Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells

Dipeptide repeat peptides on the attack Certain neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), are associated with expanded dipeptides translated from RNA transcripts of disease-associated genes (see the Perspective by West and Gitler). Kwon et al. show that the peptides encoded by the expanded repeats in the C9orf72 gene interfere with the way cells make RNA and kill cells. These effects may account for how this genetic form of ALS causes disease. Working in Drosophila, Mizielinska et al. aimed to distinguish between the effects of repeat-containing RNAs and the dipeptide repeat peptides that they encode. The findings provide evidence that dipeptide repeat proteins can cause toxicity directly. Science, this issue p. 1139 and p. 1192; see also p. 1118 Repeated structural motifs encoded by disease genes impair RNA metabolism and cause cell death. [Also see Perspective by West and Gitler] Many RNA regulatory proteins controlling pre–messenger RNA splicing contain serine:arginine (SR) repeats. Here, we found that these SR domains bound hydrogel droplets composed of fibrous polymers of the low-complexity domain of heterogeneous ribonucleoprotein A2 (hnRNPA2). Hydrogel binding was reversed upon phosphorylation of the SR domain by CDC2-like kinases 1 and 2 (CLK1/2). Mutated variants of the SR domains changing serine to glycine (SR-to-GR variants) also bound to hnRNPA2 hydrogels but were not affected by CLK1/2. When expressed in mammalian cells, these variants bound nucleoli. The translation products of the sense and antisense transcripts of the expansion repeats associated with the C9orf72 gene altered in neurodegenerative disease encode GRn and PRn repeat polypeptides. Both peptides bound to hnRNPA2 hydrogels independent of CLK1/2 activity. When applied to cultured cells, both peptides entered cells, migrated to the nucleus, bound nucleoli, and poisoned RNA biogenesis, which caused cell death.

[1]  Patrick G. Shaw,et al.  C9orf72 Nucleotide Repeat Structures Initiate Molecular Cascades of Disease , 2014, Nature.

[2]  S. McKnight,et al.  Phosphorylation-Regulated Binding of RNA Polymerase II to Fibrous Polymers of Low-Complexity Domains , 2013, Cell.

[3]  J. Rothstein,et al.  RAN proteins and RNA foci from antisense transcripts in C9ORF72 ALS and frontotemporal dementia , 2013, Proceedings of the National Academy of Sciences.

[4]  A. Isaacs,et al.  C9orf72 frontotemporal lobar degeneration is characterised by frequent neuronal sense and antisense RNA foci , 2013, Acta Neuropathologica.

[5]  Gene W. Yeo,et al.  Targeted degradation of sense and antisense C9orf72 RNA foci as therapy for ALS and frontotemporal degeneration , 2013, Proceedings of the National Academy of Sciences.

[6]  Nipun A. Mistry,et al.  RNA Toxicity from the ALS/FTD C9ORF72 Expansion Is Mitigated by Antisense Intervention , 2013, Neuron.

[7]  Ryan M. Plocinik,et al.  Partitioning RS domain phosphorylation in an SR protein through the CLK and SRPK protein kinases. , 2013, Journal of molecular biology.

[8]  E. Kremmer,et al.  The C9orf72 GGGGCC Repeat Is Translated into Aggregating Dipeptide-Repeat Proteins in FTLD/ALS , 2013, Science.

[9]  Kevin F. Bieniek,et al.  Unconventional Translation of C9ORF72 GGGGCC Expansion Generates Insoluble Polypeptides Specific to c9FTD/ALS , 2013, Neuron.

[10]  David G Hendrickson,et al.  Differential analysis of gene regulation at transcript resolution with RNA-seq , 2012, Nature Biotechnology.

[11]  Jimin Pei,et al.  Cell-free Formation of RNA Granules: Low Complexity Sequence Domains Form Dynamic Fibers within Hydrogels , 2012, Cell.

[12]  David Heckerman,et al.  A Hexanucleotide Repeat Expansion in C9ORF72 Is the Cause of Chromosome 9p21-Linked ALS-FTD , 2011, Neuron.

[13]  Bruce L. Miller,et al.  Expanded GGGGCC Hexanucleotide Repeat in Noncoding Region of C9ORF72 Causes Chromosome 9p-Linked FTD and ALS , 2011, Neuron.

[14]  Brian B. Gibbens,et al.  Non-ATG–initiated translation directed by microsatellite expansions , 2010, Proceedings of the National Academy of Sciences.

[15]  Cole Trapnell,et al.  Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. , 2010, Nature biotechnology.

[16]  Lior Pachter,et al.  Sequence Analysis , 2020, Definitions.

[17]  H. Ball,et al.  Chemical synthesis and purification of proteins: a methodology. , 2009, International journal of peptide and protein research.

[18]  Mark H. Ellisman,et al.  Hypophosphorylated SR splicing factors transiently localize around active nucleolar organizing regions in telophase daughter nuclei , 2004, The Journal of cell biology.

[19]  L. Bruijn,et al.  Unraveling the mechanisms involved in motor neuron degeneration in ALS. , 2004, Annual review of neuroscience.

[20]  David L. Spector,et al.  Nuclear speckles: a model for nuclear organelles , 2003, Nature Reviews Molecular Cell Biology.

[21]  G. Landreth,et al.  Biochemical characterization and localization of the dual specificity kinase CLK1. , 2000, Journal of cell science.

[22]  Z. Derewenda,et al.  Overcoming expression and purification problems of RhoGDI using a family of "parallel" expression vectors. , 1999, Protein expression and purification.

[23]  J. Bell,et al.  The Clk2 and Clk3 dual-specificity protein kinases regulate the intranuclear distribution of SR proteins and influence pre-mRNA splicing. , 1998, Experimental cell research.

[24]  Lin Jin,et al.  Aberrant RNA Processing in a Neurodegenerative Disease: the Cause for Absent EAAT2, a Glutamate Transporter, in Amyotrophic Lateral Sclerosis , 1998, Neuron.

[25]  T Pawson,et al.  The Clk/Sty protein kinase phosphorylates SR splicing factors and regulates their intranuclear distribution. , 1996, The EMBO journal.

[26]  M. Thiry.  Behavior of interchromatin granules during the cell cycle. , 1995, European journal of cell biology.

[27]  M. Roth,et al.  A conserved epitope on a subset of SR proteins defines a larger family of Pre-mRNA splicing factors , 1995, The Journal of cell biology.

[28]  M B Roth,et al.  SR proteins: a conserved family of pre-mRNA splicing factors. , 1992, Genes & development.

[29]  C. Murphy,et al.  A monoclonal antibody that recognizes a phosphorylated epitope stains lampbrush chromosome loops and small granules in the amphibian germinal vesicle , 1990, The Journal of cell biology.

[30]  Carl O. Pabo,et al.  Cellular uptake of the tat protein from human immunodeficiency virus , 1988, Cell.

[31]  W. Richardson,et al.  The nucleoplasmin nuclear location sequence is larger and more complex than that of SV-40 large T antigen , 1988, The Journal of cell biology.

[32]  William D. Richardson,et al.  A short amino acid sequence able to specify nuclear location , 1984, Cell.

[33]  M. Stephens.  EDF Statistics for Goodness of Fit and Some Comparisons , 1974 .