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Translation efficiency (TE) changes between shNC and shGm26917 FGSCs were quantified by ribosome profiling (Ribo-seq) and RNA-seq integration [Ribo-Seq]

ABSTRACT: Integrated analysis of Ribo-seq and RNA-seq data was performed to identify translation efficiency altered upon Gm26917 knockdown, To assess changes in translational efficiency and validate Gm26917 function.

ORGANISM(S): Mus musculus

PROVIDER: GSE304964 | GEO | 2026/06/18

REPOSITORIES: GEO

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	GSE304964_ORF_allExp.xlsx	Xlsx
	GSE304964_diffTE.xlsx	Xlsx

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Identifying differentially expressed genes (DEGs) between shNC and shGm26917 female germline stem cells (FGSCs) [RNA-Seq]

Project description:Integrated analysis of RNA-seq and Ribosome profiling and sequencing (Ribo-seq) data was performed to identify translation efficiency altered upon Gm26917 knockdown.

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Project description:METTL16 is a member of methyltransferase like (METTL) family. Unlike well-studied METTL3 and METTL14, we found a much higher percentage of METTL16 is localized in the cytosol. The subcellular distribution holds the ability to potentiate translation efficiency. Via Far-western blotting and Co-Immunoprecipitation (Co-IP) assays, we have identified the direct interactions between METTL16 and eukaryotic initiation factor 3 (eIF3) a and b. Via cross-linking immunoprecipitation and qPCR (CLIP-qPCR), we have discovered the direct associations between METTL16 and rRNAs. The METTL16-eIF3a/b and METTL16-rRNAs interactions induce the binding between eIF3 and 18S rRNA, promote the formation of 43S preinitiation complex, and eventually expedite translation initiation, the rate-limiting step of translation. To determine the exact effects of METTL16 on translation efficiency, we performed ribosome profiling (Ribo-seq) with HEK293T upon CRISPR-Cas9-induced METTL16 knockout. To guarantee the repeatability and avoid any potential off-target effects, we included 3 distint sgRNAs against METTL16.

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Determine the effects of Wdr4 on global translation efficiency via ribosome profiling (Ribo-seq)

Project description:Tumor-associated macrophages (TAMs) play a pivotal role in shaping the tumor microenvironment in hepatocellular carcinoma (HCC), significantly influencing disease progression and patient outcomes. WDR4, a tRNA-binding scaffold essential for N7-methylguanosine (m7G) methylation, has an undefined role in HCC-associated TAMs. Here, we found that WDR4 expression is markedly upregulated in HCC-infiltrating TAMs and correlates with poor prognosis in patients. Using HCC mouse models, we demonstrated that WDR4 depletion reprograms TAMs toward an anti-tumor phenotype and suppresses HCC progression. To investigate the impact of WDR4 on translational efficiency, we performed ribosome profiling (Ribo-seq) on Wdr4-deficient TAMs. To ensure reproducibility and minimize potential off-target effects, we included TAMs sorted from orthotopic HCC-bearing Wdr4 conditional knockout mice, as well as immortalized bone marrow-derived macrophages (iBMDMs) with Wdr4 knocked out using two independent sgRNAs.

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Nascent Ribo-Seq measures ribosomal loading time and reveals kinetic impact on ribosome density

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.

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Project description:We report systematical profiling of translation efficiency and mRNA stability dependent on the dynamics of poly(A)-tail length in stress conditions of human cells. In this study, we developed a new feasible method measuring poly(A)-tail length called TED-seq and applied it to investigate the change of mRNA's poly(A)-tail lengths in ER stress pharmacologically induced by thapsigargin (THAP). Combined with other global RNA analyses such as RNA-seq, Ribo-seq and PRO-seq, we observed that ER stress induced lenthening poly(A)-tail length, in particular of ER-stress-regulators, upon ER stress. More specifically, these mRNAs are translationally de-repressed and more stabilized based on increase in poly(A)-tail length. We also identified that insoluble fractions which include stress-induced RNA-granules have overall shorter length of poly(A) tail. Taken together, our data suggest that poly(A)-tail lengths are dynamically regulated and influence both translation efficiency and mRNA stability in ER stress.

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