Integrative genome-wide analysis reveals EIF3A as a key downstream regulator of translational repressor protein Musashi 2 (MSI2)

Author:

Karmakar Shilpita1,Ramirez Oscar1,Paul Kiran V1,Gupta Abhishek K1,Kumari Vandana1,Botti Valentina2,de los Mozos Igor Ruiz3ORCID,Neuenkirchen Nils4,Ross Robert J4,Karanicolas John5,Neugebauer Karla M2,Pillai Manoj M167ORCID

Affiliation:

1. Section of Hematology, Yale Cancer Center, New Haven, CT 06511, USA

2. Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06510, USA

3. Institute of Neurology, University College London and The Francis Crick Institute, London NW1 1AT, UK

4. Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06511, USA

5. Program in Molecular Therapeutics, Fox Chase Cancer Center, Philadelphia, PA 19111, USA

6. Department of Pathology, Yale University School of Medicine, New Haven, CT 06511, USA

7. Yale Stem Cell Center, Yale University School of Medicine, New Haven, CT 06510, USA

Abstract

Abstract Musashi 2 (MSI2) is an RNA binding protein (RBP) that regulates asymmetric cell division and cell fate decisions in normal and cancer stem cells. MSI2 appears to repress translation by binding to 3′ untranslated regions (3′UTRs) of mRNA, but the identity of functional targets remains unknown. Here, we used individual nucleotide resolution cross-linking and immunoprecipitation (iCLIP) to identify direct RNA binding partners of MSI2 and integrated these data with polysome profiling to obtain insights into MSI2 function. iCLIP revealed specific MSI2 binding to thousands of mRNAs largely in 3′UTRs, but translational differences were restricted to a small fraction of these transcripts, indicating that MSI2 regulation is not triggered by simple binding. Instead, the functional targets identified here were bound at higher density and contain more ‘UAG’ motifs compared to targets bound nonproductively. To further distinguish direct and indirect targets, MSI2 was acutely depleted. Surprisingly, only 50 transcripts were found to undergo translational induction on acute loss. Using complementary approaches, we determined eukaryotic translation initiation factor 3A (EIF3A) to be an immediate, direct target. We propose that MSI2 downregulation of EIF3A amplifies these effects on translation. Our results also underscore the challenges in defining functional targets of RBPs since mere binding does not imply a discernible functional interaction.

Funder

National Institutes of Health

Yale Cancer Center

Publisher

Oxford University Press (OUP)

Subject

General Medicine

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