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In vivo RNAi mediated knockdown of translation initiation factor eIF3m is associated with alterations in ribosome biogenesis [RNAseq/Riboseq]

ABSTRACT: Translation is a fundamental biological process, which defines every aspect of cell physiology. Dysregulation of protein synthesis is associated with the diseases, such as ribosomopathies, diabetes, and cancer. To examine translation dysregulation in vivo, we employed RNA interference to knockdown m-subunit of the translation initiation factor eIF3 exclusively in mouse liver. We observed an increase in the number of monosomes and multiple signs of impaired translation. We further characterized the early cellular response using transcriptome sequencing, ribosome profiling, whole proteome, and phosphoproteome analyses. The major response of hepatocytes to eIF3m deficiency was associated with the changes in mRNA expression, with almost no effect on translation efficiency for particular mRNAs. The transcription changes fell into two main categories: ribosome biogenesis (increased transcription of ribosomal proteins, dephosphorylation of eIF2α, and inhibition of rRNA processing) and cell metabolism (lipid, amino acid, nucleic acid, and drug metabolism). Overall, this work uncovers a new mode of regulation of protein synthesis mediated by the inhibition of translation initiation. It also highlights advantages of RNAi-based in vivo approach for studying regulatory network associated with essential factors involved in mammalian translation.

ORGANISM(S): Mus musculus

PROVIDER: GSE120533 | GEO | 2020/07/29

REPOSITORIES: GEO

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In vivo RNAi mediated knockdown of translation initiation factor eIF3m is associated with alterations in ribosome biogenesis (mRNA-seq)

Project description:Translation is a fundamental biological process, which defines every aspect of cell physiology. Dysregulation of protein synthesis is associated with the diseases, such as ribosomopathies, diabetes, and cancer. To examine translation dysregulation in vivo, we employed RNA interference to knockdown m-subunit of the translation initiation factor eIF3 exclusively in mouse liver. We observed an increase in the number of monosomes and multiple signs of impaired translation. We further characterized the early cellular response using transcriptome sequencing, ribosome profiling, whole proteome, and phosphoproteome analyses. The major response of hepatocytes to eIF3m deficiency was associated with the changes in mRNA expression, with almost no effect on translation efficiency for particular mRNAs. The transcription changes fell into two main categories: ribosome biogenesis (increased transcription of ribosomal proteins, dephosphorylation of eIF2α, and inhibition of rRNA processing) and cell metabolism (lipid, amino acid, nucleic acid, and drug metabolism). Overall, this work uncovers a new mode of regulation of protein synthesis mediated by the inhibition of translation initiation. It also highlights advantages of RNAi-based in vivo approach for studying regulatory network associated with essential factors involved in mammalian translation.

2020-07-29 | GSE118394 | GEO

In vivo RNAi mediated knockdown of translation initiation factor eIF3m is associated with alterations in ribosome biogenesis (rRNA-seq)

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eIF3 associates with 80S ribosomes to promote translation elongation, mitochondrial homeostasis, and muscle health

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Project description:Improper regulation of translation initiation, a vital check-point of protein synthesis in the cell, has been linked to a number of cancers. Overexpression of protein subunits of eukaryotic translation initiation factor 3 (eIF3) has been associated with increased translation of mRNAs involved in cell proliferation. In addition to playing a major role in general translation initiation by serving as a scaffold for the assembly of translation initiation complexes, eIF3 regulates translation of specific cellular mRNAs and viral RNAs. Mutations in the N-terminal Helix-Loop-Helix (HLH) RNA-binding motif of the EIF3A subunit in eIF3 interfere with Hepatitis C Virus Internal Ribosome Entry Site (IRES) mediated translation initiation in vitro. Here we use RNA-seq and ribosome profiling of engineered lentiviral HEK293T cells to show that the EIF3A HLH motif controls translation of a small set of cellular transcripts enriched in oncogenic mRNAs, including MYC.

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eIF3 PAR-CLIP in 293T cells

Project description:Regulation of protein synthesis is fundamental for all aspects of eukaryotic biology by controlling development, homeostasis, and stress responses. The 13-subunit, 800-kDa eukaryotic initiation factor 3 (eIF3) organizes initiation factor and ribosome interactions required for productive translation. However, current understanding of eIF3 function does not explain genetic evidence correlating eIF3 deregulation with tissue-specific cancers and developmental defects. Here we report the genome-wide discovery of human transcripts that interact with eIF3 using photo-activatable crosslinking and immunoprecipitation (PAR-CLIP). eIF3 binds to a highly specific programme of messenger RNAs (mRNAs) involved in cell growth control processes, including cell cycling, differentiation, and apoptosis, via the mRNA 5' untranslated region (5' UTR). Surprisingly, functional analysis of the interaction between eIF3 and two mRNAs encoding cell proliferation regulators, c-Jun and BTG1, reveals that eIF3 employs different modes of RNA stem loop binding to exert either translational activation or repression. Our findings illuminate a new role for eIF3 in governing a specialized repertoire of gene expression and suggest that binding of eIF3 to specific mRNAs could be targeted to control carcinogenesis. 293T cells were treated with 4-thiouridine and protein-RNA complexes were crosslinked, and eIF3-RNA complexes were immunoprecipitated.

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Controlling tissue patterning by translational regulation of signaling transcripts through the core translation factor eIF3c [II]

Project description:Although gene expression is tightly regulated during embryonic development, the impact of translational control has received less experimental attention. Here, we find that eukaryotic translation initiation factor-3 (eIF3) is required for Shh-mediated tissue patterning. Analysis of loss-of-function eIF3 subunit c (Eif3c) mice reveal a unique sensitivity to the Shh receptor Patched 1 (Ptch1) dosage. Genome-wide in vivo enhanced cross-linking immunoprecipitation sequence (eCLIP-seq) shows unexpected specificity for eIF3 binding to a pyrimidine rich motif present in subsets of 5’-UTRs and a corresponding change in the translation of these transcripts by ribosome profiling in Eif3c loss-of-function embryos. We further find that while Eif3c loss-of-function embryos do not show a global decrease in protein synthesis, translation of Ptch1 through this pyrimidine rich motif is specifically sensitive to eIF3 amount. Altogether, this work uncovers hidden specificity of housekeeping translation initiation machinery for the translation of key developmental signaling transcripts.

2021-09-30 | GSE183471 | GEO

Controlling tissue patterning by translational regulation of signaling transcripts through the core translation factor eIF3c [I]

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Redefining the translational status of 80S monosomes

Project description:Fully assembled ribosomes exist in two populations: polysomes and monosomes. While the former has been studied extensively, to what extent translation occurs on monosomes and its importance for overall translational output remains controversial. Here, we used ribosome profiling to examine the translational status of 80S monosomes in Saccharomyces cerevisiae. We found that the vast majority of 80S monosomes are elongating, not initiating. Further, most mRNAs exhibit some degree of monosome occupancy, with monosomes predominating on nonsense-mediated decay (NMD) targets, upstream open reading frames (uORFs), canonical ORFs shorter than ~590 nucleotides and ORFs for which the total time required to complete elongation is substantially shorter than that required for initiation. Importantly, mRNAs encoding low-abundance regulatory proteins tend to be enriched in the monosome fraction. Our data highlight the importance of monosomes for the translation of highly regulated mRNAs.Â We examined the translational status of single 80S ribosomes using ribosome profiling, and compared these monosome footprints to both polysome ribosome footprints and general ribosome profiling. RNASeq libraries were also prepared from the overall sample input.

2016-02-11 | E-GEOD-76117 | biostudies-arrayexpress

eIF3 and its mRNA-entry-channel arm contribute to the recruitment of mRNAs with long 5'-untranslated regions

Project description:Translation initiation in eukaryotes is multi-step pathway and the most regulated phase of translation. Eukaryotic initiation factor 3 is the largest and most complex of the translation initiation factors, and it contributes to events throughout the initiation pathway. In particular, eIF3 appears to play critical roles in mRNA recruitment. More recently, eIF3 has been implicated in driving the selective translation of specific classes of mRNAs. However, unraveling the mechanism of these diverse contributions — and disentangling the roles of the individual subunits of the eIF3 complex — remains challenging. We employed ribosome profiling of budding yeast cells expressing two distinct mutations targeting the eIF3 complex. These mutations either disrupt the entire complex or subunits positioned near the mRNA-entry channel of the ribosome which appear to relocate during or in response to mRNA binding and start-codon recognition. Disruption of either the entire eIF3 complex or specific targeting of these subunits affects mRNAs with long 5’-untranslated regions and whose translation is more dependent on eIF4A, eIF4B, and Ded1 but less dependent on eIF4G, eIF4E, and PABP. Disruption of the entire eIF3 complex further affects mRNAs involved in mitochondrial processes and with structured 5’-untranslated regions. Comparison of the suite of mRNAs most sensitive to both mutations with those uniquely sensitive to disruption of the entire complex sheds new light on the specific roles of individual subunits of the eIF3 complex.

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