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      Expression of Sam68 Associates with Neuronal Apoptosis and Reactive Astrocytes After Spinal Cord Injury

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          Abstract

          Src-associated in mitosis (Sam68; 68 kDa) is a novel RNA-binding protein that belongs to the signal transduction and activation of RNA family involved in various biological processes. However, the expression and roles of Sam68 in the central nervous system remain unknown. In the present study, we performed a spinal cord injury (SCI) model in adult rats and found a significant increase of Sam68 protein levels in this model, which reached a peak at day 3 and then gradually returned to normal levels at day 14 after SCI. We use immunohistochemistry analysis revealing a widespread distribution of Sam68 in the spinal cord. In addition, double-immunofluorescence staining showed that Sam68 immunoreactivity was found predominantly in neurons and astrocytes. Moreover, colocalization of Sam68/active caspase-3 has been respectively detected in neuronal nuclei, and colocalization of Sam68/PCNA has been detected in glial fibrillary acidic protein. In vitro, we found that depletion of Sam68 by short interfering RNA inhibits neuronal apoptosis and astrocyte proliferation and decreases cyclin D1 protein levels. In conclusion, this is the first study to find the Sam68 expression in SCI. Our results suggest that Sam68 might be illustrated in the apoptosis of neurons and proliferation of astrocytes after SCI. This research will provide new drug targets for clinical treatment of SCI.

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          The RNA-binding protein Sam68 modulates the alternative splicing of Bcl-x

          The RNA-binding protein Sam68 is involved in apoptosis, but its cellular mRNA targets and its mechanism of action remain unknown. We demonstrate that Sam68 binds the mRNA for Bcl-x and affects its alternative splicing. Depletion of Sam68 by RNA interference caused accumulation of antiapoptotic Bcl-x(L), whereas its up-regulation increased the levels of proapoptotic Bcl-x(s). Tyrosine phosphorylation of Sam68 by Fyn inverted this effect and favored the Bcl-x(L) splice site selection. A point mutation in the RNA-binding domain of Sam68 influenced its splicing activity and subnuclear localization. Moreover, coexpression of ASF/SF2 with Sam68, or fusion with an RS domain, counteracted Sam68 splicing activity toward Bcl-x. Finally, Sam68 interacted with heterogenous nuclear RNP (hnRNP) A1, and depletion of hnRNP A1 or mutations that impair this interaction attenuated Bcl-x(s) splicing. Our results indicate that Sam68 plays a role in the regulation of Bcl-x alternative splicing and that tyrosine phosphorylation of Sam68 by Src-like kinases can switch its role from proapoptotic to antiapoptotic in live cells.
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            Alternative splicing of the cyclin D1 proto-oncogene is regulated by the RNA-binding protein Sam68.

            Human cyclin D1 is expressed as two isoforms derived by alternate RNA splicing, termed D1a and D1b, which differ for the inclusion of intron 4 in the D1b mRNA. Both isoforms are frequently upregulated in human cancers, but cyclin D1b displays relatively higher oncogenic potential. The splicing factors that regulate alternative splicing of cyclin D1b remain unknown despite the likelihood that they contribute to cyclin D1 oncogenicity. In this study, we report that Sam68, an RNA-binding protein frequently overexpressed in prostate cancer cells, enhances splicing of cyclin D1b and supports its expression in prostate cancer cells. Chromatin immunoprecipitation and RNA coimmunoprecipitation experiments showed that Sam68 is recruited to the human CCND1 gene encoding cyclin D1 and that it binds to cyclin D1 mRNA. Transient overexpression and RNAi knockdown experiments indicated that Sam68 acts to enhance endogenous expression of cyclin D1b. Minigene reporter assays showed that Sam68 directly affected alternative splicing of CCND1 message, with a preference for the A870 allele that is known to favor cyclin D1b splicing. Sam68 interacted with the proximal region of intron 4, and its binding correlated inversely with recruitment of the spliceosomal component U1-70K. Sam68-mediated splicing was modulated by signal transduction pathways that elicit phosphorylation of Sam68 and regulate its affinity for CCND1 intron 4. Notably, Sam68 expression positively correlates with levels of cyclin D1b, but not D1a, in human prostate carcinomas. Our results identify Sam68 as the first splicing factor to affect CCND1 alternative splicing in prostate cancer cells, and suggest that increased levels of Sam68 may stimulate cyclin D1b expression in human prostate cancers.
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              The RNA-binding protein Sam68 is a multifunctional player in human cancer.

              Src associated in mitosis, of 68 kDa (Sam68) is a KH domain RNA-binding protein that belongs to the signal transduction and activation of RNA family. Although ubiquitously expressed, Sam68 plays very specialized roles in different cellular environments. In most cells, Sam68 resides in the nucleus and is involved in several steps of mRNA processing, from transcription, to alternative splicing, to nuclear export. In addition, Sam68 translocates to the cytoplasm upon cell stimulation, cell cycle transitions or viral infections, where it takes part to signaling complexes and associates with the mRNA translation machinery. Recent evidence has linked Sam68 function to the onset and progression of endocrine tumors, such as prostate and breast carcinomas. Notably, all the biochemical activities reported for Sam68 seem to be implicated in carcinogenesis. Herein, we review the recent advancement in the knowledge of Sam68 function and regulation and discuss it in the frame of its participation to neoplastic transformation and tumor progression.
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                Author and article information

                Journal
                Cellular and Molecular Neurobiology
                Cell Mol Neurobiol
                Springer Nature America, Inc
                0272-4340
                1573-6830
                April 2017
                May 28 2016
                April 2017
                : 37
                : 3
                : 487-498
                Article
                10.1007/s10571-016-0384-x
                27236696
                04f66486-7609-4217-baa4-72b2f2c0d00d
                © 2017

                http://www.springer.com/tdm

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