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 half-life, and negatively with tRNA adaptation indices. Similar results were obtained in mESCs, where the lag between IN and FP 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 a corresponding 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:ActD timecourse in HeLa cells, HeLa RNF219 KO cells, HeLa RNF219 KO cells overexpressing 3xFLAG-SBP-RNF219-WT or 3xFLAG-SBP-RNF219-mut
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to evaluate the gene expression of M2-polarized RAW264.7 cells compared with M0 RAW264.7 cells. Methods: RAW264.7 cells were polarized toward M2 using IL-4. M0 RAW264.7 cells were maintained in culture without IL-4. Total RNA of M2 and M0 RAW264.7 cells was extracted. RAW264.7 cells RNA profiles were generated by deep sequencing for two groups (M2 versus M0 RAW264.7 cells) with three samples each. Results: There were significant differences between M2 and M0 RAW264.7 cells. Conclusions: Polarization of RAW264.7 cells from M0 to M2 induces various changes at the transcription level.
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to evaluate the gene expression of M1-polarized RAW264.7 cells compared with M0 RAW264.7 cells. Methods: RAW264.7 cells were polarized toward M1 using LPS and IFN-γ. M0 RAW264.7 cells were maintained in culture without LPS and IFN-γ. Total RNA of M1 and M0 RAW264.7 cells was extracted. RAW264.7 cells RNA profiles were generated by deep sequencing for two groups (M1 versus M0 RAW264.7 cells) with three samples each. Results: There were significant differences between M1 and M0 RAW264.7 cells. Conclusions: Polarization of RAW264.7 cells from M0 to M1 induces various changes at the transcription level.
