Article

A regulated PNUTS mRNA to lncRNA splice switch mediates EMT and tumour progression

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Abstract

The contribution of lncRNAs to tumour progression and the regulatory mechanisms driving their expression are areas of intense investigation. Here, we characterize the binding of heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) to a nucleic acid structural element located in exon 12 of PNUTS (also known as PPP1R10) pre-RNA that regulates its alternative splicing. HnRNP E1 release from this structural element, following its silencing, nucleocytoplasmic translocation or in response to TGFβ, allows alternative splicing and generates a non-coding isoform of PNUTS. Functionally the lncRNA-PNUTS serves as a competitive sponge for miR-205 during epithelial–mesenchymal transition (EMT). In mesenchymal breast tumour cells and in breast tumour samples, the expression of lncRNA-PNUTS is elevated and correlates with levels of ZEB mRNAs. Thus, PNUTS is a bifunctional RNA encoding both PNUTS mRNA and lncRNA-PNUTS, each eliciting distinct biological functions. While PNUTS mRNA is ubiquitously expressed, lncRNA-PNUTS appears to be tightly regulated dependent on the status of hnRNP E1 and tumour context.

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Change history

  • Corrected online 13 November 2017

    In this Addendum, the authors include western blot data using a C-terminal PNUTS antibody. This is important in that an annotation of the alternative spliced form of PNUTS, denoted in the UCSC genome browser (https://genome.ucsc.edu/), depicts it as a non-coding RNA. However, downstream of the alternative splice site is an alternative AUG located in frame in the PNUTS ORF at position 1039. The potential for a protein product of 61 kDa being generated from this AUG was examined experimentally using a C-terminal raised antibody to PNUTS to exclude the possibility that the N-terminal deletion of the splice isoform was not the reason that the predicted 61-kDa protein was not detected in cells using an N-terminal generated antibody. The results presented here confirm our previous results using the N-terminal PNUTS antibody and originally presented in Supplementary Fig. 2b of the Article; namely, that this predicted 61-kDa product is not detectable in cells under the conditions used, even under conditions of overexpression. Figure (see PDF): lncRNA-PNUTS does not encode for a N-terminal truncated-protein product. The result of a western blot analysis of PNUTS protein expression in CaCo-2 cells upon transient lncRNA-PNUTS expression (3 days) or TGFβ treatment (1 day) is shown. The C-terminal antibody used was EPR11706 (Abcam: Ab173285; clone PPP1R10; 1/1000 dilution) raised against the C-terminal region of the PNUTS protein (amino acids 550–650). The western blot protocol and extracts used in this experiment were identical to those described in Supplementary Fig. 2 of the original Article.

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Acknowledgements

The authors are grateful to M. Gooz for help with the microscopic analysis. We also thank Y. Shao for histopathological analysis and K. Gibbs (Charleston, South Carolina USA—Biorepository & Tissue Analysis Shared Resource at Medical University of South Carolina) for providing the human tumour samples. We thank M. Bidyut for his assistance with the cloning and K. Noguchi for his help with hnRNP E1 plasmid constructs and J. Isaacs for her precious help concerning hypoxia experiments. This work was supported in part by the Cell & Molecular Imaging, Small Animal Imaging, and the Biorepository & Tissue Analysis Shared Resources, Hollings Cancer Center, Medical University of South Carolina (P30 CA138313), and the Shared Instrumentation Grant S10 OD018113. This work was also supported by grants CA555536 and CA154664 from the National Cancer Institute to P.H.H.

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Affiliations

  1. Department of Biochemistry, Medical University of South Carolina, Charleston, South Carolina 29425, USA

    • Simon Grelet
    • , Laura A. Link
    • , Breege Howley
    • , Clémence Obellianne
    • , Vamsi K. Gangaraju
    • , J. Alan Diehl
    •  & Philip H. Howe
  2. Department of Oral Health Sciences, Medical University of South Carolina, Charleston, South Carolina 29425, USA

    • Viswanathan Palanisamy
  3. Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29425, USA

    • Viswanathan Palanisamy
    • , Vamsi K. Gangaraju
    • , J. Alan Diehl
    •  & Philip H. Howe

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Contributions

The study was supervised by P.H.H. The conception and design were made by S.G. and P.H.H. The methodology was developed by S.G. and P.H.H. Acquisition and interpretation of the data was carried out by S.G. and P.H.H. The Affymetrix experiment and care of the mice was provided by B.H. Mice injections and dissection were performed by S.G., L.A.L. and C.O. The manuscript was written by S.G. and P.H.H. and all the authors contributed to its reviewing. V.P., V.K.G. and J.A.D. provided critical discussion.

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The authors declare no competing financial interests.

Corresponding author

Correspondence to Philip H. Howe.

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