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Concentration-dependent control of pyruvate kinase M mutually exclusive splicing by hnRNP proteins

Nature Structural & Molecular Biology volume 19, pages 346–354 (2012)Cite this article

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Abstract

Expression of the mammalian pyruvate kinase M (PKM) gene provides an important example of mutually exclusive splicing. We showed previously that the hnRNP proteins A1, A2 and PTB have a crucial role in this process. Here we provide evidence that concentration-dependent interactions involving a network of these proteins are sufficient to determine the outcome of PKM splicing. At high concentrations, such as those found in most cancer cells, hnRNPA1 binding to two sites in the upstream regulated exon (exon 9) orchestrates cooperative interactions leading to exon 9 exclusion. At lower concentrations, binding shifts to downstream intronic sites, such that exon 9 is included and exon 10 mainly excluded, with any mRNA including both exons degraded by nonsense-mediated decay. Together, our results provide a mechanism by which a few general factors control alternative splicing of a widely expressed transcript.

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Figure 1: Mutations of intron 9 sequences derepress exon 9 inclusion.
Figure 2: Exonic sequences are involved in PKM alternative splicing regulation.
Figure 3: hnRNPA1/A2 bind cooperatively to exonic and intronic elements.
Figure 4: Exonic hnRNPA1/A2-binding sites are crucial for exon exclusion.
Figure 5: Full-length intron 10 is required for exon 10 exclusion.
Figure 6: Two mechanisms prevent double-inclusion PKM mRNA when hnRNPA1/A2 and PTB levels are low.
Figure 7: A model for PKM mutually exclusive splicing.

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Acknowledgements

We thank T. Kashima (The Jikei University School of Medicine) for the siRNA to hnRNPs A1 and A2, E. Rosonina for help with semiquantitative PCR assays, D. Campigli Di Giammartino for discussion regarding the RNA immunoprecipitation experiments, P. Richard for help with 5′ labeling, and members of the Manley laboratory for discussions. This work was supported by US National Institutes of Health grant R01 GM048259 (J.L.M.).

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  1. Mo Chen

    Present address: Present address: Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, New York, USA.,

Authors and Affiliations

  1. Department of Biological Sciences, Columbia University, New York, New York, USA

    Mo Chen & James L Manley

  2. Cancer Biology and Genetics Program, Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

    Charles J David

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Contributions

M.C., C.J.D. and J.L.M. conceived the project. M.C. and J.L.M. designed the experiments. C.J.D. discussed the experiments and results and conducted the experiment depicted in Figure 6g. M.C. carried out the rest of the experiments. M.C. and J.L.M. wrote the paper.

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Correspondence to James L Manley.

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Chen, M., David, C. & Manley, J. Concentration-dependent control of pyruvate kinase M mutually exclusive splicing by hnRNP proteins. Nat Struct Mol Biol 19, 346–354 (2012). https://doi.org/10.1038/nsmb.2219

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