Background A common feature of non-coding repeat expansion disorders is the accumulation of RNA repeats as RNA foci in the nucleus and/or cytoplasm of affected cells. These RNA foci can be toxic by sequestering RNA-binding proteins. The reduced effective concentration of these factors can then affect various steps of post-transcriptional gene regulation, such as alternative mRNA splicing, translational regulation, mRNA transport, or mRNA stability. However, the precise step that is affected by c9orf72 repeat expansions, the major genetic cause of Amyotrophic Lateral Sclerosis, is still ill defined. Objectives We investigated the possible mechanisms wherebyc9orf72 repeats might affect cell viability by identifying proteins that bind to c9orf72repeats, and analysing their expression and subcellular distribution. Methods A 31 pure GGGGCC repeat (G4C2)31 was obtained by in vitro ligation of complementary nucleotides, and cloned into mammalian expression plasmids. Mouse motoneuronal NSC34 cells and human HeLa cells transfected with (G4C2)31 repeats were used as cellular models of ALS. Immunofluorescence analysis coupled to fluorescence in situ hybridisation (FISH) were used to assess RNA foci formation and localization of binding partners. Global protein synthesis was monitored with the SUnSET method based on puromycin incorporation. Results To get insights into the mechanisms whereby c9orf72 might induce cell toxicity, we used an in vitro-transcribed biotinylated RNA containing the (G4C2)31 repeats to identify binding proteins. Through mass spec analysis of bands excised from SDS PAGE, we were able to identify many different factors involved in post-transcriptional gene regulation. In particular, members of the hnRNP and SR family of proteins, both regulators of alternative splicing, as well as translational regulators, including initiation and elongation factors, but also Pur-alpha, Pur-beta and other translation regulatory proteins were found. The expression of (G4C2)31 repeats is sufficient to induce the formation of intra-nuclear RNA foci in NSC34 and HeLa cells. A significant, although not complete, sequestration of some of the above factors into RNA foci was observed. Most strikingly, (G4C2)31 repeat widely affects the overall distribution of Pur-alpha and its binding partner FMRP, that accumulate into intra-cytosolic granules which are positive for the expression of stress granules markers. In these conditions, global protein synthesis turned out to be decreased, as measured by the reduction in puromycin incorporation into nascent peptide chains. Discussion and conclusions Our observations show that c9orf72repeats are able to activate a stress response that lead to a general reduction of translation, and suggest that this might be dueto c9orf72 ability to bind and sequester translational regulators.
G4C2 repeat-induced translational repression in a cellular model of C9orf72 ALS.
Mauro Cozzolino;
2014
Abstract
Background A common feature of non-coding repeat expansion disorders is the accumulation of RNA repeats as RNA foci in the nucleus and/or cytoplasm of affected cells. These RNA foci can be toxic by sequestering RNA-binding proteins. The reduced effective concentration of these factors can then affect various steps of post-transcriptional gene regulation, such as alternative mRNA splicing, translational regulation, mRNA transport, or mRNA stability. However, the precise step that is affected by c9orf72 repeat expansions, the major genetic cause of Amyotrophic Lateral Sclerosis, is still ill defined. Objectives We investigated the possible mechanisms wherebyc9orf72 repeats might affect cell viability by identifying proteins that bind to c9orf72repeats, and analysing their expression and subcellular distribution. Methods A 31 pure GGGGCC repeat (G4C2)31 was obtained by in vitro ligation of complementary nucleotides, and cloned into mammalian expression plasmids. Mouse motoneuronal NSC34 cells and human HeLa cells transfected with (G4C2)31 repeats were used as cellular models of ALS. Immunofluorescence analysis coupled to fluorescence in situ hybridisation (FISH) were used to assess RNA foci formation and localization of binding partners. Global protein synthesis was monitored with the SUnSET method based on puromycin incorporation. Results To get insights into the mechanisms whereby c9orf72 might induce cell toxicity, we used an in vitro-transcribed biotinylated RNA containing the (G4C2)31 repeats to identify binding proteins. Through mass spec analysis of bands excised from SDS PAGE, we were able to identify many different factors involved in post-transcriptional gene regulation. In particular, members of the hnRNP and SR family of proteins, both regulators of alternative splicing, as well as translational regulators, including initiation and elongation factors, but also Pur-alpha, Pur-beta and other translation regulatory proteins were found. The expression of (G4C2)31 repeats is sufficient to induce the formation of intra-nuclear RNA foci in NSC34 and HeLa cells. A significant, although not complete, sequestration of some of the above factors into RNA foci was observed. Most strikingly, (G4C2)31 repeat widely affects the overall distribution of Pur-alpha and its binding partner FMRP, that accumulate into intra-cytosolic granules which are positive for the expression of stress granules markers. In these conditions, global protein synthesis turned out to be decreased, as measured by the reduction in puromycin incorporation into nascent peptide chains. Discussion and conclusions Our observations show that c9orf72repeats are able to activate a stress response that lead to a general reduction of translation, and suggest that this might be dueto c9orf72 ability to bind and sequester translational regulators.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
