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Muscleblind participates in RNA toxicity of expanded CAG and CUG repeats in Caenorhabditis elegans

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Abstract

We have utilized Caenorhabditis elegans as a model to investigate the toxicity and underlying mechanism of untranslated CAG repeats in comparison to CUG repeats. Our results indicate that CAG repeats can be toxic at the RNA level in a length-dependent manner, similar to that of CUG repeats. Both CAG and CUG repeats of toxic length form nuclear foci and co-localize with C. elegans muscleblind (CeMBL), implying that CeMBL may play a role in repeat RNA toxicity. Consistently, the phenotypes of worms expressing toxic CAG and CUG repeats, including shortened life span and reduced motility rate, were partially reversed by CeMbl over-expression. These results provide the first experimental evidence to show that the RNA toxicity induced by expanded CAG and CUG repeats can be mediated, at least in part, through the functional alteration of muscleblind in worms.

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Acknowledgments

We are grateful to Dr. Y.-C. Wu (National Taiwan University) for providing myo-3::gfp and unc-54::mcherry plasmids and for discussion and reading the manuscript and to Dr. C.-S. Chen (National Cheng Kung University) for providing L4440::bre3 plasmid. This work was supported by the following grants from the National Science Council of Taiwan: NSC 95-2320-B-040-040-MY2 and NSC-97-2320-B-040-013-MY3 to H. Pan and NSC 96-2320-B-194-006-MY3 to K.-M. Hsiao.

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Authors and Affiliations

  1. Department of Biomedical Sciences, Chung Shan Medical University, Taichung, 402, Taiwan

    Li-Chun Wang & Huichin Pan

  2. Institute of Biotechnology, National Cheng Kung University, Tainan, 701, Taiwan

    Kuan-Yu Chen

  3. Department of Medical Research, Chung Shan Medical University Hospital, Taichung, 402, Taiwan

    Huichin Pan

  4. Institute of Molecular Biology, National Chung Cheng University, Chia-Yi, 621, Taiwan

    Chia-Chieh Wu, Po-Hsuan Chen, Yuan-Ting Liao, Chin Li, Min-Lang Huang & Kuang-Ming Hsiao

  5. Department of Life Science, National Chung Cheng University, 168, University Road, Min-Hsiung, Chia-Yi, 62102, Taiwan, ROC

    Chin Li, Min-Lang Huang & Kuang-Ming Hsiao

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  1. Li-Chun Wang
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Correspondence to Kuang-Ming Hsiao.

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L.-C. Wang, K.-Y. Chen and H. Pan contributed equally to this paper.

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Suppl. Fig. 1

Muscle phenotype of CUG125 worms. (A) Electron microscopy images of GFP and CUG125 muscles. mcb, muscle cell body; M, M-line; db, dense body; bm, basement membrane; hyp, hypodermis; c, cuticle; Bar, 1 μm. (B) Dense body alignment in muscles of GFP and CUG125 worms. Anti-DEB-1 staining of dense bodies is shown as red spots. Arrow indicates the dense body within a muscle cell. Arrowhead indicates the dense body at the cell boundary. Bar, 10 μm (JPEG 327 kb)

Suppl. Fig. 2

Untranslated CAG and CUG repeats are not translated into polyQ protein. To investigate if CAG and CUG transgenes were translated into polyQ protein, Western blot analysis was performed using antibody against polyQ protein (Millipore, MAB1574) at a dilution of 1:3000 and total protein extracted from CAG83, CUG83 or Q78 transgenic worms. Q78 worms were generated by injection of myo3::gfp(Q78) plasmid (which contains CAG78 repeats in the coding region of gfp gene driven by the myo3 promoter) and were used as a positive control. 50 μg of total protein was loaded per well. The result indicates that untranslated CAG125 or CUG125 repeats in the transgenes do not produce a protein recognized by the antibody (JPEG 318 kb)

Suppl. Fig. 3

Reduced bre-3 gene expression by RNAi does not cause obvious effect on the life span of GFP, CUG83, and CAG83 worms. More than 30 transgenic worms at the F2 generation treated with bre-3(RNAi) or empty vector were assayed (JPEG 446 kb)

Suppl. Fig. 4

Reduced expression of deb-1a and deb-1c in CUG125, CAG125, and CeMbl(RNAi) worms. Semi-quantitative RT-PCR analyses of the deb-1a and deb-1c RNA level in GFP, CAG125 and CUG125 worms (left panel) or in control(RNAi) and CeMbl(RNAi) worms (right panel) were performed. Representative RT-PCR products were run on a 1% agarose gel and are shown in the figure. Actin served as an internal control for normalization (JPEG 285 kb)

Suppl. Fig. 5

The expression level of CeMbl was reduced in CAG125 worms. QPCR analysis of CeMbl RNA was performed using three cDNA preparations transcribed from one RNA sample. The RNA sample was collected from several CAG125 transgenic lines. In this experiment, the CeMbl RNA expression level in CAG125 worms is approximately 80% of that in GFP worms (P=0.001) (JPEG 190 kb)

Suppl. Fig. 6

Over-expression of CeMbl slightly increased the expression level of the gfp transcript containing CAG125 or CUG125 repeats. QPCR analysis of gfp transcript was performed using three cDNA preparations transcribed from one RNA sample. The RNA sample was collected from several CAG125 or CUG125 transgenic lines with or without co-expression of CeMbl-a. In this experiment, the average gfp RNA level in CUG125 and CAG125 worms co-expressing CeMbl is approximately 1.4 fold of that in control worms (JPEG 199 kb)

Suppl. Fig. 7

Either CeMbl-a or CeMbl-b over-expression can not reverse the (A) muscle morphology and (B) brood size of CUG125 and CAG125 worms. At least 30 worms of F3 to F4 generation from three independent injections were analyzed. Statistical analysis was performed using a student’s t-test. There is no significant difference between worms expressing untranslated CUG125 or CAG125 with or without CeMbl over-expression (JPEG 477 kb)

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Wang, LC., Chen, KY., Pan, H. et al. Muscleblind participates in RNA toxicity of expanded CAG and CUG repeats in Caenorhabditis elegans . Cell. Mol. Life Sci. 68, 1255–1267 (2011). https://doi.org/10.1007/s00018-010-0522-4

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