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. 2001 Jul 1;29(13):2766-71.
doi: 10.1093/nar/29.13.2766.

In vivo co-localisation of MBNL protein with DMPK expanded-repeat transcripts

M Fardaei  1 , K Larkin, J D Brook, M G Hamshere
Affiliations

In vivo co-localisation of MBNL protein with DMPK expanded-repeat transcripts

M Fardaei et al. Nucleic Acids Res. .

Abstract

Myotonic dystrophy (DM1) is the most common form of adult muscular dystrophy and is inherited as an autosomal dominant trait. The genetic basis of DM1 is the expansion of a CTG repeat in the 3' untranslated region of a protein kinase gene (DMPK). The molecular mechanism by which this expanded repeat produces the pathophysiology of DM1 remains unknown. Transcripts from the expanded allele accumulate as foci in the nucleus of DM1 cells and it has been suggested that these transcript foci sequester cellular proteins that are required for normal nuclear function. We have investigated the role of three RNA-binding proteins, CUG-BP, hnRNP C and MBNL, as possible sequestered factors. Using a combination of indirect immunofluorescence to detect endogenous proteins and overexpression of proteins with green fluorescent protein (GFP) tags we have shown that CUG-BP and hnRNP C do not co-localise with expanded repeat foci in DM1 cell lines. However, GFP-tagged MBNL does itself form foci in DM1 cell lines and co-localises with the foci of expanded repeat transcripts. GFP-tagged MBNL does not appear as foci in non-DM1 cell lines. This work provides further support for the involvement of MBNL in DM1.

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Figures

Figure 1
Figure 1
Distribution of the CUG-BP in control and DM1 fibroblast cells. Indirect immunofluorescence with mAb 3B1 (anti CUG-BP) of (A) control and (B) DM1 fibroblast cells.
Figure 2
Figure 2
Quantification of endogenous and GFP-tagged proteins in DM1 cells. Indirect immunofluorescence or fluorescence from GFP-tagged proteins was quantified using digital images in which mean intensities were estimated per unit pixel. There was no significant difference between the levels of protein at the transcript foci and in the background for CUG-BP or hnRNP C. The level of GFP/MBNL in the transcript foci was significantly higher than background in DM1 cells. (number of transcript foci: 92 P < 0.01, t-test).
Figure 3
Figure 3
Distribution of hnRNP C in control and DM1 fibroblast cells.Endogenous hnRNP C was detected using mAb 4F4 (anti-hnRNP C) in (A) control and (B) DM1 cells.
Figure 4
Figure 4
Transient expression of GFP-tagged proteins in control and DM1 fibroblast cells. (A–D) Cells transfected with GFP/CUG-BP, (E–H) cells transfected with GFP/hnRNP C and (I–L) cells transfected with GFP/MBNL. (A, E and I) Control fibroblasts, (B–D, F–H and J–L) DM1 cell lines. (B, F and J) GFP-tagged protein distribution in DM1 fibroblast cells. (C, G and K) Location of DMPK expanded transcripts using in-situ hybridization with (CAG)10-Cy3 probe. (D, H and L) Merged images which show that GFP/CUG-BP (D) and GFP/hnRNP C (H) do not co-localise with foci of expanded repeat transcripts. The merge image of GFP/MBNL and the (CAG)10-Cy3 probe (L) shows that GFP/MBNL co-localises with the foci of expanded DMPK transcripts.
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