GSE193962
poly-PR expression invokes massive changes in cellular transcriptome and leads to upregulation of RQC genes at transcriptional level
Hexanucleotide expansion mutations in C9ORF72 are a cause of familial amyotrophic lateral sclerosis. We previously reported that long arginine-rich dipeptide repeats (DPR), mimicking abnormal proteins expressed from the hexanucleotide expansion, caused translation stalling when expressed in cell culture models. Whether this stalling provides a mechanism of pathogenicity remains to be determined. Here we explored the molecular features of the stalling and examined whether known regulatory mechanisms of ribosome quality control (RQC) are involved in efforts to sense and resolve the stalls. The most well-known RQC mechanisms come largely from studies of mRNA lacking stop codons, whereby ribosomes become stalled at the poly-adenylate sequence in the 3`UTR.  Here we find that arginine-containing DPRs lead to stalling in a length dependent manner, with lengths longer than 40 repeats to invoke strong stalling. Mutational screening of 40×Gly-Xxx DPRs finds that stalling is most pronounced where Xxx are positively charged amino acids. Through a  genome-wide CRISPR screen we find that genes that regulate poly-adenylate readthrough respond differently to the readthrough of arginine-rich DPRs. Indeed, we find evidence that DPR-mediated stalling has no natural regulatory responses even though the stalls may be sensed, as evidenced by an upregulation of RQC gene expression. These findings therefore implicate Arg-rich DPR-mediated stalled ribosomes as posing a particular danger to cellular health and viability. This dataset contains the RNA-sequencing data used to support the conclusions from this study. Overall design: To determine whether poly-adenylate-mediated stalling induces similar gene expression responses as poly-PR mediated stalling, we performed transcriptome analysis. We created three stable HEK293T cell lines expressing GFP-P2A-ChFP (negative control), GFP-P2A-poly-adenylate and GFP-P2A-102×PR under a Tet-inducible promoter. HEK293K cell lines harbouring the constructs under the Tet-on expression system were induced for expression by adding 500 ng/µl doxycycline to the cells for 48 h prior to analysis. RNA-seq analysis was performed for both uninduced cells and cells with 48-h induced expression. Triplicate samples were prepared. Count tables were generated in Galaxy.org using HISAT2 algorithm, and the list of differentially expressed genes were generated with DESeq2 package in R.