The MicroRNA Biology of the Mammalian Nucleus

Abstract

MicroRNAs (miRNAs) are a class of genome-encoded small RNAs that are primarily considered to be post-transcriptional negative regulators of gene expression acting in the cytoplasm. Over a decade of research has focused on this canonical paradigm of miRNA function, with many success stories. Indeed, miRNAs have been identified that act as master regulators of a myriad of cellular processes, and many miRNAs are promising therapeutic targets or disease biomarkers. However, it is becoming increasingly apparent that the canonical view of miRNA function is incomplete. Several lines of evidence now point to additional functions for miRNAs in the nucleus of the mammalian cell. The majority of cellular miRNAs are present in both the nucleus and the cytoplasm, and certain miRNAs show specific nuclear enrichment. Additionally, some miRNAs colocalize with sub-nuclear structures such as the nucleolus and chromatin. Multiple components of the miRNA processing machinery are present in the nuclear compartment and are shuttled back and forth across the nuclear envelope. In the nucleus, miRNAs act to regulate the stability of nuclear transcripts, induce epigenetic alterations that either silence or activate transcription at specific gene promoters, and modulate cotranscriptional alternative splicing events. Nuclear miRNA-directed gene regulation constitutes a departure from the prevailing view of miRNA function and as such, warrants detailed further investigation.

Figure 1

Nucleocytoplasmic shuttling of miRNAs. miRNAs are initially transcribed as long pri-miRNA transcripts in the nucleus. A complex of DGCR8 and DROSHA recognizes hairpin structures located on the pri-miRNA. DROSHA liberates the pre-miRNA by cleaving at the base of the hairpin (indicated by black arrowheads). Export of the pre-miRNA through the Nuclear Pore Complex (NPC) is mediated by the karyopherin XPO5. In the cytoplasm, the pre-miRNA is further processed by the RISC loading complex (RLC) which consists of an Argonaute protein (AGO2 is depicted here), DICER1, TARBP2, and other factors (which include the cytoplasm-restricted HSP90AA1, TSN, TSNAX, AHA1, FKBP4, CDC37, and PTGES3). The loop sequence of the hairpin is removed by DICER1 cleavage (indicated by black arrowheads). Subsequently, TARBP2 facilitates the loading of the RNA duplex into AGO2. The mature form of the miRNA is shown in red whereas the passenger strand (miRNA*) is shown in blue. Exogenous RNA duplexes (i.e., siRNAs) can also enter AGO2 at this stage in the processing pathway. One of the two strands is retained in AGO2 and the other degraded. The loaded cytoplasmic RISC (cRISC) complex, which contains the mature miRNA species and the silencing factor TNRC6A, is now capable of binding to cytoplasmic target transcripts. In the case of high miRNA-target complementarity AGO2 cleaves at the point indicated by the black arrowhead. Alternatively, cRISC can be imported into the nucleus by XPO1 or IPO8. TRNC6A acts as a navigator protein during this process. Nuclear RISC (nRISC) maintains a similar composition to cRISC although or may also exist as AGO2-miRNA complex alone (not depicted). The nRISC complex may bind additional nuclear factors or the Argonaute protein may form a distinct multi-protein complex (e.g., RITS, not depicted). nRISC binds to complementary nuclear transcripts or, in the absence of nuclear targets, be exported to the cytoplasm in a process facilitate by XPO1. Differential accumulation of miRNAs in the cytoplasm or nucleus is, in part, determined by the location of target transcripts.

Figure 2

Gene regulatory mechanisms of nuclear miRNAs. miRNAs can induce Post-Transcriptional Gene Silencing (PTGS) of a target transcript via the nuclear RNA Induced Silencing Complex (nRISC). (a) Silencing can occur via AGO2-mediated target slicing leading to transcript degradation (indicated by black arrowhead), or (b) by a slicer-independent mechanism. (c) Transcriptional Gene Silencing (TGS) occurs when a miRNA directs the RNA-Induced Transcriptional Silencing complex (RITS) to low-copy promoter RNA (pRNA) transcripts. RITS consists of chromatin remodeling activities (HDAC1, EHMT2, and EZH2) in addition to the DNA-methyltransferase, DNMT3A which facilitate the transition from a transcriptionally active chromatin structure to silent heterochromatin. Several putative mechanisms of Transcriptional Gene Activation (TGA) have been proposed. (d) At certain loci, lncRNAs silence gene expression by recruiting transcriptional repressors (e.g., Polycomb Repressive Complex 2, PRC2). miRNA-mediated silencing of the lncRNA disrupts the recruitment of silencing factors leading to activation of the target loci. (e) Alternatively, miRNAs may induce TGA by recruiting a protein complex containing transcriptional activators, as cleavage of the pRNA is not necessarily required for activation to occur. (f) miRNAs can influence alternate splicing decisions at specific exons. miRNA-mediated modulating of the chromatin landscape at the targeted exon effects the rate of RNAPII procession. Faster RNAPII procession through open chromatin promotes exon exclusion whereas slower RNAPII procession through compacted chromatin favors exon inclusion.