ago) protein, a key com- imported into the nucleus to regulate Email: [email protected] ponent of the RNA-induced silencing gene expression. In support, a number of and [email protected] complex (RISC) complex, and guides it miRNAs have been recently identified to www.landesbioscience.com RNA Biology 1 regulate gene expression in the nucleus (RNAa) (see recent review by Portnoy can readily activate E-Cadherin (CDH1) by binding to the promoter of targeted et al.11). Mutation analysis showed that and cold-shock domain-containing protein genes.5-8 In our recent work, by using a dsRNAs with mismatches between dsRNA C2 (CSDC2), which contain putative combination of computational prediction and targeted promoter sequences retained miR-373 target sites with at least 80% and experimental validation, we identified the ability to induce gene expression,20,21 sequence complementarity in their pro- miRNAs highly complementary to pro- indicating that RNAa does not require moters. Furthermore, gene activation by moter sequences (also known as pro- perfect complementarity between the miR-373 is Dicer dependent and involves moter-targeting miRNAs) which can guide RNA and target sequences, a recruitment of RNAP II at the target activate gene expression in both human feature reminiscent of miRNA targeting promoter. and mouse cells.6,7 mRNA sequence. This observation led Kim et al.8 subsequently reported Promoter targeting miRNAs. We and to the hypothesis that endogenously another promoter-targeting miRNA, others showed that synthetic double- expressed miRNAs might also trigger miR-320. Computational analysis revealed stranded RNA (dsRNA) targeting gene RNAa. We supported this notion by that miR-320 is among one of the 9 promoters, also known as small activating identifying miR-373 as the first example mature miRNAs which exhibited perfect RNA (saRNA), activate gene expression of a promoter-targeting miRNA in human sequence complementarity with gene pro- via a process known as RNA activation cells7 (Table 1). We showed that miR-373 moters and is transcribed from the © 2012 Landes Bioscience. Do not distribute. 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. Pre-miRNAs are then exported to the cytoplasm by Exportin 5 (XPO5)/CRM1 and are processed into mature miRNAs by Dicer. One of the strands is preferentially incorporated into the Argonaute (Ago) protein, a component of the miRISC complex. (A) Classical function of miRNA-mediated post-transcriptional inhibition by 3 ‘UTR targeting. (B and C) In order for miRNAs to function in the nucleus, Ago-miRNA complex is imported into the nucleus by binding to Importin 8 (Imp8). Promoter-targeted miRNA complexed with Ago binds to chromosomal DNA sequences or nascent cognate transcripts derived from promoters. During RNAa, recruitment of chromatin modifying proteins (CMPs) leads to increased H3K4 methylation thereby activates transcription at the targeted promoter (B). In TGS, recruitment of CMPs leads to increased H3K9/K27 methylation thereby inhibits transcription at the targeted promoter (C). RNAa: RNA activation; TGS: transcriptional gene silencing; CMPs: chromatin modifying proteins; miRISC: miRNA-containing RNA induced silencing complex 2 RNA Biology Volume 9 Issue 3 Table 1. Promoter-targeting miRNAs miRNA Species Validated target promoter(s) Gene regulatory effect Reference miR-373 Human CDH1, CSDC2 Activation 7 miR-744, miR-1186, miR-466d-3p Mouse Ccnb1 Activation 6 miR-320 Human POLR3D Silencing 8 miR-423–5p Human PR, IGSF1 Silencing 5 miR-372, miR-373, miR-520c-3p Human PR Silencing 5 promoter of POLR3D gene. miR-320 proof-of-principle demonstration for small chromatin modifying proteins to activate levels correlated inversely with POLR3D RNA-mediated gene regulation studies at transcription (Fig. 1). expression in different tissues examined the well-characterized PR locus. However, Given the observation that mouse phy- and transfection of miR-320 mimics due to the lack of functional evidence of siological Ccnb1 expression depends on induced gene silencing of POLR3D, miR-423-5p, the endogenous functions of the miRNA pathway and the fact that implying that miR-320 targets the pro- this miRNA in mediating TGS still needs Ccnb1 is an essential protein that drives moter of POLR3D and directs transcrip- to be further examined in other cell types mitotic cell cycle entry, it is expected that tional gene silencing (TGS) of POLR3D and/or other physiological conditions. perturbation of such intricate relationship in cis (Table 1). Following transfection may have profound functional conse- of miR-320, enrichment of Ago1 and Ccnb1 Promoter-targeting miRNAs quences. Indeed, transient overexpression H3K27me3 was observed at the POLR3D of Ccnb1 promoter targeting miRNAs promoter. mR-320 also induced enrich- In our recent work by Huang et al.,6 we enhanced in vitro cell proliferation and ment of EZH2, a histone methyltrans- identified miRNAs (miR-744, miR-1186, promoted mitosis in the short-term. ferase, suggesting miR-320 mediated TGS miR-466d-3p) which are highly comple- Surprisingly, stable expression of these of POLR3D associated with epigenetic mentary to sites in the mouse Cyclin B1 miRNAs in mouse prostate cancer cells changes. (Ccnb1) promoter and can activate Ccnb1 disrupted global chromosome stability © 20125 Landes Bioscience. Very recently, Younger et al. identified expression (Table 1). In an attempt to and suppressed in vivo tumorigenecity. multiple exogenous miRNA mimics identify miRNAs that may have gene Collectively, this work provides the first (miR-423-5p, miR-372, miR-373, miR- activating roles in an endogenous context, example of physiologically relevant RNAa 520c-3p) that inhibit the expression of Ccnb1 came out of the initial screen as and demonstrated that miRNAs have progesterone receptor (PR), a locus well- one of the genes that were downregulated nuclear function to positively impact gene characterized for smallDo RNA mediated notby depletion ofdistribute. Drosha and Dicer. In silico transcription. What is more, cancer cells gene regulation (Table 1). Consistent with miRNA target prediction conducted on may exploit such mechanism to gain a their previous reports using perfectly a 1-kb promoter region of the mouse growth advantage. Identifying additional matched dsRNAs, they showed that TGS Ccnb1 gene identified 21 potential targets examples will provide insights into con- at the PR promoter mediated by miR-423- for 20 miRNAs.