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TIMP1 intron 3 retention is a marker of colon cancer progression controlled by hnRNPA1

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Abstract

We previously reported a 40-transcripts signature marking the normal mucosa to colorectal adenocarcinoma transition. Eight of these mRNAs also showed splicing alterations, including a specific intron 3 retention in tissue metalloprotease inhibitor I (TIMP1), which decreased during the early steps of colorectal cancer progression. To decipher the mechanism of intron 3 retention/splicing, we first searched for putative RNA binding protein binding sites onto the TIMP1 sequence. We identified potential serine arginine rich splicing factor 1 (SRSF1) and heterogeneous nuclear RiboNucleoProtein A1 (hnRNPA1) binding sites at the end of intron 3 and the beginning of exon 4, respectively. RNA immunoprecipitation showed that hnRNPA1, but not SRSF1 could bind to the corresponding region in TIMP1 pre-mRNA in live cells. Furthermore, using a TIMP1-based ex vivo minigene approach, together with a plasmon resonance in vitro RNA binding assay, we confirmed that hnRNPA1 could indeed bind to wild type TIMP1 exon 4 pre-mRNA and control TMP1 intron 3 splicing, the interaction being abolished in presence of a mutant sequence that disrupted this site. These results indicated that hnRNPA1, upon binding to TIMP1 exon 4, was a positive regulator of intron 3 splicing. We propose that this TIMP1-intron 3 + transcript belongs to the class of nuclear transcripts with “detained” introns, an abundant molecular class, including in cancer.

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Data availability

Sequences of PCR primers will be made available on request.

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Acknowledgements

The author wish to thank the Université de Brest for housing.

Funding

This work was supported by the INSERM, the Université de Brest and the Ligue contre le cancer comité du Finistère; M. Flodrops was recipient of a doctoral fellowship from the Ligue contre le cancer (Grant No. RPH18240NN). A. Busson was recipient of a grant from the BioIntelligence program; L. Corcos was partially apointed by Brest University Hospital.

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  1. UMR1078 INSERM, Université de Brest, Génétique Génomique Fonctionnelle et Biotechnologies, 22 avenue Camille Desmoulins, 29200, Brest, France

    Marion Flodrops, Adeline Busson, Pascal Trouvé, Chandran Ka, Brigitte Simon, Danielle Arzur, Catherine Le Jossic-Corcos & Laurent Corcos

  2. Sir William Dunn School of Pathology, University of Oxford, Oxford, England

    Gwendal Dujardin

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Contributions

MF prepared and performed the experiments, analysed the results and drafted the manuscript; GD conceived the experiments and drafted the manuscript; AB performed some of the PCR-based experiments; PT prepared and performed the plasmon resonance experiments; CK participated in minigene engineering; BS and DA provided overall technical expertise and participated in transfection experiments; CLJ-C conceived the experiments and drafted the manuscript; LC concieved the study, analyzed the results, drafted the manuscript, and obtained financial support.

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Correspondence to Laurent Corcos.

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The authors declare having no conflict of interest.

Ethics approval

These procedures were approved by the Ethics committee of Brest University Hospital.

Informed consent

A written informed consent form was elaborated together with the Ethics Committee of Brest University Hospital (headed by Pr. J.M. Boles). Patients had then given their consent to have RNA from their CRC tumors being analyzed for this project (Reference [7] of the present manuscript), and to prepare publication of results with respect to ethics regulation. The inform consent form was returned to the Anatomy and Pathology department of Brest University Hospital that handled and housed the tissue fragments.

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11033_2020_5375_MOESM1_ESM.pdf

Supplementary Figure 1: Structure of the pMG1.1_polR2G-TIMP1 minigene. The upper part shows the TIMP1 genomic cassette, with the numbering starting at the first base of the cassette and ending at the last (708 bp). Positions of the putative SRSF1 (343 bp) and hnRNPA1 (412 bp) binding sites are shown. The lower part shows the pMG1.1_polR2G-TIMP1 minigene plasmid with the wild type TIMP1 or the mutated TIMP1 exon 4 sequence (inserted nucleotides in red). The white boxes on each side of the TIMP1 sequence are from the polR2G genomic sequence [37]. (PDF 86 kb)

11033_2020_5375_MOESM2_ESM.pdf

Supplementary Figure 2: Effects of hnRNPA1 and SRSF1 siRNAs. The mean FC of SRSF1 expression was 0.34 (± 0.14) following siSRSF1 transfection, and the mean FC of hnRNPA1 expression was 0.19 (± 0.15) following sihnRNPA1 transfection. Parallel effects were observed at the protein levels. (PDF 382 kb)

Supplementary file3 (PDF 592 kb)

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Flodrops, M., Dujardin, G., Busson, A. et al. TIMP1 intron 3 retention is a marker of colon cancer progression controlled by hnRNPA1. Mol Biol Rep 47, 3031–3040 (2020). https://doi.org/10.1007/s11033-020-05375-w

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