RNA-binding protein SAM68 interacts with endocytic proteins and actin cytoskeleton modulators
DOI:
https://doi.org/10.15407/dopovidi2020.05.103Keywords:
alternative splicing, ITSN1, SAM68, SRSF1Abstract
SAM68 is a nuclear RNA-binding protein involved in the regulation of mRNA processing. SAM68 overexpression is observed in multiple types of cancer. Recently, the possible link between RNA-binding protein SAM68 and scaffold protein ITSN1 has been identified. The aim of the study was to confirm the probability of direct binding bet ween SAM68 and ITSN, analyze the effect of ITSN1 on SAM68-mediated alternative splicing, and identify novel SAM68 partners among endocytic proteins and actin cytoskeleton modulators. The interactions were revealed in pull-down assays using purified recombinant proteins or cell lysates. ITSN1 knockdown in HeLa cells was performed using the shRNA approach. The expression of isoforms produced by alternative splicing was measured using RT-PCR. It was demonstrated that SAM68 directly interacted with ITSN1 in vitro. Next, it was found that ITSN1 knockdown in HeLa cells induced SRSF1 intron 3 retention increasing the expression of the proto-oncogenic isoform of SRSF1 by three times. It was also shown that SH3 domains of AMPH1, BIN1, CTTN1, TKS4, and TKS5 precipitated SAM68 from lysate of 293 cells. As a result, SAM68 directly binds to ITSN1 and interacts with endocytic proteins and actin cytoskeleton modulators, whereas SAM68-mediated splicing in HeLa cells may be regulated by ITSN1.
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Sanchez-Jimenez, F. & Sanchez-Margalet, V. (2013). Role of Sam68 in post-transcriptional gene regulation. Int. J. Mol. Sci., 14, No. 12, pp. 23402-23419. Doi: https://doi.org/10.3390/ijms141223402
Frisone, P., Pradella, D., Di Matteo, A., Belloni, E., Ghigna, C. & Paronetto, M. P. (2015). SAM68: Signal Transduction and RNA Metabolism in Human Cancer. Biomed. Res. Int., 2015, 528954. Doi: https://doi.org/10.1155/2015/528954
Najib, S., Martin-Romero, C., Gonzalez-Yanes, C. & Sanchez-Margalet, V. (2005). Role of Sam68 as an adaptor protein in signal transduction. Cell. Mol. Life Sci., 62, No. 1, pp. 36-43. Doi: https://doi.org/10.1007/s00018-004-4309-3
Asbach, B., Ludwig, C., Saksela, K. & Wagner, R. (2012). Comprehensive analysis of interactions between the Src-associated protein in mitosis of 68 kDa and the human Src-homology 3 proteome. PLoS One, 7, No. 6, e38540. Doi: https://doi.org/10.1371/journal.pone.0038540
Pankivskyi, S., Senchenko, N., Busko, P. & Rynditch, A. (2019). Scaffold proteins ITSN1 and ITSN2 interact with nuclear RNA-binding proteins. Вiopolymers and Cell, 35, No. 2, pp. 81-90. Doi: https://doi.org/10.7124/bc.000999
Herrero-Garcia, E. & O’Bryan, J. P. (2017). Intersectin scaffold proteins and their role in cell signaling and endocytosis. Biochim. Biophys. Acta. Mol. Cell. Res., 1864, No. 1, pp. 23-30. Doi: https://doi.org/10.1016/j.bbamcr.2016.10.005
Alvisi, G., Paolini, L., Contarini, A., Zambarda, C., Di Antonio, V., Colosini, A., & Radeghieri, A. (2018). Intersectin goes nuclear: secret life of an endocytic protein. Biochem. J., 475, No. 8, pp. 1455-1472. Doi: https://doi.org/10.1042/BCJ20170897
Moffat, J., Grueneberg, D. A., Yang, X., Kim, S. Y., Kloepfer, A. M., Hinkle, G. & Root, D. E. (2006). A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell, 124, No. 6, pp. 1283-1298. Doi: https://doi.org/10.1016/j.cell.2006.01.040
Livak, K. J. & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 25, No. 4, pp. 402-408. Doi: https://doi.org/10.1006/meth.2001.1262
Das, S. & Krainer, A. R. (2014). Emerging functions of SRSF1, splicing factor and oncoprotein, in RNA metabolism and cancer. Mol. Cancer Res., 12, No. 9, pp. 1195-1204. Doi: https://doi.org/10.1158/1541-7786.MCR-14-0131
Lareau, L. F., Inada, M., Green, R. E., Wengrod, J. C. & Brenner, S. E. (2007). Unproductive splicing of SR genes associated with highly conserved and ultraconserved DNA elements. Nature, 446, No. 7138, pp. 926-929. Doi: https://doi.org/10.1038/nature05676
Valacca, C., Bonomi, S., Buratti, E., Pedrotti, S., Baralle, F. E., Sette, C. & Biamonti, G. (2010). Sam68 regulates EMT through alternative splicing-activated nonsense-mediated mRNA decay of the SF2/ASF proto-oncogene. J. Cell Biol., 191, No. 1, pp. 87-99. Doi: https://doi.org/10.1083/jcb.201001073
Jeganathan, N., Predescu, D., Zhang, J., Sha, F., Bardita, C., Patel, M. & Predescu, S. (2016). Rac1-mediated cytoskeleton rearrangements induced by intersectin-1s deficiency promotes lung cancer cell proliferation, migration and metastasis. Mol. Cancer, 15, No. 1, 59. Doi: https://doi.org/10.1186/s12943-016-0543-1
Bardita, C., Predescu, D. N., Sha, F., Patel, M., Balaji, G. & Predescu, S. A. (2015). Endocytic deficiency induced by ITSN-1s knockdown alters the Smad2/3-Erk1/2 signaling balance downstream of Alk5. J. Cell. Sci., 128, No. 8, pp. 1528-1541. Doi: https://doi.org/10.1242/jcs.163030
Carman, P. J. & Dominguez, R. (2018). BAR domain proteins-a linkage between cellular membranes, signaling pathways, and the actin cytoskeleton. Biophys. Rev., 10, No. 6, pp. 1587-1604. Doi: https://doi.org/10.1007/s12551-018-0467-7
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