Cytoplasmic and Nuclear Retained DMPK mRNAs Are Targets for RNA Interference in Myotonic Dystrophy Cells

Abstract

Small interfering RNA (siRNA) duplexes induce the specific cleavage of target RNAs in mammalian cells. Their involvement in down-regulation of gene expression is termed RNA interference (RNAi). It is widely believed that RNAi predominates in the cytoplasm. We report here the co-existence of cytoplasmic and nuclear RNAi phenomena in primary human myotonic dystrophy type 1 (DM1) cells by targeting myotonic dystrophy protein kinase (DMPK) mRNAs. Heterozygote DM1 myoblasts from a human DM1 fetus produce a nuclear retained mutant DMPK transcript with large CUG repeats (∼3,200) from one allele of the DMPK gene and a wild type transcript with 18 CUG repeats, thus providing for both a nuclear and cytoplasmic expression profile to be evaluated. We demonstrate here for the first time down-regulation of the endogenous nuclear retained mutant DMPK mRNAs targeted with lentivirus-delivered short hairpin RNAs (shRNAs). This nuclear RNAi(-like) phenomenon was not observed when synthetic siRNAs were delivered by cationic lipids, suggesting either a link between processing of the shRNA and nuclear import or a separate pathway for processing shRNAs in the nuclei. Our observation of simultaneous RNAi on both cytoplasmic and nuclear retained DMPK has important implications for post-transcriptional gene regulation in both compartments of mammalian cells. Small interfering RNA (siRNA) duplexes induce the specific cleavage of target RNAs in mammalian cells. Their involvement in down-regulation of gene expression is termed RNA interference (RNAi). It is widely believed that RNAi predominates in the cytoplasm. We report here the co-existence of cytoplasmic and nuclear RNAi phenomena in primary human myotonic dystrophy type 1 (DM1) cells by targeting myotonic dystrophy protein kinase (DMPK) mRNAs. Heterozygote DM1 myoblasts from a human DM1 fetus produce a nuclear retained mutant DMPK transcript with large CUG repeats (∼3,200) from one allele of the DMPK gene and a wild type transcript with 18 CUG repeats, thus providing for both a nuclear and cytoplasmic expression profile to be evaluated. We demonstrate here for the first time down-regulation of the endogenous nuclear retained mutant DMPK mRNAs targeted with lentivirus-delivered short hairpin RNAs (shRNAs). This nuclear RNAi(-like) phenomenon was not observed when synthetic siRNAs were delivered by cationic lipids, suggesting either a link between processing of the shRNA and nuclear import or a separate pathway for processing shRNAs in the nuclei. Our observation of simultaneous RNAi on both cytoplasmic and nuclear retained DMPK has important implications for post-transcriptional gene regulation in both compartments of mammalian cells. Small interfering RNAs (siRNA(s)) 1The abbreviations used are: siRNA, small interfering RNA; wt, wild type; mt, mutant; DM1, myotonic dystrophy type 1; DMPK, DM protein kinase; shRNA, small hairpin RNA; RISC, RNA-induced silencing complex; UTR, untranslated region; DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; eGFP, enhanced GFP; MOPS, 4-morpholinepropanesulfonic acid; ssiDM10, synthetic siRNA DM10; HIV, human immunodeficiency virus. 1The abbreviations used are: siRNA, small interfering RNA; wt, wild type; mt, mutant; DM1, myotonic dystrophy type 1; DMPK, DM protein kinase; shRNA, small hairpin RNA; RISC, RNA-induced silencing complex; UTR, untranslated region; DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; eGFP, enhanced GFP; MOPS, 4-morpholinepropanesulfonic acid; ssiDM10, synthetic siRNA DM10; HIV, human immunodeficiency virus. have been shown to direct sequence-specific inhibition of gene expression in mammalian cells (1Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8018) Google Scholar). siRNAs are RNA duplexes of 21–23 nucleotides with ∼2 nucleotide 3′-overhangs that can induce degradation of their homologous target mRNAs without eliciting interferon responses in mammalian cells. The degradation of the target mRNAs occurs at the post-transcriptional level and is termed post-transcriptional gene silencing, one of the RNA interference (RNAi) pathways. RNAi requires incorporation of one of the short RNA strands into the RNA-induced silencing complex (RISC), wherein the sequence serves as a guide for identification of the targeted RNAs through base pairing. Argonaute 2 (Ago2) in RISC cleaves the target at a single site within the target mRNA through the PIWI domain (2Song J.J. Smith S.K. Hannon G.J. Joshua-Tor L. Science. 2004; 305: 1434-1437Crossref PubMed Scopus (1064) Google Scholar, 3Liu J. Carmell M.A. Rivas F.V. Marsden C.G. Thomson J.M. Song J.J. Hammond S.M. Joshua-Tor L. Hannon G.J. Science. 2004; 305: 1437-1441Crossref PubMed Scopus (1983) Google Scholar, 4Meister G. Landthaler M. Patkaniowska A. Dorsett Y. Teng G. Tuschl T. Mol. Cell. 2004; 15: 185-197Abstract Full Text Full Text PDF PubMed Scopus (1409) Google Scholar), subsequently degrading the target. Because most protein components of RNAi, including Ago2 and Dicer, assemble and function in the cytoplasm (5Carmell M.A. Xuan Z. Zhang M.Q. Hannon G.J. Genes Dev. 2002; 16: 2733-2742Crossref PubMed Scopus (683) Google Scholar, 6Tabara H. Yigit E. Siomi H. Mello C.C. Cell. 2002; 109: 861-871Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, 7Provost P. Dishart D. Doucet J. Frendewey D. Samuelsson B. Radmark O. EMBO J. 2002; 21: 5864-5874Crossref PubMed Scopus (365) Google Scholar, 8Zeng Y. Cullen B.R. RNA (N. Y.). 2002; 8: 855-860Crossref PubMed Scopus (224) Google Scholar) it is widely believed that RNAi only occurs in the cytoplasm. Several lines of evidence have indicated that RNAi(-like) phenomena may also occur in the nucleus in addition to the well characterized cytoplasmic mechanism. siRNAs have been shown to initiate transcriptional gene silencing by targeting DNA sequences in the nucleus of fission yeast (9Verdel A. Jia S. Gerber S. Sugiyama T. Gygi S. Grewal S.I. Moazed D. Science. 2004; 303: 672-676Crossref PubMed Scopus (936) Google Scholar), flies (10Pal-Bhadra M. Leibovitch B.A. Gandhi S.G. Rao M. Bhadra U. Birchler J.A. Elgin S.C. Science. 2004; 303: 669-672Crossref PubMed Scopus (554) Google Scholar), and human cells (11Kawasaki H. Taira K. Nature. 2004; 431: 211-217Crossref PubMed Scopus (315) Google Scholar, 12Morris K.V. Chan S.W. Jacobsen S.E. Looney D.J. Science. 2004; 305: 1289-1292Crossref PubMed Scopus (787) Google Scholar). Moreover, in Caenorhabditis elegans, nuclear proteins are reportedly required for RNAi (13Dudley N.R. Labbe J.C. Goldstein B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4191-4196Crossref PubMed Scopus (106) Google Scholar). Previous studies have also indirectly indicated nuclear RNAi(-like) pathways in animals (4Meister G. Landthaler M. Patkaniowska A. Dorsett Y. Teng G. Tuschl T. Mol. Cell. 2004; 15: 185-197Abstract Full Text Full Text PDF PubMed Scopus (1409) Google Scholar, 10Pal-Bhadra M. Leibovitch B.A. Gandhi S.G. Rao M. Bhadra U. Birchler J.A. Elgin S.C. Science. 2004; 303: 669-672Crossref PubMed Scopus (554) Google Scholar, 14Bosher J.M. Dufourcq P. Sookhareea S. Labouesse M. Genetics. 1999; 153: 1245-1256Crossref PubMed Google Scholar, 15Shinagawa T. Ishii S. Genes Dev. 2003; 17: 1340-1345Crossref PubMed Scopus (107) Google Scholar), and a recent study demonstrated that the nuclear 7SK RNA can be degraded by siRNAs (16Robb G.B. Brown K.M. Khurana J. Rana T.M. Nat. Struct. Mol. Biol. 2005; 12: 133-137Crossref PubMed Scopus (254) Google Scholar). Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by a large unstable CTG expansion in the 3′-UTR of the DMPK (myotonic dystrophy protein kinase) gene (17Brook J.D. McCurrach M.E. Harley H.G. Buckler A.J. Church D. Aburatani H. Hunter K. Stanton V.P. Thirion J.P. Hudson T. et al.Cell. 1992; 69: 385Abstract Full Text PDF PubMed Scopus (10) Google Scholar). Mutant (mt) transcripts harboring the large CUG repeats are fully transcribed and polyadenylated, but remain trapped in the nucleus (18Davis B.M. McCurrach M.E. Taneja K.L. Singer R.H. Housman D.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7388-7393Crossref PubMed Scopus (369) Google Scholar). Complete nuclear retention of mt DMPK mRNAs with large CUG repeats is believed to be one of the most important pathological features of DM1 (19Mankodi A. Thornton C.A. Curr. Opin. Neurol. 2002; 15: 545-552Crossref PubMed Scopus (78) Google Scholar). Because DM1 cells express both a normal cytoplasmically localized DMPK mRNA and a mt nuclear retained version of this mRNA, this transcript represents a good target to determine whether or not the same siRNAs can target both transcripts for degradation. Using heterozygous DM1 myoblasts, we show reduction of both nuclear retained mt DMPK mRNAs containing long CUG repeats and cytoplasmic wt DMPK mRNAs that do not have long CUG repeats. The co-existence of nuclear and cytoplasmic RNAi in humans suggests that components of the RNAi machinery exist in both compartments. Finally, we demonstrate here that the same short hairpin RNA transcripts can function in both compartments, suggesting either nuclear Dicing or import of Diced siRNAs into the nucleus. These findings have important implications for applications of siRNAs in mammalian gene regulation. Primary Human Muscle Cell Cultures—DM1 and normal control myoblasts were obtained from the quadriceps of 15-week-old aborted fetuses. Skeletal muscle biopsies were approved by Laval University and the CHUL ethical committees. Myoblasts were grown and differentiation was carried out as described previously (20Langlois M.A. Lee N.S. Rossi J.J. Puymirat J. Mol. Ther. 2003; 7: 670-680Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). In Situ Hybridization and Confocal Microscopy—Myoblasts grown on glass coverslips were hybridized with a PNA Cy3-(CAG)5 probe to detect mt DMPK mRNAs as described in the Singer laboratory protocols (20Langlois M.A. Lee N.S. Rossi J.J. Puymirat J. Mol. Ther. 2003; 7: 670-680Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Nuclei were visualized by inclusion of 4′,6-diamidino-2-phenylindole (DAPI) in the mounting solution. Samples were observed using confocal microscopy (Zeiss LSM 510), and optical sections were obtained at optical Z resolution. Amira software was used to process images and of the confocal of shRNAs into Their and siRNA to DM1 and are to nucleotides and of the human muscle DMPK mRNA shRNAs were of a human using the was used as a for N.S. T. G. H. A. P. Rossi J. Nat. 2002; PubMed Scopus Google Scholar). The the sequences of the of the and the the sequences of the of the and shRNA and and a of was using to containing were into the with The shRNA were by DNA shRNA in were with and into the site of the S. J. J. J.A. Mol. Ther. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). cells were with the for and and with were and for with DM1 myoblasts of cells in were by of synthetic normal was with and with siRNAs was using either to the or at a K.V. Chan S.W. Jacobsen S.E. Looney D.J. Science. 2004; 305: 1289-1292Crossref PubMed Scopus (787) Google Scholar). differentiation was carried out (20Langlois M.A. Lee N.S. Rossi J.J. Puymirat J. Mol. Ther. 2003; 7: 670-680Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). was by of with the shRNA DMPK mRNA was using either (20Langlois M.A. Lee N.S. Rossi J.J. Puymirat J. Mol. Ther. 2003; 7: 670-680Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) or or RNAs and were in of by or of was to the and RNA was to of target DMPK of RNA was in a and was carried out as previously described (20Langlois M.A. Lee N.S. Rossi J.J. Puymirat J. Mol. Ther. 2003; 7: 670-680Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). was on expression within type using the level of normal transcripts in the control cells without shRNA as the base of was as described previously N.S. T. G. H. A. P. Rossi J. Nat. 2002; PubMed Scopus Google Scholar). cells were grown to in a and into differentiation of The was in and the protein was by protein of protein were on a and to a from was used as a probe D. D. Taneja K. Puymirat J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). DMPK protein were to the proteins to the Cell myoblasts were into nuclear and cytoplasmic by in Y. K. Lee S. EMBO J. 2002; 21: PubMed Scopus Google Scholar). RNAs from the nuclear and the cytoplasmic were with 2 of as described of RNA from were and of nuclear and cytoplasmic RNAs were in the DMPK mRNA were to mRNAs on the same and of and were to probe to be of probe of the DMPK probe of or of probe The were for and on of probe was to a in a were with The was and for in for nuclear nuclear of and of RNAs were as were carried out for in the of of to specific between wt and mt DMPK RNAs were from the described Hybridization was carried out in the same as described previously (20Langlois M.A. Lee N.S. Rossi J.J. Puymirat J. Mol. Ther. 2003; 7: 670-680Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), for of the DNA with of The probe to mt DMPK mRNA but not to wt as by not were in and carried out with or without in not DMPK mRNAs were with transcripts in DM1 myoblasts have CTG repeats in one allele of the DMPK gene and 18 CTG repeats in the The of the transcripts were by and not The DM1 myoblasts were from the quadriceps of a 15-week-old DM1 fetus and of the myoblasts for in differentiation DMPK expression but not in in using a DNA probe to the of the repeats that mt DMPK mRNAs are localized in the nucleus of DM1 myoblasts and normal transcripts are in the cytoplasm are localized in the cytoplasm of normal myoblasts, as demonstrated previously by (18Davis B.M. McCurrach M.E. Taneja K.L. Singer R.H. Housman D.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7388-7393Crossref PubMed Scopus (369) Google Scholar, K.L. McCurrach M. M. Housman D. Singer R.H. J. Cell Biol. PubMed Scopus Google Scholar, K.L. PubMed Scopus Google Scholar, M. K. J.D. 2001; PubMed Scopus Google Scholar). the of mt DMPK confocal was fluorescent in on the DM1 cells. DMPK mRNAs were localized the nucleus to the nuclear The of mt and wt DMPK mRNAs be using fluorescent in have the same base but only DMPK mRNAs in the DM1 cells show nuclear DMPK mRNAs in the DM1 cells differentiation to study nuclear RNAi was used to shRNAs targeting DMPK mRNAs control of the human into the DM1 myoblasts S. J. J. J.A. Mol. Ther. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). gene by a was also as a shRNAs were are to the sequences of the DMPK mRNA and one is targeted to a site in the 3′-UTR previously shown to be to cleavage (20Langlois M.A. Lee N.S. Rossi J.J. Puymirat J. Mol. Ther. 2003; 7: 670-680Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), and one is to sequence shRNA were into DM1 myoblasts of expression be obtained differentiation their S.M. M. L. B. S.M. M. Ther. 2003; PubMed Scopus Google Scholar). The cells were by The expression of shRNAs was by using the RNAs from cells with that to the strands but not to shRNAs were from the and were into siRNAs as the function of shRNA in DM1 myoblasts, the RNA was from primary and the level of DMPK mRNA was by using a DMPK probe DMPK mRNA between and CUG repeats and is from the mt DMPK transcripts containing long CUG repeats Myoblasts were to in the of for to RNA to induce the expression of DMPK mRNA D. T. O. J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). of mt transcripts were in DM1 mt DMPK transcripts with large CUG are the normal transcripts not shRNAs and the most down-regulation of normal DMPK mRNAs and in to control and also mt DMPK transcripts that are localized in the nucleus and and The shRNA the 3′-UTR only a in expression of both wt and mt DMPK transcripts and The shRNA control not in DMPK mRNA that phenomenon occurs not only with cytoplasmic transcripts but also with nuclear that nuclear transcripts are by we nuclear and cytoplasmic of DM1 cells the shRNAs The mt DMPK mRNAs were observed in the nucleus retained mt mRNAs were by both shRNAs and and In the normal transcripts in the nuclear were also by shRNAs and shRNAs and the normal transcripts in the cytoplasm and We not detect cleavage from the nuclear targeted suggesting that the RNAs are degraded The in DMPK obtained from this a as that with the in and The shRNAs and were the most and a but These show that shRNAs both nuclear and cytoplasmic the of RNAi(-like) process in both compartments of human cells. Because only the normal DMPK transcripts can be in the cytoplasm of DM1 myoblasts, we a to the of DMPK protein from normal shRNAs and DMPK protein by and to the control The of DMPK protein were by in DM1 cells shRNA These are with the reduction in mRNA with and nuclear RNAi(-like) pathway of shRNAs mt DMPK we DM1 cells with synthetic siRNA using The first on a from the protein and the nuclear has previously been to the nuclear import of siRNAs K.V. Chan S.W. Jacobsen S.E. Looney D.J. Science. 2004; 305: 1289-1292Crossref PubMed Scopus (787) Google Scholar, G. 2003; PubMed Scopus Google Scholar), the on nuclear and has been to siRNAs to target cells A. P. 2004; PubMed Scopus Google Scholar). of into the nucleus or cytoplasm using or we used and we confocal microscopy and the to using Amira software and delivered to the cytoplasm delivered siRNAs into both the nuclear and cytoplasmic compartments whether nuclear down-regulation of mt DMPK transcripts were by we on RNAs from the DM1 cells by or Using a to the to DM1 cells the level of wt DMPK mRNAs but not mt DMPK transcripts the level of mt DMPK mRNAs well as wt DMPK in the DM1 cells mutant of in the of the down-regulation of wt or mt transcripts when either or was sequence for both the nuclear and cytoplasmic Using a of and in the for was by S. S. G. P. G. 2004; PubMed Scopus Google Scholar), we not detect the of either transcript and These that the nuclear expression of shRNAs produce in the nucleus of primary human cells when the target is retained in the nucleus. for the observed reduction in mRNA be a in transcriptional gene silencing can be by RNAs N.R. Goldstein B. Curr. Opin. Mol. Ther. 2003; Google Scholar), and it has been that transcriptional gene silencing can be by siRNAs and shRNAs in human cells (11Kawasaki H. Taira K. Nature. 2004; 431: 211-217Crossref PubMed Scopus (315) Google Scholar, 12Morris K.V. Chan S.W. Jacobsen S.E. Looney D.J. Science. 2004; 305: 1289-1292Crossref PubMed Scopus (787) Google Scholar). We carried out nuclear using from DM1 myoblasts the shRNAs shRNAs and show of wt and mt DMPK transcripts but transcriptional normal for both mt and wt DMPK and The mRNA in both the nucleus and the cytoplasm of DM1 cells is thus not a of but is to post-transcriptional gene RNAi in both the nuclear and cytoplasmic compartments. We were to a reduction in the nuclear retained mt DMPK mRNAs in DM1 evidence that RNAi pathways in the cytoplasm. protein components of the RISC are cytoplasmic (5Carmell M.A. Xuan Z. Zhang M.Q. Hannon G.J. Genes Dev. 2002; 16: 2733-2742Crossref PubMed Scopus (683) Google Scholar, 6Tabara H. Yigit E. Siomi H. Mello C.C. Cell. 2002; 109: 861-871Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, 7Provost P. Dishart D. Doucet J. Frendewey D. Samuelsson B. Radmark O. EMBO J. 2002; 21: 5864-5874Crossref PubMed Scopus (365) Google Scholar, 8Zeng Y. Cullen B.R. RNA (N. Y.). 2002; 8: 855-860Crossref PubMed Scopus (224) Google Scholar). In a separate study that only cytoplasmic transcripts be targeted by RNAi Y. Cullen B.R. RNA (N. Y.). 2002; 8: 855-860Crossref PubMed Scopus (224) Google Scholar), reduction in nuclear transcripts was this was to RNAi on transcripts in the process of The mt DMPK transcripts targeted in this study are in the and B.M. McCurrach M.E. Taneja K.L. Singer R.H. Housman D.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7388-7393Crossref PubMed Scopus (369) Google Scholar) and are by the of the of the mt DMPK we that the observed down-regulation is in the nucleus and is not with of the mt DMPK through the nuclear the of study also demonstrated direct targeting of 7SK small nuclear RNA in human cells by and siRNAs also RNAi on nuclear retained (16Robb G.B. Brown K.M. Khurana J. Rana T.M. Nat. Struct. Mol. Biol. 2005; 12: 133-137Crossref PubMed Scopus (254) Google Scholar). RISC components be in the nuclear as well as cytoplasmic compartments (16Robb G.B. Brown K.M. Khurana J. Rana T.M. Nat. Struct. Mol. Biol. 2005; 12: 133-137Crossref PubMed Scopus (254) Google Scholar). with a nuclear RNAi(-like) pathway to in human cells. of studies have also indicated that phenomena may occur in the nucleus of human cells in addition to the well characterized cytoplasmic mechanism. nuclear RNAi pathway was by a study that shRNAs the control of the are in silencing targeted transcripts Nat. 2002; PubMed Scopus Google Scholar). In elegans, the containing can be targeted by RNAi J.M. Dufourcq P. Sookhareea S. Labouesse M. Genetics. 1999; 153: 1245-1256Crossref PubMed Google Scholar). siRNAs have been shown to initiate transcriptional gene silencing by targeting DNA sequences in the nucleus of fission yeast (9Verdel A. Jia S. Gerber S. Sugiyama T. Gygi S. Grewal S.I. Moazed D. Science. 2004; 303: 672-676Crossref PubMed Scopus (936) Google Scholar), flies (10Pal-Bhadra M. Leibovitch B.A. Gandhi S.G. Rao M. Bhadra U. Birchler J.A. Elgin S.C. Science. 2004; 303: 669-672Crossref PubMed Scopus (554) Google Scholar), and human cells (11Kawasaki H. Taira K. Nature. 2004; 431: 211-217Crossref PubMed Scopus (315) Google Scholar, 12Morris K.V. Chan S.W. Jacobsen S.E. Looney D.J. Science. 2004; 305: 1289-1292Crossref PubMed Scopus (787) Google Scholar). Moreover, in elegans, nuclear proteins are reportedly required for RNAi (13Dudley N.R. Labbe J.C. Goldstein B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4191-4196Crossref PubMed Scopus (106) Google Scholar). Previous studies have also indirectly indicated nuclear RNAi(-like) pathways in animals (4Meister G. Landthaler M. Patkaniowska A. Dorsett Y. Teng G. Tuschl T. Mol. Cell. 2004; 15: 185-197Abstract Full Text Full Text PDF PubMed Scopus (1409) Google Scholar, 10Pal-Bhadra M. Leibovitch B.A. Gandhi S.G. Rao M. Bhadra U. Birchler J.A. Elgin S.C. Science. 2004; 303: 669-672Crossref PubMed Scopus (554) Google Scholar, 14Bosher J.M. Dufourcq P. Sookhareea S. Labouesse M. Genetics. 1999; 153: 1245-1256Crossref PubMed Google Scholar, 15Shinagawa T. Ishii S. Genes Dev. 2003; 17: 1340-1345Crossref PubMed Scopus (107) Google Scholar). These studies that nuclear RNAi(-like) phenomena occur in a of are between and the Rana and (16Robb G.B. Brown K.M. Khurana J. Rana T.M. Nat. Struct. Mol. Biol. 2005; 12: 133-137Crossref PubMed Scopus (254) Google Scholar) of nuclear RNAi, both used in human cells. The mt DMPK transcripts we targeted with shRNAs are large nuclear retained mRNAs transcribed by RNA the 7SK or RNAs targeted by Rana and (16Robb G.B. Brown K.M. Khurana J. Rana T.M. Nat. Struct. Mol. Biol. 2005; 12: 133-137Crossref PubMed Scopus (254) Google Scholar) are small nuclear RNAs transcribed by We demonstrated that the same shRNAs from can target both nuclear and cytoplasmic observed of nuclear using synthetic In observation used primary human cells human study with their (16Robb G.B. Brown K.M. Khurana J. Rana T.M. Nat. Struct. Mol. Biol. 2005; 12: 133-137Crossref PubMed Scopus (254) Google Scholar) that nuclear RNAi(-like) can target a of transcripts in of human cells and may thus be a The that shRNAs can direct nuclear RNA the of whether the shRNAs are into siRNAs in the nucleus or are in the cytoplasm and into the nucleus. The processing of the shRNAs a cleavage or processing of the this time we have not been to between Because siRNAs delivered to the nucleus by the can also nuclear RNAi, can function in the nucleus as by studies (4Meister G. Landthaler M. Patkaniowska A. Dorsett Y. Teng G. Tuschl T. Mol. Cell. 2004; 15: 185-197Abstract Full Text Full Text PDF PubMed Scopus (1409) Google Scholar, G.B. Brown K.M. Khurana J. Rana T.M. Nat. Struct. Mol. Biol. 2005; 12: 133-137Crossref PubMed Scopus (254) Google Scholar). of cytoplasmic RNAi is have Argonaute protein containing the of a domain and PIWI T. A. S. 2003; PubMed Scopus Google Scholar). of the human proteins have been shown to be both nuclear and cytoplasmic and (16Robb G.B. Brown K.M. Khurana J. Rana T.M. Nat. Struct. Mol. Biol. 2005; 12: 133-137Crossref PubMed Scopus (254) Google a of RISC is in the nucleus. It to be components of the RNAi machinery exist in both compartments. for nuclear phenomenon be to of cytoplasmic components of RISC containing siRNAs when the nuclear is In study a is cells are not the of In addition we observed targeting of mt DMPK transcripts with lentivirus-delivered shRNAs or synthetic siRNAs but not with cationic synthetic This is in to G.B. Brown K.M. Khurana J. Rana T.M. Nat. Struct. Mol. Biol. 2005; 12: 133-137Crossref PubMed Scopus (254) Google cationic of synthetic siRNAs in nuclear The in and that of Rana and (16Robb G.B. Brown K.M. Khurana J. Rana T.M. Nat. Struct. Mol. Biol. 2005; 12: 133-137Crossref PubMed Scopus (254) Google Scholar) is not it is that be a for of cytoplasmic siRNAs as has been for of siRNA transcriptional gene silencing (11Kawasaki H. Taira K. Nature. 2004; 431: 211-217Crossref PubMed Scopus (315) Google Scholar, 12Morris K.V. Chan S.W. Jacobsen S.E. Looney D.J. Science. 2004; 305: 1289-1292Crossref PubMed Scopus (787) Google Scholar). Finally, nuclear RNAi(-like) pathways in human primary cells using shRNAs can target nuclear retained mt DMPK RNAs containing long CUG repeats that is believed to be one of the most important pathological features of This has important implications for myotonic dystrophy or nuclear We and for their We also of for providing for providing the and and of for We also for and for