Cytoplasmic and Nuclear Retained DMPK mRNAs Are Targets for RNA Interference in Myotonic Dystrophy 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)) 1 have been shown to direct sequence-specific inhibition of gene expression in mammalian cells (1). siRNAs are RNA duplexes of 21-23 nucleotides with ϳ2 nucleotide 3Ј-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 (2)(3)(4), subsequently degrading the target. Because most protein components of RNAi, including Ago2 and Dicer, assemble and function in the cytoplasm (5)(6)(7)(8) 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 (9), flies (10), and human cells (11,12). Moreover, in Caenorhabditis elegans, nuclear proteins are reportedly required for RNAi (13). Previous studies have also indirectly indicated nuclear RNAi(-like) pathways in animals (4,10,14,15), and a recent study demonstrated that the nuclear 7SK RNA can be degraded by siR-NAs (16).
Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by a large unstable CTG expansion in the 3Ј-UTR of the DMPK (myotonic dystrophy protein kinase) gene (17). Mutant (mt) transcripts harboring the large CUG repeats are fully transcribed and polyadenylated, but remain trapped in the nucleus (18). Complete nuclear retention of mt DMPK mRNAs with large CUG repeats is believed to be one of the most important pathological features of DM1 (19). 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.

MATERIALS AND METHODS
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 (20).
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 (20). Nuclei were visualized by inclusion of 4Ј,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 construct three-dimensional surface models of the confocal image data stacks.
shRNA constructs in pCR2.1 vectors were digested with BamHI-EcoRV and subcloned into the BamHI-SmaI site of the pHIV7 vector (22). 293T cells were co-transfected with the pHIV7, pCMV-Rev, pGP (coding for gag and pol), and pCMV-G (coding VSV-G). Infectious lentivirus particles pseudotyped with VSV-G were collected after 48 h and incubated for 30 min with DM1 myoblasts of low passage (P Ͻ 6). Transduced cells expressing eGFP encoded in pHIV7 were selected by fluorescence-activated cell sorting 3 days later.
For transfection of synthetic siRNAs, normal culture medium was replaced with Opti-MEM (Invitrogen) medium, and transfection with siRNAs (25 nM) was performed using either Lipofectamine 2000 (Invitrogen), according to the manufacturer's specifications, or MPG peptide at a 3:1 charge ratio (12). One day after transfection, differentiation was carried out (20). Transfection efficiency was determined by cotransfection of GFP plasmids with the shRNA constructs.
DMPK mRNA Extraction-RNA extraction was performed using either proteinase K digestion (20) or PARIS TM (Ambion). After digestion or PARIS treatment, total RNAs and DNAs were recovered in 10 -100 l of water by LiCl or standard acid phenol-chloroform extraction. One ml of TRIzol (Invitrogen) was then added to the samples, and RNA was extracted according to manufacturer specifications.
Northern Blotting-For detection of target DMPK transcripts, 10 g of total RNA was loaded in a 1.0% agarose gel and Northern hybridization was carried out as previously described (20). Sample normalization was based on cyclophilin expression levels within each cell type using the level of normal transcripts in the control cells without shRNA treatment as the base line. For detection of shRNAs, Northern hybridization was performed as described previously (21).
Western Blotting-DM1 cells were grown to confluence in a 100-mm dish and scraped into differentiation medium after 48 h of growth. The cell pellet was washed twice in Hanks' buffered salt solution, and the protein content was quantified by BCA protein assay (Pierce). Forty micrograms of protein were electrophoresed on a SDS-polyacrylamide gel and electroblotted to a nylon membrane. Anti-DMPK polyclonal antibody (a gift from Dr. Lubov Timchenko, Baylor College) was used as a probe (23). DMPK protein levels were normalized to the total proteins transferred to the nylon membrane.
Cell Fractions-Differentiated myoblasts were separated into nuclear and cytoplasmic fractions by centrifugation following lysis in digitonin buffer (24). Total RNAs from the nuclear pellets and the cytoplasmic fractions were extracted with 2 ml of proteinase K buffer as described earlier. Five micrograms of RNA from each fraction were formaldehyde and 1ϫ MOPS. Equivalent amounts of nuclear and cytoplasmic RNAs were loaded in the gels. DMPK mRNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase mRNAs on the same membrane.
Slot Blots and Nuclear Run-on Assays-Three microliters of 5 M NaOH and water were added to each probe to be 50 l; 125 ng of CTG-80 probe (240 bp), 727 ng of the DMPK probe (1,454 bp), 5 g of pBluescript KSϩ vector, or 350 ng of cyclophilin probe (700 bp). The probes were then boiled for 5 min and cooled on ice. After adding 50 l of 4 M ammonium acetate, each probe was applied to a nylon filter inserted in a slot-blot apparatus. After gentle suction, slots were washed twice with 2ϫ SSC. The nylon filter was removed and UV light cross-linked for 3 min in preparation for nuclear run-on assays.
For nuclear run-on assays, isolation of nuclei and synthesis of radiolabeled nascent RNAs were performed as follows. Transcriptions were carried out for 50 min in the presence of 200 Ci of [␣-32 P]ATP/sample to maintain similar specific activities between wt and mt DMPK transcripts. Total labeled RNAs were extracted from the samples described above. Hybridization was carried out overnight in the same Northern blot hybridization buffer as described previously (20), except for replacement of the salmon sperm DNA with 200 g/ml of Escherichia coli tRNA. The CTG-80 probe specifically binds to mt DMPK mRNA but not to wt transcripts, as verified by Northern blotting (data not shown). All assays were performed in duplicate and carried out simultaneously with or without 400 g/ml ␣-amanitin, which blocked transcription in all samples (data not shown). DMPK mRNAs were normalized with cyclophilin transcripts in each sample.

RESULTS
Heterozygous DM1 myoblasts have ϳ3,200 CTG repeats in one allele of the DMPK gene and 18 CTG repeats in the other (Fig. 1A). The size differences of the transcripts were verified by Northern (Fig. 1B) and Southern hybridization (data not shown). The DM1 myoblasts were isolated from the quadriceps of a 15-week-old aborted, congenitally affected DM1 fetus (see "Materials and Methods"). Culturing of the myoblasts for 48 h in differentiation medium allowed DMPK expression but did not result in myoblast fusion (Fig. 1C). Fluorescent in situ hybridization using a Cy3-labeled DNA probe to the region upstream of the repeats shows that mt DMPK mRNAs are localized in the nucleus of DM1 myoblasts and normal (wt) transcripts are visible in the cytoplasm (Fig. 1C, right), whereas they are localized exclusively in the cytoplasm of normal myoblasts, as demonstrated previously by several groups (18,(25)(26)(27). To investigate the subnuclear localization of mt DMPK transcripts, confocal microscope imaging was performed following fluorescent in situ hybridization on the differentiated DM1 cells. DMPK mRNAs were localized inside the nucleus distal to the nuclear envelope (Fig. 1D). The localization of mt and wt DMPK mRNAs cannot be distinguished using fluorescent in situ hybridization, because they have the same base composition, but only DMPK mRNAs in the differentiated DM1 cells show nuclear signals. Therefore, DMPK mRNAs in the DM1 cells following differentiation represent an excellent model system to study nuclear RNAi A lentivirus-based vector (pHIV7) was used to deliver shR-NAs targeting DMPK mRNAs under control of the human U6 Pol III promoter into the DM1 myoblasts (22). An eGFP gene driven by a CMV promoter was also incorporated as a selection marker ( Fig. 2A). Four shRNAs were designed. Two are complementary to the coding sequences of the DMPK mRNA (DM10 and DM130), one is targeted to a site in the 3Ј-UTR previously shown to be accessible to ribozyme cleavage (DM1892) (20), and one is directed to an irrelevant sequence ( Fig. 2A, CTRL IR). shRNA constructs were efficiently transduced into cultured DM1 myoblasts (Fig. 2B). High levels of transgene expression could be obtained throughout myoblast differentiation following their genome integration (28). The cells expressing eGFP were then sorted by flow cytometry. The expression of shRNAs was assessed by Northern hybridization using the total RNAs isolated from sorted cells with sense probes that hybridize to the antisense strands but not to targets. All shRNAs were expressed from the lentiviral vectors, and they were processed into ϳ21-nucleotide siRNAs as expected (Fig. 2B).
To investigate the function of shRNA constructs in DM1 myoblasts, the total RNA was extracted from transduced primary myoblast cultures, and the level of DMPK mRNA was determined by Northern hybridization using a DMPK cDNA probe (Fig. 3A). Normal DMPK mRNA contains between 5 and 37 CUG repeats and is easily distinguished from the mt DMPK transcripts containing long CUG repeats (Fig. 1B). Myoblasts were allowed to differentiate in the absence of growth factors for 48 h prior to RNA extraction to induce the expression of DMPK mRNA (29). Steady-state levels of mt transcripts were elevated in congenital DM1 cells, because mt DMPK transcripts with large CUG repeat expansions are more stable than the normal transcripts (data not shown). shRNAs DM10 and DM130 showed the most effective down-regulation of normal (wt) DMPK mRNAs (64.2 Ϯ 3.5 and 74.5 Ϯ 2.3% decreases in expression, respectively, relative to control expression) (Fig. 3,  A and B). Unexpectedly, they also effectively down-regulated mt DMPK transcripts that are localized exclusively in the nucleus (46.6 Ϯ 2.4 and 57.6 Ϯ 6.4%) (Fig. 3, A and B). The shRNA directed against the 3Ј-UTR (DM1892) showed only a moderate decrease in expression of both wt and mt DMPK transcripts (26.5 Ϯ 2.4 and 15.1 Ϯ 3.3%). The irrelevant shRNA control did not elicit any significant change in total DMPK mRNA levels. Taken together our data suggest that an RNAilike phenomenon occurs not only with cytoplasmic transcripts but also with nuclear transcripts.
To confirm that nuclear transcripts are affected by shRNAs, we performed nuclear and cytoplasmic fractionation of differentiated DM1 cells stably expressing the shRNAs (Fig. 3, C-E). The mt DMPK mRNAs were observed exclusively in the nucleus (Fig. 3C). Nuclear retained mt mRNAs were reduced effectively by both shRNAs DM10 and DM130 (51.5 Ϯ 6.6 and 48.8 Ϯ 3.9%) (Fig. 3). In addition, the normal transcripts re-maining in the nuclear fraction were also reduced by these shRNAs (47.2 Ϯ 5.7 and 46.2 Ϯ 4.0%). As expected, shRNAs DM10 and DM130 down-regulated the normal transcripts found in the cytoplasm (77.8 Ϯ 1.3 and 73.6 Ϯ 1.5%). We could not detect any intermediate size cleavage products from the nuclear targeted transcripts, suggesting that the RNAs are rapidly degraded following cleavage. The changes in DMPK levels obtained from this fractionation experiment showed a similar trend as that seen with the Northern blot in (Fig. 3, A  and B). The shRNAs DM10 and DM130 were the most effective and DM1892 had a weak but significant effect. These results show that shRNAs reduce both nuclear and cytoplasmic transcripts, demonstrating the existence of an RNAi(-like) process in both compartments of human cells.
Because only the normal DMPK transcripts can be translated in the cytoplasm of DM1 myoblasts, we performed a Western blot to measure the levels of DMPK protein translated from these normal transcripts. shRNAs DM10 and DM130 reduced DMPK protein levels by 55.9 Ϯ 3.2 and 73.2 Ϯ 3.0%, respectively, relative to the control (Fig. 3, F-H). The levels of DMPK protein were lowered by 30.8 Ϯ 4.7% in DM1 cells expressing shRNA DM1892. These results are consistent with the reduction in mRNA levels determined with Northern blotting (Fig. 3, A and B).
To confirm nuclear RNAi(-like) pathway of shRNAs against mt DMPK mRNAs, we transfected DM1 cells with synthetic siRNA DM10 (ssiDM10) using two different methods. The first methodology relied on MPG, a fusion peptide derived from the HIV type-1 gp41 transmembrane protein and the SV40 nuclear localization peptide, which has previously been reported to facilitate the nuclear import of siRNAs (12, 30), whereas the second method relied on conventional Lipofectamine 2000, which lacks nuclear specificity and has been reported to generally deliver siRNAs to target cells (31). To confirm delivery of ssiDM10 into the nucleus or cytoplasm using MPG or Lipofectamine 2000, we used Cy3-labeled ssiDM10. After transfection and differentiation, we performed confocal microscopy and then processed the image stacks to construct three-dimensional models using Amira software (Fig. 4, B and C). Lipofectamine delivered ssiDM10 mainly to the cytoplasm (Fig. 4B), whereas MPG delivered siRNAs into both the nuclear and cytoplasmic compartments (Fig. 4C). To examine whether nuclear downregulation of mt DMPK transcripts were affected by different delivery methods, we performed Northern hybridization analyses on RNAs from the DM1 cells transfected by MPG or Lipofectamine. Using a modified Lipofectamine 2000 to increase the transfection efficiency to DM1 cells (ϳ65%), ssiDM10 effectively diminished the level of wt DMPK mRNAs (ϳ70%) but not mt DMPK transcripts (Fig. 4A). However, ssiDM10 effectively diminished the level of mt DMPK mRNAs (ϳ75%) (as well as wt DMPK mRNAs) in the DM1 cells transfected via MPG (Fig. 4A). A mutant form of ssiDM10 (mt ssiDM10), which contains four mismatches in the middle of the antisense strand, showed no down-regulation of wt or mt transcripts when either MPG or Lipofectamine was used, demonstrating sequence specificity for both the nuclear and cytoplas-mic mechanisms. Using a mixture of MPG and Lipofectamine, in which the efficiency for ssiRNA delivery was made really poor by disrupting membrane organization (32), we did not detect the knockdown of either transcript (Fig. 4A, lanes 6 and  7). These data suggest that the initial nuclear expression of shRNAs produce an interference-like effect in the nucleus of primary human cells when the target message is generally retained in the nucleus.
An alternative explanation for the observed reduction in steady-state mRNA levels could be a decrease in transcription, because transcriptional gene silencing can be initiated by double-stranded RNAs (33), and it has recently been reported that transcriptional gene silencing can be induced by siRNAs and shRNAs in human cells (11,12). We therefore carried out nuclear run-on experiments using nuclei isolated from transduced DM1 myoblasts expressing the shRNAs (Fig. 5). Cells expressing shRNAs DM10 and DM130 show reduced amounts of wt and mt DMPK transcripts (Fig. 5A), but transcriptional initiation appears normal for both mt and wt DMPK (Fig. 5, B and C). The reduced mRNA levels seen in both the nucleus and the cytoplasm of DM1 cells is thus not a result of altered transcription but is due to post-transcriptional gene silencing. Taken together our data strongly support RNAi mechanisms in both the nuclear and cytoplasmic compartments. DISCUSSION We were surprised to find a significant reduction in the nuclear retained mt DMPK mRNAs in DM1 cells, because existing evidence indicates that RNAi pathways operate primarily in the cytoplasm. Most protein components of the RISC are cytoplasmic (5)(6)(7)(8). In addition, a separate study concluded that only cytoplasmic transcripts could be targeted by RNAi (8), although some reduction in nuclear transcripts was apparent, this was attributed to RNAi acting on transcripts in the process of being exported. The mt DMPK transcripts targeted in this study are located exclusively in the nucleoplasm (Fig.  1D and Ref. 18) and are clearly down-regulated by the appropriate shRNAs. Given our observations of the intracellular compartmentalization of the mt DMPK RNAs, we believe that the observed down-regulation is occurring in the nucleus and is not associated with transport of the mt DMPK through the nuclear pores. During the course of our work another study also demonstrated direct targeting of 7SK small nuclear RNA in human cells by siRNAs, and other siRNAs could also trigger RNAi on endogenous, nuclear retained U6snRNA (16). Moreover some RISC components could be found in the nuclear as well as cytoplasmic compartments (16). Taken together with our results, a nuclear RNAi(-like) pathway appears to operate in human cells.
A number of indirect studies have also indicated that RNAi-(-like) phenomena may occur in the nucleus of human cells in addition to the well characterized cytoplasmic mechanism. A nuclear RNAi pathway was predicted by a study demonstrating that shRNAs under the control of the U6ϩ27 promoter, although exclusively nuclear, are functionally active in silencing targeted transcripts (34). In C. elegans, the intron containing pre-mRNAs can be targeted by RNAi (14). siRNAs have been shown to initiate transcriptional gene silencing by targeting DNA sequences in the nucleus of fission yeast (9), flies (10), and human cells (11,12). Moreover, in C. elegans, nuclear proteins are reportedly required for RNAi (13). Previous studies have also indirectly indicated nuclear RNAi(-like) pathways in animals (4,10,14,15). These studies suggest that nuclear RNAi(-like) phenomena occur in a variety of animals.
There are some notable differences between our results and the Rana and co-workers (16) demonstration of nuclear RNAi, even though both used in human cells. The mt DMPK transcripts we targeted with shRNAs are large nuclear retained We demonstrated that the same shRNAs expressed from lentiviral vectors can simultaneously target both nuclear and cytoplasmic transcripts, whereas they observed knockdown of exclusive nuclear targets using synthetic siRNA. In addition, our observation used differentiated primary human cells rather than human cell lines. Taken together, our study along with their results (16) suggest that nuclear RNAi(-like) mechanism can target a variety of different transcripts in several types of human cells and may thus be a general phenomenon.
The demonstration that shRNAs can direct nuclear RNA destruction raises the question of whether the shRNAs are processed into siRNAs directly in the nucleus or are processed in the cytoplasm and transported back into the nucleus. The Approximately four times more volume of nuclear extracts than cytoplasmic extracts was loaded in the gel to achieve equal loading of total RNAs. D, hybridization of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in A. E, quantification of DMPK mRNAs normalized to glyceraldehyde-3-phosphate dehydrogenase. Normalizations were also carried out relative to total 28 and 18 S rRNA intensities using ethidium bromide staining to confirm cell fractionation purity (data not shown). Standard error bars are based on three independent experiments. F, Western blotting of whole cell extracts using anti-DMPK antibody. Cells were allowed to differentiate for 48 h prior to harvesting. Only normal DMPK mRNA contributes to DMPK protein levels because the mutant mRNA is nuclear retained and not translated. G, Ponceau red staining of the membrane prior to blotting with a rabbit anti-DMPK antibody. Samples were normalized to total sample protein levels. H, quantification of DMPK protein normalized to Ponceau red staining. Standard error bars are based on four independent experiments.

FIG. 4. Effect of different delivery methods of siRNAs on down-regulation of wt and mt DMPK mRNAs in DM1 cells.
A, expression of wt and mt DMPK mRNAs in DM1 cells transfected with wt or mt ssiDM10 using Lipofectamine 2000 (cytoplasmic (Cy)) or MPG (nuclear (Nu)). mt siDM10 contains four mismatches to the target in the middle of the antisense strand and was used as a control for specificity of RNAi. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. A combination of Lipofectamine and MPG was also used as a negative control as described in the text. An RNA marker (M) was used as a size indicator. One representative Northern assay from two independent experiments is depicted. GFP plasmids were co-transfected with the siR-NAs to monitor transfection efficiency in the DM1 cells. B and C, localization of Cy3labeled siRNAs by different transfection methods using fluorescent microscopy. Cytoplasmic delivery of siRNAs was accomplished by Lipofectamine 2000 (B). MPG can deliver some siRNAs to the nucleus (ϳ35%) (C). DAPI staining was used to identify the nucleus. Scale bars ϭ 10 m.
processing of the shRNAs could take place via a Dicer cleavage mechanism or via endonucleolytic processing of the loop. At this time we have not been able to distinguish differences between these possibilities. Because siRNAs delivered to the nucleus by the MPG peptide can also trigger nuclear RNAi, they can apparently function in the nucleus as suggested by other studies (4,16). A major component of cytoplasmic RNAi is Ago2. Humans have seven additional Argonaute (Ago) protein family members all containing the common architecture of a central PAZ domain and C-terminal PIWI motif (35). Two of the eight human Ago proteins have been shown to be both nuclear and cytoplasmic (Ago1 and Ago 2) (16); therefore, a major component of RISC is in the nucleus. It remains to be determined what other components of the RNAi machinery exist in both cellular compartments.
One possible mechanism for nuclear RNAi-related phenomenon would be to propose redistribution of cytoplasmic components of RISC containing siRNAs during mitosis when the nuclear membrane is disrupted. In our study such a mechanism is unlikely, because our differentiated cells are not dividing during the course of our experiments. In addition we observed targeting of mt DMPK transcripts with lentivirusdelivered shRNAs or MPG-delivered synthetic siRNAs but not with cationic lipid-delivered synthetic siRNAs. This last result is in contrast to Ref. 16 where cationic lipid delivery of syn-thetic siRNAs resulted in nuclear RNAi. The difference in our result and that of Rana and co-workers (16) is not known; however, it is possible that mitotic activity could be a requirement for redistribution of cytoplasmic siRNAs as has been proposed for conflicting results of siRNA induced transcriptional gene silencing (11,12).
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 DM1. This has important implications for future myotonic dystrophy treatment or other nuclear diseases.
FIG. 5. Nuclear run-on assays. A, Northern blot showing the specificity of the CTG (80) probe for the mt DMPK transcripts. B, nuclear run-on assays performed in duplicate. De novo transcribed labeled RNA from the transduced myoblasts was hybridized to the following probes: a probe with 80 CTG ((CTG)80) repeats, the DMPK coding sequence cDNA (DMPK), the pBluescript KSϩ vector (Plasmid DNA), and cyclophilin cDNA (Cyclophilin). Transcription reactions in the presence of 500 g/ml ␣-amanitin were also performed in parallel as negative controls, and no signals were present in these slot blots (data not shown). C, quantification of DMPK mRNAs normalized to cyclophilin transcripts in each sample. Standard error bars are based on three independent experiments.