We identified the - to our knowledge - first RNA binding protein without helicase activity that promotes translation of its target mRNAs. The CCHC-Zinc Finger, Nucleic Acid Binding Protein (CNBP) is a eukaryote-conserved nucleic-acid binding protein required in mammals for embryonic development. It contains seven CCHC-type zinc-finger domains and was suggested to act as a nucleic acid chaperone, as well as a transcription factor. Here, we identify all CNBP isoforms as cytoplasmic, messenger RNA (mRNA)-binding proteins. Using PAR-CLIP, we mapped its binding sites on RNA at nucleotide-level resolution on a genome-wide scale and found that CNBP interacted with 4178 mRNAs in a human cell line, preferentially at a G-rich motif close to the AUG start codon on mature mRNAs. Loss- and gain-of-function analyses coupled with system-wide RNA and protein quantification revealed that CNBP did not affect RNA abundance, but rather promoted translation of its targets. This is consistent with an RNA chaperone function of CNBP helping to resolve secondary structures, thus promoting translation. The impact of sequence-specific translational regulatory events controlled by RBPs cannot be studied by standard mRNA quantification at a systems-wide scale. Thus, the study of translational regulation affords our group the opportunity to become experts in state-of-the-art methods measuring translation efficiency, including ribosome profiling (Ingolia et al., 2009) and/or approaches to measuring protein abundance, including SILAC and iTRAQ (Ross et al., 2004; Schwanhausser et al., 2009). Combined with the biochemical assays established for the study of CNBP these protocols will allow us to dissect the molecular function of one of the most abundant RBPs in mammalian cells and tissues, YBX1, which we are implicating in translational regulation processes.

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2
Fiscal Year
2017
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Arthritis, Musculoskeletal, Skin Dis
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Gogakos, Tasos; Brown, Miguel; Garzia, Aitor et al. (2017) Characterizing Expression and Processing of Precursor and Mature Human tRNAs by Hydro-tRNAseq and PAR-CLIP. Cell Rep 20:1463-1475
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Mattijssen, Sandy; Arimbasseri, Aneeshkumar G; Iben, James R et al. (2017) LARP4 mRNA codon-tRNA match contributes to LARP4 activity for ribosomal protein mRNA poly(A) tail length protection. Elife 6:
Benhalevy, Daniel; Gupta, Sanjay K; Danan, Charles H et al. (2017) The Human CCHC-type Zinc Finger Nucleic Acid-Binding Protein Binds G-Rich Elements in Target mRNA Coding Sequences and Promotes Translation. Cell Rep 18:2979-2990
Arimbasseri, Aneeshkumar G; Iben, James; Wei, Fan-Yan et al. (2016) Evolving specificity of tRNA 3-methyl-cytidine-32 (m3C32) modification: a subset of tRNAsSer requires N6-isopentenylation of A37. RNA 22:1400-10
Arimbasseri, Aneeshkumar G; Blewett, Nathan H; Iben, James R et al. (2015) RNA Polymerase III Output Is Functionally Linked to tRNA Dimethyl-G26 Modification. PLoS Genet 11:e1005671