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Analysis of total RNA and ribosome-associated RNA in HTT KO MEF versus control.

ABSTRACT: Analysis of total RNA and ribosome-associated RNA in HTT KO MEF versus control.

PROVIDER: PRJNA863124 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR20689800_1.fastq.gz	Fastqsanger.gz
	SRR20689800_2.fastq.gz	Fastqsanger.gz
	SRR20689801_1.fastq.gz	Fastqsanger.gz
	SRR20689801_2.fastq.gz	Fastqsanger.gz
	SRR20689790_1.fastq.gz	Fastqsanger.gz

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Project description:Purpose: We previously highlighted that the wild-type protein Huntingtin (HTT) interacts with the ribosome in mouse embryonic fibroblasts (MEF). We previously showed that HTT does not control control new protein synthesis at a global level. Hence, we sought to determine whether HTT regulates translation of specific mRNA subsets. To this end, we performed a translatome analysis in HTT-deleted versus control samples. Methods: From HTT-deleted and control MEF, we isolated total cellular RNA via Trizol/chloroform extraction. From matching samples, we performed ribosome purification and isolated ribosome-associated RNA via Trizol/chloroform extraction of ribosome pellets. From n=3 independent pairs of replicates for total RNA and n=3 independent pairs of replicates for ribosome-associated RNA, libraries were prepared and sequenced (Genewiz) (total 12 samples). Read alignment and counting were performed using STAR and HTSeqCount softwares. We performed differential expression analysis comparing HTT-deleted samples to control samples for total RNA on one hand, and HTT-deleted samples to control samples for ribosome-associated RNA on the other hand. We did so using EdgeR with RUVseq normalization based on residuals (k=2). Results: For each RNA hit, we analysed differential association to the ribosome normalized to differential expression, by comparing HTT-deleted and control samples. From this analysis, we identified 115 genes displaying differential ribosome association upon HTT deletion, but for which total RNA expression level was unchanged (cut-offs: false discovery rate (FDR) p- value < 0.05, |log2 fold-change| > 1). Among the pool of translationally-only regulated hits, we found genes with decreased association with the ribosome in absence of HTT (Rxfp1, Kng1, Cxcl9, Trpm6, Wnt2b); and with increased association with the ribosome in absence of HTT (Trpm2, Tox2, Ip6k3, Csf3r, Tlr1). Conclusions: Our results highlight differential ribosome association at a single transcript level upon HTT deletion. Altogether, these data support the hypothesis that HTT regulates the translation of a specific subset of mRNA.

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Analysis of total RNA and ribosome-associated RNA in HTT KO MEF versus control.

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