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Nuclear Micrornas and Their Unconventional Role in Regulating Non-Coding Rnas

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Nuclear Micrornas and Their Unconventional Role in Regulating Non-Coding Rnas Protein Cell 2013, 4(5): 325–330 DOI 10.1007/s13238-013-3001-5 Protein & Cell MINI-REVIEW Nuclear microRNAs and their unconventional role in regulating non-coding RNAs Hongwei Liang, Junfeng Zhang, Ke Zen, Chen-Yu Zhang, Xi Chen Jiangsu Engineering Research Center for microRNA Biology and Biotechnology, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, China Correspondence: [email protected] (X. Chen), [email protected] (C. Zhang) Received January 2, 2013 Accepted February 22, 2013 Cell ABSTRACT (Ambros, 2004; Bartel, 2004). The pri-miRNA is then cleaved & into a precursor miRNA (pre-miRNA; 60–80 nucleotides) by MicroRNAs (miRNAs) are small non-coding RNAs (ncR- the nuclear microprocessor complex formed by Drosha and NAs) that are involved in post-transcriptional gene regula- DGCR8 (Ambros, 2004; Bartel, 2004). The pre-miRNA is then tion. It has long been assumed that miRNAs exert their transported by Exportin-5 in a RanGTP-dependent manner roles only in the cytoplasm, where they recognize their into the cytoplasm, where it is further cleaved into a double- Protein target protein-coding messenger RNAs (mRNAs), and stranded miRNA/miRNA* duplex by Dicer (Ambros, 2004; result in translational repression or target mRNA degrada- Bartel, 2004). Finally, one strand of the duplex (mature miRNA) tion. Recent studies, however, have revealed that mature is incorporated into the RNA-induced silencing complex (RISC) miRNAs can also be transported from the cytoplasm to (Ambros, 2004; Bartel, 2004). It is generally accepted that miR- the nucleus and that these nuclear miRNAs can function NAs negatively regulate protein-coding gene expression at the in an unconventional manner to regulate the biogenesis post-transcriptional level, by a mechanism of either translation- and functions of ncRNAs (including miRNAs and long al repression or direct mRNA degradation (Ambros, 2004; Bar- ncRNAs), adding a new layer of complexity to our under- tel, 2004). In this manner, miRNAs play crucial regulatory roles standing of gene regulation. In this review, we summarize in a wide range of biological processes, including cellular pro- recent literature on the working model of these unconven- liferation and differentiation, migration, apoptosis, development tional miRNAs and speculate on their biological signifi- and metabolism (Ambros, 2004; Bartel, 2004). In addition, cance. We have every reason to believe that these novel altered expression of miRNAs contributes to the pathogenesis models of miRNA function will become a major research of many human diseases (Esquela-Kerscher and Slack, 2006; topic in gene regulation in eukaryotes. van Rooij and Olson, 2007; Tang et al., 2008). Recent studies suggest that miRNAs work in a much more KEYWORDS microRNA, nuclear microRNA, non-coding sophisticated way than was initially assumed. The fi nding that RNA, nucleus, Argonaute, Exportin, Importin significant amounts of mature miRNAs are localized in the nucleus (Meister et al., 2004; Politz et al., 2006; Hwang et al., 2007; Foldes-Papp et al., 2009; Liao et al., 2010; Jeffries et INTRODUCTION al., 2011) raises the interesting possibility that miRNAs may MicroRNAs (miRNAs) are a class of naturally occurring, single- function as gene regulators in the nucleus, through a mecha- stranded, small, non-coding RNAs (ncRNAs) of 22 nucleotides nism other than classic post-transcriptional repression. Indeed, in length that play pivotal roles in post-transcriptional gene recent studies by our (Tang et al., 2012) and other research regulation (Ambros, 2004; Bartel, 2004). Research during groups (Hansen et al., 2011; Zisoulis et al., 2012) demonstrate the past decade has identified major factors participating in that some miRNAs can act in an unconventional manner to miRNA biogenesis and has established the basic principles of regulate the biogenesis and function of ncRNAs, including miRNA function. In conventional linear processing pathways, miRNAs and long ncRNAs. These intriguing discoveries lead an miRNA gene is fi rst transcribed by RNA polymerase II or to a novel model of miRNA function and add new complexity RNA polymerase III into a primary miRNA transcript (pri-miRNA) to the gene regulatory network. In this review, we summarize © Higher Education Press and Springer-Verlag Berlin Heidelberg 2013 May 2013 | Volume 4 | Issue 5 | 325 MINI-REVIEW Hongwei Liang et al. recent literature on these unconventional miRNAs and hypoth- PRESENCE OF ARGONAUTE PROTEINS IN THE esize about their biological signifi cance. NUCLEUS Consistent with the observation that mature miRNAs are PRESENCE OF miRNAs IN THE NUCLEUS concentrated in the nucleus, studies have also reported the In the conventional pathway of miRNA processing, miRNAs localization of Argonaute proteins in the human cell nucleus. are transported out of the nucleus by the specific nuclear Robb et al. fi rst showed the presence of active Argonaute pro- transport receptor Exportin-5 and eventually mature in the cy- teins in nuclear compartments (Robb et al., 2005). In addition, toplasm, where they silence target gene expression by inhibit- Weinmann et al. detected AGO2 in the nucleus of HeLa cells ing protein translation or degrading mRNA molecules (Ambros, (Weinmann et al., 2009). Notably, it has been shown that Argo- 2004; Bartel, 2004). Thus, according to this canonical model, naute proteins are directly associated with nuclear miRNAs in miRNAs only exert their post-transcriptional regulatory roles in Caenorhabditis elegans (C. elegans) (Zisoulis et al., 2012) and the cytoplasmic compartment. However, evidence has accu- human cells (Hansen et al., 2011; Zisoulis et al., 2012). Be- mulated demonstrating that certain mature miRNAs can re-en- cause Argonaute proteins are the catalytic center of RISC, their ter the nucleus, and, in some cases, are even more abundant presence in the nucleus may imply that they have biological in the nucleus than in the cytoplasm (Meister et al., 2004; Politz relevance in the nucleus. We speculate that cells may transfer et al., 2006; Hwang et al., 2007; Foldes-Papp et al., 2009; Liao not only miRNAs but also miRNA effectors to the nucleus to et al., 2010; Jeffries et al., 2011). enhance the function of miRNAs. From this point of view, Ar- The fi rst miRNA identifi ed in the cell nucleus was miR-21. gonaute proteins may play a critical role in stabilizing miRNAs Cell Employing a Northern blotting assay, Meister et al. analyzed in the nucleus, and therefore only the miRNAs that are associ- & total RNA isolated from the nuclear and cytoplasmic fractions ated with Argonautes are stable and have biological function of HeLa cells and found that approximately 20% of mature after they enter into the nucleus, whereas the non-Argonaute- miR-21 was located in the nucleus (Meister et al., 2004). The bound miRNAs may be simply degraded in the nucleus. fi rst visible evidence for the existence of mature miRNAs in the nucleus was provided by Politz et al. (2006), who character- NUCLEAR-CYTOPLASMIC TRANSPORT OF Protein ized the intracellular localization of miR-206 in a rat myogenic miRNAs AND ARGONAUTE PROTEINS cell line through in situ hybridization. Their results indicated that miR-206 was not only distributed throughout the cytoplasm The above fi ndings strongly argue that there are some mecha- but also concentrated in the nucleus (Politz et al., 2006). Using nisms in eukaryotes to guide the cytoplasmic-nuclear transport high-throughput profi ling technology, such as microarrays and of miRNAs and Argonaute proteins. Generally, the nuclear–cy- deep sequencing, researchers compared the levels of mature toplasmic transport of proteins and RNAs through nuclear pore miRNAs in both cytoplasmic and nuclear fractions and reported complexes (NPCs) is mediated by Importins and Exportins that many miRNAs were located in both the cytoplasm and the (Kohler and Hurt, 2007; Katahira and Yoneda, 2011; Lee et al., nucleus (Liao et al., 2010; Jeffries et al., 2011). These results 2011). The interactions between transport factors and cargo indicated that the presence of mature miRNAs in the nucleus is molecules are regulated by the small GTPase Ran (Kohler a general phenomenon in mammalian cells. Foldes-Papp et al. and Hurt, 2007; Katahira and Yoneda, 2011; Lee et al., 2011). provided unequivocal proof for the transport of miRNAs from In the nucleus, Ran is present in the GTP-bound form, while in the cytoplasm to the nucleus. Applying superquencher mo- the cytoplasm, it is present in the GDP-bound form. Importins lecular beacon (SQMB) probes, they detected single-stranded and Exportins differ diametrically in the way they harness the miR-122 in living cells by hybridization without immobilization RanGTP gradient: Importins bind their cargo at a low RanGTP (Foldes-Papp et al., 2009). They observed that the binding level in the cytoplasm and release cargo upon binding to of fluorescent SQMB to miR-122, which initially occurred in RanGTP in the nucleus. In contrast, Exportins recruit their the cytoplasm, was increased in the nucleus of human liver cargo at high RanGTP levels in the nucleus, and the ternary cells in a time-dependent manner, implying the traffi cking of a RanGTP-Exportin-cargo complex passes through the NPC cytoplasmic miRNA into the cell nucleus (Foldes-Papp et al., into the cytoplasm, where GTPase activation triggers disas- 2009). Perhaps the most systematic studies in this fi eld were sembly of the export complex. Furthermore, different Importins performed by Hwang et al., who elucidated for the fi rst time and Exportins vary greatly in their substrate range (Kohler and one possible mechanism through which mature miRNAs could Hurt, 2007; Katahira and Yoneda, 2011; Lee et al., 2011). For re-enter the cell nucleus (Hwang et al., 2007). These authors example, pre-miRNAs are exported to the cytoplasm primarily showed that human miR-29b was predominantly localized in by Exportin-5, whereas tRNAs are exported by Exportin-t. the nucleus, whereas other miR-29 family members, such as Castanotto et al.
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