The MicroRNA Biology of the Mammalian Nucleus-论文阅读讨论-ReadPaper - 轻松读论文 | 专业翻译 | 一键引文

DOI: 10.1038/mtna.2014.40

Thomas C. Roberts

Molecular therapy. Nucleic acids

Jan 2014

146被引用

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摘要原文

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. 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. Canonical miRNA Biogenesis and FunctionMicroribonucleic acids (microRNAs, miRNAs, miRs) are short (~22 nucleotide), single-stranded, genome-encoded RNA molecules. miRNAs are generated by the cleavage of precursor hairpins in two sequential processing reactions. Initially, miRNAs are transcribed as long primary-miRNA (pri-miRNA) transcripts which are cleaved in the nucleus by the enzyme DROSHA (Drosha) to liberate the precursor-miRNA (pre-miRNA) hairpin.1Lee Y Ahn C Han J Choi H Kim J Yim J et al.The nuclear RNase III Drosha initiates microRNA processing.Nature. 2003; 425: 415-419Crossref PubMed Scopus (3924) Google Scholar The pre-miRNA is subsequently exported from the nucleus in a process mediated by the karyopherin XPO5 (Exportin-5).2Lund E Güttinger S Calado A Dahlberg JE Kutay U Nuclear export of microRNA precursors.Science. 2004; 303: 95-98Crossref PubMed Scopus (2040) Google Scholar Further processing by the enzyme DICER1 (Dicer) in the cytoplasm removes the loop sequence from the hairpin to produce an RNA duplex analogous to a small interfering RNA (siRNA).3Bernstein E Caudy AA Hammond SM Hannon GJ Role for a bidentate ribonuclease in the initiation step of RNA interference.Nature. 2001; 409: 363-366Crossref PubMed Scopus (3733) Google Scholar The double-stranded RNA duplex is then passed to an Argonaute protein (e.g., AGO2, Argonaute-2) and one strand is discarded, leaving only the mature miRNA species.The primary function of miRNAs appears to be the execution of endogenous RNA interference (RNAi) (i.e., the post-transcriptional regulation of gene expression).4Filipowicz W Jaskiewicz L Kolb FA Pillai RS Post-transcriptional gene silencing by siRNAs and miRNAs.Curr Opin Struct Biol. 2005; 15: 331-341Crossref PubMed Scopus (435) Google Scholar The mature miRNA acts to guide the ribonucleoprotein complex RISC (RNA-induced silencing complex) to target mRNA transcripts at cytoplasmic Processing-bodies (P-bodies).5Liu J Valencia-Sanchez MA Hannon GJ Parker R MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies.Nat Cell Biol. 2005; 7: 719-723Crossref PubMed Scopus (952) Google Scholar,6Liu J Rivas FV Wohlschlegel J Yates 3rd JR Parker R Hannon GJ A role for the P-body component GW182 in microRNA function.Nat Cell Biol. 2005; 7: 1261-1266Crossref PubMed Scopus (507) Google Scholar Canonically, miRNAs function by binding in the 3′ untranslated region (3′ UTR) of a target mRNA, typically forming an imperfect duplex. While multiple mismatches are tolerated between a miRNA and its target, high complementarity in the miRNA “seed” region (i.e., nucleotides 2–7) is generally required for effective target recognition.7Lewis BP Shih IH Jones-Rhoades MW Bartel DP Burge CB Prediction of mammalian microRNA targets.Cell. 2003; 115: 787-798Abstract Full Text Full Text PDF PubMed Scopus (4171) Google Scholar The degree of complementarity between the miRNA and its cognate target determines the fate of the targeted RNA. High levels of complementarity result in cleavage at a specific nucleotide position via the “slicer” activity of AGO2.8Liu J Carmell MA Rivas FV Marsden CG Thomson JM Song JJ et al.Argonaute2 is the catalytic engine of mammalian RNAi.Science. 2004; 305: 1437-1441Crossref PubMed Scopus (2009) Google Scholar Lower levels of complementarity lead to gene silencing via alternate mechanisms. Early studies showed that miRNAs induced translational repression whereby protein expression was reduced while mRNA levels were unaffected. Subsequently, other studies have shown that miRNAs can also induce mRNA decay by slicer-independent mechanisms.9Zeng Y Yi R Cullen BR MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms.Proc Natl Acad Sci USA. 2003; 100: 9779-9784Crossref PubMed Scopus (715) Google Scholar,10Guo H Ingolia NT Weissman JS Bartel DP Mammalian microRNAs predominantly act to decrease target mRNA levels.Nature. 2010; 466: 835-840Crossref PubMed Scopus (3060) Google Scholar,11Lim LP Lau NC Garrett-Engele P Grimson A Schelter JM Castle J et al.Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs.Nature. 2005; 433: 769-773Crossref PubMed Scopus (3950) Google Scholar Importantly, there are numerous exceptions to the canonical modes of miRNA biogenesis and function.12Roberts TC Wood MJ Non-canonical microRNA biogenesis and function.Applied RNAi: From Fundamental Research to Therapeutic Applications. Caister Academic Press, Norfolk, UK.2014: pp. 18-pp. 42Google ScholarmiRNAs Are Present in the NucleusThe prevailing view is that miRNAs function to regulate mRNA stability and translation in the cytoplasm. However, multiple studies have detected miRNAs in the nuclear compartment. For example, miR-21 was detected in both nuclear and cytoplasmic HeLa cell extracts by northern blot as early as 2004.13Meister G Landthaler M Patkaniowska A Dorsett Y Teng G Tuschl T Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs.Mol Cell. 2004; 15: 185-197Abstract Full Text Full Text PDF PubMed Scopus (1428) Google Scholar Subsequently, Hwang and coworkers showed that miR-29b is predominantly nuclear localized in the nuclei of HeLa and 3T3 cells, whereas the related miRNA, miR-29a, is mainly located in the cytoplasm.14Hwang HW Wentzel EA Mendell JT A hexanucleotide element directs microRNA nuclear import.Science. 2007; 315: 97-100Crossref PubMed Scopus (540) Google Scholar A key difference between these two miRNAs is the presence of a hexanucleotide sequence (AGUGUU) at the 3′ terminus of miR-29b. Transplantation of this motif onto an siRNA targeted against luciferase was sufficient to bias its cellular localization towards the nucleus, thereby demonstrating that this sequence is a bona fide nuclear localization signal.14Hwang HW Wentzel EA Mendell JT A hexanucleotide element directs microRNA nuclear import.Science. 2007; 315: 97-100Crossref PubMed Scopus (540) Google ScholarSystematic analyses of the sub-cellular distribution of miRNAs suggest that the majority of miRNAs are present in both nuclear and cytoplasmic compartments.15Liao JY Ma LM Guo YH Zhang YC Zhou H Shao P et al.Deep sequencing of human nuclear and cytoplasmic small RNAs reveals an unexpectedly complex subcellular distribution of miRNAs and tRNA 3’ trailers.PLoS ONE. 2010; 5: e10563Crossref PubMed Scopus (231) Google Scholar,16Park CW Zeng Y Zhang X Subramanian S Steer CJ Mature microRNAs identified in highly purified nuclei from HCT116 colon cancer cells.RNA Biol. 2010; 7: 606-614Crossref PubMed Scopus (51) Google Scholar,17Jeffries CD Fried HM Perkins DO Nuclear and cytoplasmic localization of neural stem cell microRNAs.RNA. 2011; 17: 675-686Crossref PubMed Scopus (87) Google Scholar,18Khudayberdiev SA Zampa F Rajman M Schratt G A comprehensive characterization of the nuclear microRNA repertoire of post-mitotic neurons.Front Mol Neurosci. 2013; 6: 43Crossref PubMed Scopus (54) Google Scholar Importantly, the use of microarrays with probes that preclude the binding of pre-miRNA hairpins confirmed the presence of mature miRNA species in the nucleus (as opposed to DROSHA processing products).16Park CW Zeng Y Zhang X Subramanian S Steer CJ Mature microRNAs identified in highly purified nuclei from HCT116 colon cancer cells.RNA Biol. 2010; 7: 606-614Crossref PubMed Scopus (51) Google Scholar Small RNA sequencing of nuclear and cytoplasmic fractions from human nasopharyngeal carcinoma cells revealed substantial overlap between miRNAs found in either location (nuclear: 339 miRNAs, cytoplasmic: 324 miRNAs, 300 miRNAs overlap).15Liao JY Ma LM Guo YH Zhang YC Zhou H Shao P et al.Deep sequencing of human nuclear and cytoplasmic small RNAs reveals an unexpectedly complex subcellular distribution of miRNAs and tRNA 3’ trailers.PLoS ONE. 2010; 5: e10563Crossref PubMed Scopus (231) Google Scholar Similarly, Gagnon et al. reported that ~75% of cellular miRNAs are present in both the nucleus and the cytoplasm of HeLa cells.19Gagnon KT Li L Chu Y Janowski BA Corey DR RNAi factors are present and active in human cell nuclei.Cell Rep. 2014; 6: 211-221Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar Using a combination of microarray analysis and small RNA deep sequencing, Khudayberdiev et al. investigated nuclear miRNAs in rat primary cortical neurons.18Khudayberdiev SA Zampa F Rajman M Schratt G A comprehensive characterization of the nuclear microRNA repertoire of post-mitotic neurons.Front Mol Neurosci. 2013; 6: 43Crossref PubMed Scopus (54) Google Scholar In general, each miRNA was found to be approximately two- to fourfold less abundant in the nucleus relative to the cytoplasm, although two miRNAs (miR-25 and miR-92a) showed clear nuclear enrichment. Expression levels of miRNAs in the nucleus declined during the process of post-mitotic neuronal development18Khudayberdiev SA Zampa F Rajman M Schratt G A comprehensive characterization of the nuclear microRNA repertoire of post-mitotic neurons.Front Mol Neurosci. 2013; 6: 43Crossref PubMed Scopus (54) Google Scholar suggesting that nuclear miRNAs might be important for maintaining the undifferentiated state, and that the global down-regulation of nuclear miRNAs is important for cortical development.Conflicting results have been reported regarding the localization of miR-29b. Nuclear enrichment of miR-29b was confirmed in 5-8F cells15Liao JY Ma LM Guo YH Zhang YC Zhou H Shao P et al.Deep sequencing of human nuclear and cytoplasmic small RNAs reveals an unexpectedly complex subcellular distribution of miRNAs and tRNA 3’ trailers.PLoS ONE. 2010; 5: e10563Crossref PubMed Scopus (231) Google Scholar but not in HCT116 colorectal carcinoma cells16Park CW Zeng Y Zhang X Subramanian S Steer CJ Mature microRNAs identified in highly purified nuclei from HCT116 colon cancer cells.RNA Biol. 2010; 7: 606-614Crossref PubMed Scopus (51) Google Scholar suggesting that the machinery involved in the motif-dependent nuclear import of miR-29b is differentially active between cell-types. While Liao et al. identified a number of nuclear-enriched miRNAs in addition to miR-29b (e.g., miR-1, miR-15, miR-32, and miR-148a/b), the hexanucleotide motif identified by Hwang et al.14Hwang HW Wentzel EA Mendell JT A hexanucleotide element directs microRNA nuclear import.Science. 2007; 315: 97-100Crossref PubMed Scopus (540) Google Scholar was not found to be over-represented among these nuclear miRNAs,15Liao JY Ma LM Guo YH Zhang YC Zhou H Shao P et al.Deep sequencing of human nuclear and cytoplasmic small RNAs reveals an unexpectedly complex subcellular distribution of miRNAs and tRNA 3’ trailers.PLoS ONE. 2010; 5: e10563Crossref PubMed Scopus (231) Google Scholar indicating that distinct mechanisms are responsible for their differential sub-cellular localizations.Some miRNAs have also been found to colocalize with specific substructures within the nucleus. A study by Politz et al. utilizing in situ hybridization in rat L6 myoblasts found that miR-206 (which is primarily expressed in skeletal muscle and regulates the process of myogenic differentiation)20Kim HK Lee YS Sivaprasad U Malhotra A Dutta A Muscle-specific microRNA miR-206 promotes muscle differentiation.J Cell Biol. 2006; 174: 677-687Crossref PubMed Scopus (636) Google Scholar was expressed in the cytoplasm but was also concentrated in the granular component of the nucleolus.21Politz JC Zhang F Pederson T MicroRNA-206 colocalizes with ribosome-rich regions in both the nucleolus and cytoplasm of rat myogenic cells.Proc Natl Acad Sci USA. 2006; 103: 18957-18962Crossref PubMed Scopus (126) Google Scholar A follow-up miRNA profiling study showed that many mature and precursor miRNAs (mainly nonmuscle specific) localize to the nucleolus of rat myoblasts, suggesting that they may be involved in cellular housekeeping functions.22Politz JC Hogan EM Pederson T MicroRNAs with a nucleolar location.RNA. 2009; 15: 1705-1715Crossref PubMed Scopus (142) Google Scholar Additionally, variations in the nucleolar localization of miRNAs were observed between cells in the same cultures, suggesting that sub-nuclear miRNA localization may be a transient (and perhaps regulated) process. Remarkably, the induction of cell stress by the transfection/electroporation of foreign nucleic acids, or by infection with influenza A virus, induced a shift in the sub-cellular distribution of nucleolar miRNAs to a more cytoplasmic location in HeLa cells.23Li ZF Liang YM Lau PN Shen W Wang DK Cheung WT et al.Dynamic localisation of mature microRNAs in Human nucleoli is influenced by exogenous genetic materials.PLoS ONE. 2013; 8: e70869Crossref PubMed Scopus (46) Google Scholar Taken together, these studies suggest that (i) the nucleolus is a site of storage for miRNAs which remain inactive until released by cell stress, and/or (ii) that nucleolar miRNAs function in a manner distinct from the canonical cytoplasmic RISC paradigm. Given that the nucleolus is the site of ribosomal gene transcription, ribosomal RNA maturation and RNA editing, it is possible that nuclear miRNAs participate in the regulation of these processes, or are themselves subject to RNA editing.24Nishikura K Functions and regulation of RNA editing by ADAR deaminases.Annu Rev BioChem. 2010; 79: 321-349Crossref PubMed Scopus (812) Google Scholar,25Kawahara Y Zinshteyn B Sethupathy P Iizasa H Hatzigeorgiou AG Nishikura K Redirection of silencing targets by adenosine-to-inosine editing of miRNAs.Science. 2007; 315: 1137-1140Crossref PubMed Scopus (621) Google Scholar,26Humphreys DT Hynes CJ Patel HR Wei GH Cannon L Fatkin D et al.Complexity of murine cardiomyocyte miRNA biogenesis, sequence variant expression and function.PLoS ONE. 2012; 7: e30933Crossref PubMed Scopus (67) Google ScholarmiRNA Pathway Components Shuttle Between Cytoplasm and NucleusMultiple studies have identified components of the miRNA processing pathway in the nucleus. All four human Argonaute proteins and other RNAi factors (i.e., DICER1, TARBP2 and TNRC6A (GW182)) have been detected by western blot in nuclear lysates from a variety of human cell lines.19Gagnon KT Li L Chu Y Janowski BA Corey DR RNAi factors are present and active in human cell nuclei.Cell Rep. 2014; 6: 211-221Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar,27Robb GB Brown KM Khurana J Rana TM Specific and potent RNAi in the nucleus of human cells.Nat Struct Mol Biol. 2005; 12: 133-137Crossref PubMed Scopus (258) Google Scholar,28Rüdel S Flatley A Weinmann L Kremmer E Meister G A multifunctional human Argonaute2-specific monoclonal antibody.RNA. 2008; 14: 1244-1253Crossref PubMed Scopus (116) Google Scholar,29Chu Y Yue X Younger ST Janowski BA Corey DR Involvement of argonaute proteins in gene silencing and activation by RNAs complementary to a non-coding transcript at the progesterone receptor promoter.Nucleic Acids Res. 2010; 38: 7736-7748Crossref PubMed Scopus (135) Google Scholar Nuclear extracts retain the catalytic activities of AGO2 and DICER1 in vitro19Gagnon KT Li L Chu Y Janowski BA Corey DR RNAi factors are present and active in human cell nuclei.Cell Rep. 2014; 6: 211-221Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar,27Robb GB Brown KM Khurana J Rana TM Specific and potent RNAi in the nucleus of human cells.Nat Struct Mol Biol. 2005; 12: 133-137Crossref PubMed Scopus (258) Google Scholar suggesting that active RISC complexes are present in the nucleus. There have been conflicting reports regarding the composition of the nuclear RISC complex. Using fluorescence correlation spectroscopy, Ohrt et al. showed that two distinct forms of RISC exist in the nuclear and cytoplasmic compartments (nRISC and cRISC respectively).30Ohrt T Mütze J Staroske W Weinmann L Höck J Crell K et al.Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy reveal the cytoplasmic origination of loaded nuclear RISC in vivo in human cells.Nucleic Acids Res. 2008; 36: 6439-6449Crossref PubMed Scopus (144) Google Scholar The size of these complexes was markedly different. cRISC was determined to be ~3 MDa consistent with previous estimates,31Chendrimada TP Finn KJ Ji X Baillat D Gregory RI Liebhaber SA et al.MicroRNA silencing through RISC recruitment of eIF6.Nature. 2007; 447: 823-828Crossref PubMed Scopus (383) Google Scholar whereas nRISC was much smaller at ~150 kDa (which is close to the size of AGO2 alone). Asymmetric siRNA strand incorporation was found to be similar between cRISC and nRISC suggesting a common loading mechanism.30Ohrt T Mütze J Staroske W Weinmann L Höck J Crell K et al.Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy reveal the cytoplasmic origination of loaded nuclear RISC in vivo in human cells.Nucleic Acids Res. 2008; 36: 6439-6449Crossref PubMed Scopus (144) Google ScholarIn contrast, several studies have reported that Argonaute proteins form multi-protein complexes in the nucleus. The RNAi factors AGO2, TNRC6A, DICER1, and TARBP2 were shown to be directly associated by pairwise coimmunoprecipitation in HeLa cell nuclei.19Gagnon KT Li L Chu Y Janowski BA Corey DR RNAi factors are present and active in human cell nuclei.Cell Rep. 2014; 6: 211-221Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar All four proteins were detected in a high molecular weight fraction following size exclusion chromatographic separation of nuclear lysates, suggesting that the nuclear RNAi machinery forms an intact protein complex.19Gagnon KT Li L Chu Y Janowski BA Corey DR RNAi factors are present and active in human cell nuclei.Cell Rep. 2014; 6: 211-221Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar Similarly, TNRC6A and AGO2 were found to colocalize with miRNAs, and form punctate foci in the nucleus.32Nishi K Nishi A Nagasawa T Ui-Tei K Human TNRC6A is an Argonaute-navigator protein for microRNA-mediated gene silencing in the nucleus.RNA. 2013; 19: 17-35Crossref PubMed Scopus (106) Google Scholar In further support, it was shown that both AGO1 and AGO2 are present in the chromatin fraction of HeLa nuclear extracts, and coprecipitate with multiple RNAi factors (TNRC6A, TNRC6B, hnRNPs, RNA helicases) and epigenetic modifier proteins (CBX3, TIF1B, SUV39H1, EHMT2).33Ameyar-Zazoua M Rachez C Souidi M Robin P Fritsch L Young R et al.Argonaute proteins couple chromatin silencing to alternative splicing.Nat Struct Mol Biol. 2012; 19: 998-1004Crossref PubMed Scopus (204) Google ScholarThe translocation of endogenous miRNAs from cytoplasm to nucleus has been visualized directly by Földes-Papp et al. who utilized optoporation to selectively permeabilize single cells to superquencher molecular probes complementary to the mature form of miR-122.34Földes-Papp Z König K Studier H Bückle R Breunig HG Uchugonova A et al.Trafficking of mature miRNA-122 into the nucleus of live liver cells.Curr Pharm Biotechnol. 2009; 10: 569-578Crossref PubMed Scopus (66) Google Scholar miR-122 probes were initially localized to the cytoplasm, but fluorescence signal was also detected in the nucleus ~5 minutes post-optoporation, thereby demonstrating nuclear import.34Földes-Papp Z König K Studier H Bückle R Breunig HG Uchugonova A et al.Trafficking of mature miRNA-122 into the nucleus of live liver cells.Curr Pharm Biotechnol. 2009; 10: 569-578Crossref PubMed Scopus (66) Google Scholar Furthermore, the delivery of siRNAs directly to the cytoplasm by microinjection led to efficient silencing of the nuclear-retained RN7SK transcript, suggesting that RISC is loaded in the cytoplasm and subsequently shuttled to the nucleus.30Ohrt T Mütze J Staroske W Weinmann L Höck J Crell K et al.Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy reveal the cytoplasmic origination of loaded nuclear RISC in vivo in human cells.Nucleic Acids Res. 2008; 36: 6439-6449Crossref PubMed Scopus (144) Google Scholar In further support, multiple RISC loading factors (i.e., HSP90AA1, TSN, TSNAX, AHA1, FKBP4, CDC37, and PTGES3) are restricted to the cytoplasm, and in vitro loading of RISC with radiolabeled duplex RNAs is impaired in nuclear extracts.19Gagnon KT Li L Chu Y Janowski BA Corey DR RNAi factors are present and active in human cell nuclei.Cell Rep. 2014; 6: 211-221Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar Together these observations strongly suggest that RISC loading occurs exclusively in the cytoplasm prior to nuclear import. The spatial separation of miRNA processing and RISC loading between nucleus and cytoplasm permits tighter control of flux through the miRNA processing pathway, thereby avoiding toxicity associated with pathway oversaturation.35Grimm D Streetz KL Jopling CL Storm TA Pandey K Davis CR et al.Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways.Nature. 2006; 441: 537-541Crossref PubMed Scopus (1352) Google ScholarThe observations that (i) mature miRNAs are present in the nucleus, and (ii) nuclear RISC is deficient with respect to miRNA loading, necessitate the existence of cellular machinery for shuttling RISC across the nuclear envelope. Such mechanisms have been identified in C. elegans through genetic screens,36Guang S Bochner AF Pavelec DM Burkhart KB Harding S Lachowiec J et al.An Argonaute transports siRNAs from the cytoplasm to the nucleus.Science. 2008; 321: 537-541Crossref PubMed Scopus (238) Google Scholar and in Tetrahymena,37Noto T Kurth HM Kataoka K Aronica L DeSouza LV Siu KW et al.The Tetrahymena argonaute-binding protein Giw1p directs a mature argonaute-siRNA complex to the nucleus.Cell. 2010; 140: 692-703Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar but were unknown in mammalian cells until recently. The translocation of molecules through the Nuclear Pore Complex (NPC) is mediated by a family of proteins called the karyopherins (for example, the nuclear export of pre-miRNA hairpins by the karyopherin XPO5, as mentioned above).38Mosammaparast N Pemberton LF Karyopherins: from nuclear-transport mediators to nuclear-function regulators.Trends Cell Biol. 2004; 14: 547-556Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar Several studies have identified karyopherins that are involved in the transport of RISC between the nucleus and the cytoplasm in mammals. For example, Castanotto et al. showed that XPO1 (Exportin-1) facilitates the transport of mature miRNAs from the cytoplasm to the nucleus in human cells.39Castanotto D Lingeman R Riggs AD Rossi JJ CRM1 mediates nuclear-cytoplasmic shuttling of mature microRNAs.Proc Natl Acad Sci USA. 2009; 106: 21655-21659Crossref PubMed Scopus (86) Google Scholar Inhibition of XPO1 with Leptomycin B prevented the accumulation of miRNAs in the nucleus, and synthetic dicer substrate siRNAs competed with endogenous miRNAs for nuclear import. Furthermore, coimmunoprecipitation studies showed that XPO1 is associated with the RISC components (AGO1, AGO2, DICER1, RHA, TNRC6B) and the transcriptional regulators (EZH2, TOP2A, and MTA) suggesting that XPO1 may facilitate the import of intact protein complexes loaded with miRNAs.39Castanotto D Lingeman R Riggs AD Rossi JJ CRM1 mediates nuclear-cytoplasmic shuttling of mature microRNAs.Proc Natl Acad Sci USA. 2009; 106: 21655-21659Crossref PubMed Scopus (86) Google Scholar,40Till S Lejeune E Thermann R Bortfeld M Hothorn M Enderle D et al.A conserved motif in Argonaute-interacting proteins mediates functional interactions through the Argonaute PIWI domain.Nat Struct Mol Biol. 2007; 14: 897-903Crossref PubMed Scopus (184) Google Scholar Interestingly, the P-body-associated RNAi factor, TNRC6A, acts as a navigator protein that facilitates the shuttling of AGO2 to and from the nucleus in conjunction with XPO1.32Nishi K Nishi A Nagasawa T Ui-Tei K Human TNRC6A is an Argonaute-navigator protein for microRNA-mediated gene silencing in the nucleus.RNA. 2013; 19: 17-35Crossref PubMed Scopus (106) Google Scholar The TNRC6A protein contains both nuclear import and export signals and was shown to mediate nRISC export in an XPO1-dependent manner, suggesting that this karyopherin regulates both nuclear import and export of miRNAs.32Nishi K Nishi A Nagasawa T Ui-Tei K Human TNRC6A is an Argonaute-navigator protein for microRNA-mediated gene silencing in the nucleus.RNA. 2013; 19: 17-35Crossref PubMed Scopus (106) Google ScholarThe karyopherin IPO8 (Importin-8) has also been implicated in the nuclear import of miRNAs. In HeLa cells, IPO8 was found to associate with AGO2 in the nucleus and in P-bodies in the cytoplasm.41Weinmann L Höck J Ivacevic T Ohrt T Mütze J Schwille P et al.Importin 8 is a gene silencing factor that targets argonaute proteins to distinct mRNAs.Cell. 2009; 136: 496-507Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar siRNA-mediated knockdown of IPO8 resulted in a reduction of AGO2 in the nucleus while the total cellular concentration of AGO2 was unchanged, indicating a shift in the sub-cellular distribution of AGO2. Importantly, some residual AGO2 protein remained in the nucleus after IPO8 knockdown consistent with multiple redundant cellular pathways for Argonaute-nucleocytoplasmic shuttling.41Weinmann L Höck J Ivacevic T Ohrt T Mütze J Schwille P et al.Importin 8 is a gene silencing factor that targets argonaute proteins to distinct mRNAs.Cell. 2009; 136: 496-507Abstract Full Text Full Text PDF PubMed Scopus (251) Google ScholarSeveral lines of evidence suggest that sub-cellular miRNA localization may be determined by the location of their target transcript(s). For example, Berezhna et al. showed that an siRNA targeting RN7SK was predominantly localized to the nucleus, whereas an siRNA targeting the cytoplasmic hepatitis C virus replicon RNA was retained in the cytoplasm.42Berezhna SY Supekova L Supek F Schultz PG Deniz AA siRNA in human cells selectively localizes to target RNA sites.Proc Natl Acad Sci USA. 2006; 103: 7682-7687Crossref PubMed Scopus (76) Google Scholar Separately, Ahlenstiel et al. showed that complexes of AGO1 and an siRNA targeting the 5′ LTR region of the integrated HIV provirus were localized to the nucleus of HIV-infected cells but not in HIV-naïve cells.43Ahlenstiel CL Lim HG Cooper DA Ishida T Kelleher AD Suzuki K Direct evidence of nuclear Argonaute distribution during transcriptional silencing links the actin cytoskeleton to nuclear RNAi machinery in human cells.Nucleic Acids Res. 2012; 40: 1579-1595Crossref PubMed Scopus (53) Google Scholar Intriguingly, these studies suggest that siRNAs/miRNAs become “trapped” and accumulate in the specific sub-cellular compartment in which their cognate targets reside. Nuclear accumulation of Argonaute-bound small RNAs is also dependent on the duration of interaction with the target transcript as a RN7SK-targeted siRNA with central mismatches (which inhibit the slicer activity of AGO2 and more closely mimic endogenous miRNA structure) showed inc

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The MicroRNA Biology of the Mammalian Nucleus-论文阅读讨论-ReadPaper - 轻松读论文 | 专业翻译 | 一键引文 | 图表同屏 (2025)

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