Nuclear miRNAs: Gene Regulation Activities

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs which contribute to the regulation of many physiological and pathological processes. Conventionally, miRNAs perform their activity in the cytoplasm where they regulate gene expression by interacting in a sequence-specific manner with mature messenger RNAs. Recent studies point to the presence of mature miRNAs in the nucleus. This review summarizes current findings regarding the molecular activities of nuclear miRNAs. These molecules can regulate gene expression at the transcriptional level by directly binding DNA on the promoter or the enhancer of regulated genes. miRNAs recruit different protein complexes to these regions, resulting in activation or repression of transcription, through a number of molecular mechanisms. Hematopoiesis is presented as a paradigmatic biological process whereby nuclear miRNAs possess a relevant regulatory role. Nuclear miRNAs can influence gene expression by affecting nuclear mRNA processing and by regulating pri-miRNA maturation, thus impacting the biogenesis of miRNAs themselves. Overall, nuclear miRNAs are biologically active molecules that can be critical for the fine tuning of gene expression and deserve further studies in a number of physiological and pathological conditions.

Keywords: RNA processing; gene regulation; hematopoiesis; miRNAs; nuclear localization; transcriptional control.

Figure 1

Biogenesis of miRNAs and their return to the nucleus. The biogenesis of miRNAs is mediated by several steps. (1) miRNA genes are transcribed by RNA Pol-II or Pol-III into primary miRNAs (pri-miRNAs) and then cleaved by Drosha and DGCR8 into precursor miRNAs (pre-miRNAs). They are then exported into the cytoplasm with the help of Exportin 5 and RanGTP. (2) The pre-miRNAs are cleaved into miRNA duplexes by DICER in the cytoplasm and associated with the Argonaute proteins (AGO), forming the mature miRNA. (3) Part of the mature miRNAs are translocated into the nucleus with the help of Importin 8 and TNRC6. (4) AGO2 and TNRC6 can be transported into the cytoplasm via Exportin 1 (also called XPO1 or chromosomal region maintenance 1 or CRM1). miRNA, microRNA; RNA Pol-II/III, RNA polymerase II/III; pri-miRNA, miRNA primary transcript; pre-miRNA, miRNA precursor; AGO2, Argonaute 2; TNRC6A, trinucleotide repeat containing 6; TRBP, transactivation response-RNA-binding protein.

Figure 2

The activating function of nuclear miRNAs. (A) miRNA-mediated gene promoter regulation. (1) Direct interaction between miRNAs and complementary sequences on target gene promoters with the presence of AGO. This interaction allows an activator protein complex to be located in the vicinity of the targeted promoter region, whose chromatin structure is enriched with activator markers such as H3K4me3. RNA Pol-II is recruited and the gene is transcribed. (2) The miRNA-AGO-TBP complex interacts directly with the TATA box motif present on the promoter recruiting HMT and HAT. Chromatin is modified, activating markers such as H3K4me3, the H3ac and H4ac are increased, and RNA Pol-II is recruited to initiate transcription. (3) RNA Pol-II transcribes the promoter into promoter-associated RNA (pRNA). AGO mediates miRNA binding to pRNA, recruits HMT by producing active histone modifications, such as H3K4me3 with RNA polymerase II enrichment. (B) Transcription regulation through interaction with enhancers (1). A miRNA is transcribed from its gene located near enhancer loci. The mature miRNA forms a complex with AGO and p300/CBP, inducing active chromatin markers in the enhancer regions, and RNA Pol-II call. Thus, the enhancer is transcribed. (2) Next, the eRNA binds to p300 and other proteins to activate the target gene promoter. miRNAs, microRNAs; RNA Pol-II, RNA polymerase II; H3, histone H3; AGO, Argonaute 1 or Argonaute 2; TBP, TATA-Box-binding protein; eRNA, enhancer RNA; HAT, histone acetyltransferase; HMT, histone methyltransferase; pRNA, promoter-associated RNA.

Figure 3

The suppressive function of nuclear miRNAs. miRNAs can inhibit gene expression at transcriptional level. The miRNA-AGO1 complex recruits a protein complex consisting of YY1, DICER1, SUZ12, EZH2 that promotes inhibitory chromatin modifications with increased H3K27me3 and H3K9me3 markers. This can occur through direct binding to the promoter via sites complementary to the seed region (1) or through binding to the pRNA, a promoter associated non-coding transcript (2). In this way, miRNA leads to a decrease in RNA Pol-II and the suppression of target genes. YY1, Yin Yang 1 transcription factor; EZH2, Enhancer of Zeste Homolog 2; SUZ12, Suppressor of Zeste 12 protein homolog; TSS, transcription start site, pRNA, a promoter-associated RNA.

Figure 4

Graphical sketch of an experimental procedure used for the identification of nuclear miRNA genomic targets and epigenetic status at these sites. Transfection of fluorophore (i.e., Cy5)-labeled mimic miRNA allows for the visualization by confocal microscopy of its subcellular localization. In our model, Cy5-labeled mimic miR-223 (red signals) enters in the nucleus of myeloid cells after retinoic acid-induced differentiation. βtubulin (green signals) marks the cell cytosol [42]. Within this methodological approach, antibodies against Cy5 (α-Cy5) and against histone marks (α-H3K4me3 and α-H3K27me3) can be utilized to immunoprecipitate chromatin, enabling the identification across the whole genome through ChIP-sequencing of the complementary sequences targeted by nuclear miRNA and the epigenetic status at these sites. Portions of the figure were created with BioRender.com and Servier Medical Art, licensed under CC BY 4.0. For details, see text.