Project description:Mouse testes samples from P8, P10, adult, and Ythdc2-WLA homozygous mutant mice were subjected to CLIP assays to generate YTHDC2-RNA interaction maps.
Project description:mTRAN proteins are are plant specific-components of the mitoribosome. To asses of loss-of-function mtran1 mtran2 double mutants show decreased translation rates, we performed ribosome footprinting coupled to RNA-seq (Ribo-seq).
Project description:Expression of 5'-tRFCys is significantly upregulated during breast cancer metastasis and suppression of 5'-tRFCys significantly reduced metastatic lung colonization of breast cancer. We identified Nucleolin (Ncl) as the direct binding partner of 5'-tRFCys. To identify the molecular mechanism underlying 5'-tRFCys promotion of breast cancer, we performed Ncl HITS-CLIP, RNA-Seq, Ribosome profiling (Ribo-Seq) upon inhibition of 5'-tRFCys. These analyses reveal that 5'-tRFCys enhances Ncl binding to a subset of transcripts, resulting in increased transcript stability of those transcripts and expression.
Project description:mRNAs are generally assumed to be loaded instantly with ribosomes upon entry into the cytoplasm. To measure ribosome density on nascent mRNA, we developed nascent Ribo-Seq (nRibo-Seq) by combining Ribo-Seq with progressive 4-thiouridine labelling. In mouse macrophages, we experimentally determined, for the first time, the lag between the appearance of nascent RNA and its association with ribosomes, which was calculated to be 20 - 22 min for bulk mRNA, and approximated the time it takes for mRNAs to be fully loaded with ribosomes to be 41 - 44 min. Notably, ribosomal loading time is adapted to gene function as rapid loading was observed with highly regulated genes. The lag and ribosomal loading time correlate positively with ORF size and mRNA half-life, and negatively with tRNA adaptation index. Similar results were obtained in mouse embryonic stem cells, where the lag in ribosome loading was even more pronounced with 35 - 38 min. We validated our measurements after stimulation of macrophages with lipopolysaccharide, where the lag between cytoplasmic and translated mRNA leads to uncoupling between input and ribosome-protected fragments. Uncoupling is stronger for mRNAs with long ORFs or half-lives, a finding we also confirmed at the level of protein production by nascent chain proteomics. As a consequence of the lag in ribosome loading, ribosome density measurements are distorted when performed under conditions where mRNA levels are far from steady state expression, and transcriptional changes affect ribosome density in a passive way. This study uncovers an unexpected and considerable lag in ribosome loading, and provides guidelines for the interpretation of Ribo-Seq data taking passive effects on ribosome density into account.
Project description:In order to identify YTHDC2 downstream genes, we performed RNA-seq in WT and YTHDC2 knockout H1975 cells and analyzed the changed mRNAs.