A human microprotein that interacts with the mRNA decapping complex

Abstract

Proteomic detection of non-annotated microproteins indicates the translation of hundreds of small open reading frames (smORFs) in human cells, but whether these microproteins are functional or not is unknown. Here, we report the discovery and characterization of a 7-kDa human microprotein we named non-annotated P-body dissociating polypeptide (NoBody). NoBody interacts with mRNA decapping proteins, which remove the 5′ cap from mRNAs to promote 5′-to-3′ decay. Decapping proteins participate in mRNA turnover and nonsense-mediated decay (NMD). NoBody localizes to mRNA-decay-associated RNA–protein granules called P-bodies. Modulation of NoBody levels reveals that its abundance is anticorrelated with cellular P-body numbers and alters the steady-state levels of a cellular NMD substrate. These results implicate NoBody as a novel component of the mRNA decapping complex and demonstrate potential functionality of a newly discovered microprotein.

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Figure 1: The LOC550643/LINC01420 gene encodes the NoBody peptide in a short open reading frame (sORF).
Figure 2: NoBody enriches a complex of proteins involved in mRNA decapping and crosslinks to EDC4.
Figure 3: Sequence dependence of NoBody–EDC4 co-precipitation.
Figure 4: NoBody localizes to P-bodies and its expression level is inversely correlated with P-body numbers.
Figure 5: Perturbation of NoBody expression modulates levels of a nonsense-mediated decay substrate in cells.

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Acknowledgements

We thank A. Ting for the generous use of her Zeiss Axio Observer inverted confocal microscope. This study was supported by a George E. Hewitt Foundation for medical research Postdoctoral Fellowship (Q.C.), NIH (R01 GM102491, A.S.), the NCI Cancer Center Support Grant P30 (CA014195 MASS core, A.S.), The Leona M. and Harry B. Helmsley Charitable Trust grant (#2012-PG-MED002, A.S.), and Dr. Frederick Paulsen Chair/Ferring Pharmaceuticals (A.S.). S.A.S. was supported by Yale University West Campus start-up funds, an American Cancer Society Institutional Research Grant Individual Award for New Investigators from the Yale Cancer Center (IRG-58-012-57) and a NIH Ruth L. Kirchstein postdoctoral fellowship (1F32GM099408). N.G.D. was supported by an Anderson Endowed Postdoctoral Fellowship in the Biological Sciences.

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Contributions

S.A.S. and A.S. conceived the project. N.G.D., J.M., L.W., Q.C., K.H.L., S.A.S. and A.S. designed the experiments. S.A.S. conducted the majority of the experiments and data analyses, including the identification of NoBody, the discovery of NoBody-binding partners, cellular imaging, and NMD studies. N.G.D. performed NoBody purification and in vitro EDC4 co-purification and crosslinking. J.M. generated NoBody expression constructs, and J.M. and Q.C. carried out the cellular EDC4-NoBody crosslinking experiments. L.W. assisted with identification of NoBody binding partners and co-immunoprecipitation experiments. K.H.L. carried out cellular imaging. E.O.C. and J.L.-A. provided advice and experimental help with NMD studies. B.A.B. provided proteomics assistance. N.G.D., A.S. and S.A.S. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Alan Saghatelian or Sarah A Slavoff.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1 and 2, Supplementary Figures 1–16 and Supplementary Note. (PDF 2619 kb)

Supplementary Dataset 1

Immunoprecipitation mass spectrometry data for NoBody pulldowns with anti-FLAG antibody. (XLSX 360 kb)

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D'Lima, N., Ma, J., Winkler, L. et al. A human microprotein that interacts with the mRNA decapping complex. Nat Chem Biol 13, 174–180 (2017). https://doi.org/10.1038/nchembio.2249

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