Ribosome Profiling Analysis of In Vivo Translation

The conserved Musashi (Msi) family of RNA binding proteins are expressed in stem/progenitor and cancer cells, but generally absent from differentiated cells, consistent with a role in cell state regulation. We found that Msi genes are rarely mutated but frequently overexpressed in human cancers and are associated with an epithelial-luminal cell state. Using ribosome profiling and RNA-seq analysis, we found that Msi proteins regulate translation of genes implicated in epithelial cell biology and epithelial-to-mesenchymal transition (EMT), and promote an epithelial splicing pattern. Overexpression of Msi proteins inhibited the translation of Jagged1, a factor required for EMT, and repressed EMT in cell culture and in mammary gland in vivo. Knockdown of Msis in epithelial cancer cells promoted loss of epithelial identity. Our results show that mammalian Msi proteins contribute to an epithelial gene expression program in neural and mammary cell types.

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TRIBE editing reveals specific mRNA targets of eIF4E-BP in Drosophila and in mammals

10.1101/2020.02.24.962852 ◽

2020 ◽

Cited By ~ 2

Author(s):

Hua Jin ◽

Daxiang Na ◽

Reazur Rahman ◽

Weijin Xu ◽

Allegra Fieldsend ◽

...

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Growth Control ◽

Ribosome Profiling ◽

Translational Efficiency ◽

Mtor Inhibition ◽

Mrna Targets ◽

Specific Mrnas ◽

Specific Mrna

Abstract4E-BP (eIF4E-BP) represses translation initiation by binding to the 5’cap-binding protein eIF4E and inhibiting its activity. Although 4E-BP has been shown to be important in growth control, stress response, cancer, neuronal activity and mammalian circadian rhythms, it is not understood how it preferentially represses a subset of mRNAs. We successfully used hyperTRIBE (Targets of RNA-binding proteins identified by editing) to identify in vivo 4E-BP mRNA targets in both Drosophila and mammals under conditions known to activate 4E-BP. The protein associates with specific mRNAs, and ribosome profiling data show that mTOR inhibition changes the translational efficiency of 4E-BP TRIBE targets compared to non-targets. In both systems, these targets have specific motifs and are enriched in translation-related pathways, which correlate well with the known activity of 4E-BP and suggest that it modulates the binding specificity of eIF4E and contributes to mTOR translational specificity.

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Ribosome elongation kinetics of consecutively charged residues are coupled to electrostatic force

10.1101/2021.08.04.455055 ◽

2021 ◽

Author(s):

Sarah E Leininger ◽

Judith Rodriguez ◽

Quyen V Vu ◽

Yang Jiang ◽

Ma Suan Li ◽

...

Keyword(s):

Amino Acid ◽

Conformational Changes ◽

Electrostatic Force ◽

Ribosome Profiling ◽

Peptide Bond Formation ◽

Translation Speed ◽

Charged Residues ◽

Nascent Chain ◽

Chain Conformations

The speed of protein synthesis can dramatically change when consecutively charged residues are incorporated into an elongating nascent protein by the ribosome. The molecular origins of this class of allosteric coupling remain unknown. We demonstrate, using multi-scale simulations, that positively charged residues generate large forces that pull the P-site amino acid away from the A-site amino acid. Negatively charged residues generate forces of similar magnitude but opposite direction. And that these conformational changes, respectively, raise and lower the transition state barrier height to peptide bond formation, explaining how charged residues mechanochemically alter translation speed. This mechanochemical mechanism is consistent with in vivo ribosome profiling data exhibiting a proportionality between translation speed and the number of charged residues, experimental data characterizing nascent chain conformations, and a previously published cryo-EM structure of a ribosome-nascent chain complex containing consecutive lysines. These results expand the role of mechanochemistry in translation, and provide a framework for interpreting experimental results on translation speed.

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RiboTools: a Galaxy toolbox for qualitative ribosome profiling analysis

Bioinformatics ◽

10.1093/bioinformatics/btv174 ◽

2015 ◽

Vol 31 (15) ◽

pp. 2586-2588 ◽

Cited By ~ 27

Author(s):

Rachel Legendre ◽

Agnès Baudin-Baillieu ◽

Isabelle Hatin ◽

Olivier Namy

Keyword(s):

Ribosome Profiling ◽

Profiling Analysis

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Multimapping confounds ribosome profiling analysis: A case‐study of the Hsp90 molecular chaperone

Proteins Structure Function and Bioinformatics ◽

10.1002/prot.25766 ◽

2019 ◽

Vol 88 (1) ◽

pp. 57-68 ◽

Cited By ~ 3

Author(s):

Jackson C. Halpin ◽

Radhika Jangi ◽

Timothy O. Street

Keyword(s):

Molecular Chaperone ◽

Ribosome Profiling ◽

Profiling Analysis

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Ribosome profiling analysis of eEF3-depleted Saccharomyces cerevisiae

Scientific Reports ◽

10.1038/s41598-019-39403-y ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 7

Author(s):

Villu Kasari ◽

Tõnu Margus ◽

Gemma C. Atkinson ◽

Marcus J. O. Johansson ◽

Vasili Hauryliuk

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosome Profiling ◽

Profiling Analysis

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Fidelity of translation initiation is required for coordinated respiratory complex assembly

Science Advances ◽

10.1126/sciadv.aay2118 ◽

2019 ◽

Vol 5 (12) ◽

pp. eaay2118 ◽

Cited By ~ 5

Author(s):

Danielle L. Rudler ◽

Laetitia A. Hughes ◽

Kara L. Perks ◽

Tara R. Richman ◽

Irina Kuznetsova ◽

...

Keyword(s):

Translation Initiation ◽

Mitochondrial Protein ◽

Molecular Machines ◽

Ribosome Profiling ◽

Untranslated Regions ◽

Mitochondrial Protein Synthesis ◽

Mitochondrial Translation ◽

Initiation Factors ◽

Mitochondrial Ribosomes

Mammalian mitochondrial ribosomes are unique molecular machines that translate 11 leaderless mRNAs; however, it is not clear how mitoribosomes initiate translation, since mitochondrial mRNAs lack untranslated regions. Mitochondrial translation initiation shares similarities with prokaryotes, such as the formation of a ternary complex of fMet-tRNAMet, mRNA and the 28S subunit, but differs in the requirements for initiation factors. Mitochondria have two initiation factors: MTIF2, which closes the decoding center and stabilizes the binding of the fMet-tRNAMet to the leaderless mRNAs, and MTIF3, whose role is not clear. We show that MTIF3 is essential for survival and that heart- and skeletal muscle–specific loss of MTIF3 causes cardiomyopathy. We identify increased but uncoordinated mitochondrial protein synthesis in mice lacking MTIF3, resulting in loss of specific respiratory complexes. Ribosome profiling shows that MTIF3 is required for recognition and regulation of translation initiation of mitochondrial mRNAs and for coordinated assembly of OXPHOS complexes in vivo.

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