Project description:FTSJ1 is a conserved human 2’-O-methyltransferase (Nm-MTase) that modifies several transfer RNAs (tRNAs) at position 32 and the wobble position 34 in the AntiCodon Loop (ACL). Its loss of function has been linked to Non-Syndromic X-Linked Intellectual Disability (NSXLID), and more recently to cancers. However, the molecular mechanisms underlying these pathologies are currently unclear. Here we report a novel FTSJ1 pathogenic variant from a NSXLID patient. Using blood cells derived from this patient and other affected individuals carrying FTSJ1 mutations, we performed an unbiased and comprehensive RiboMethSeq analysis to map the ribose methylation (Nm) on all tRNAs and identify novel targets. In addition, we performed a transcriptome analysis in these cells and found that several genes previously associated with intellectual disability and cancers were deregulated. We also found changes in the miRNA population that suggest potential cross-regulation of some miRNAs with these key mRNA targets. Finally, we show that differentiation of FTSJ1-depleted human neuronal progenitor cells (NPC) into neurons displays long and thin spine neurites compared to control cells. These defects are also observed in Drosophila and are associated with long term memory deficit in this organism. Altogether, our study adds insight into FTSJ1 pathologies in human by the identification of novel FTSJ1 targets and the defect in neuron morphology.
Project description:We report the application of RNA-sequencing for high-throughput profiling of transcriptomes in PC9 cells transfected with stably expressed FTSJ1.
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.
Project description:We conducted ribosome profiling and mRNA-seq using mouse skeletal muscle tissues and 293T cells (NTC control and DROSHA KO) with Hiseq 2500
Project description:Upf1, Upf2, and Upf3 are the central regulators of nonsense-mediated mRNA decay (NMD), the eukaryotic mRNA quality control pathway generally triggered when a premature termination codon is recognized by the ribosome. The NMD-related functions of the Upf proteins likely commence while these factors are ribosome-associated, but little is known of the timing of their ribosome binding, their specificity for ribosomes translating NMD substrates, or the nature and role of any ribosome:Upf complexes. Here, we have elucidated details of the ribosome-associated steps of NMD. By combining yeast genetics with selective ribosome profiling and co-sedimentation analyses of polysomes with wild-type and mutant Upf proteins, our approaches have identified distinct states of ribosome:Upf association. All three Upf factors manifest progressive polysome association as mRNA translation proceeds, but these events appear to be preceded by formation of a Upf1:80S complex as mRNAs initiate translation. This complex is likely executing an early mRNA surveillance function.