SR proteins, or SRSF (serine/arginine-rich splicing factor), are a family of proteins that are involved the assembly of the spliceosome, which is involved in RNA processing and alternative splicing [2,4,6]. The splicing process includes the removal of non-coding introns and alternative splicing of exons from pre-mRNA transcripts. Multiple mature mRNA transcripts can formed from a single gene by including or excluding different combinations of exons [4,6].
In humans, there are 12 different SR proteins. Each SR protein is composed of two domains: the RNA recognition motif (RRM) and the RS domain. The RRM is involved in binding RNA while the RS domain is involved in binding other proteins [1,6]. A SRSF may activate or repress splicing depending on the nucleotide location that it is bound to an mRNA transcript . Errors that occur during RNA processing and alternative splicing, as well as mutations in genes that encode for SRSFs (e.g. SRSF3), can result in a disease phenotype. Recent studies have found recurring mutations in genes encoding SRSFs. These mutations were shown to result in altered splicing patterns of their targets in human tumor samples [2,5].
While it has been previously demonstrated that mutations in SR proteins affect the splicing patterns of their targets, little is known about how changes in SRSF expression effects the splicing of other SR proteins. Due to prominent role of SR proteins in alternative splicing, it was predicted that changes in the expression of SRSF3 would result in the production of alternatively spliced products in other SR proteins as well as CLK1, another target of SRSF3.
Materials and Methods
Four samples were created by transfecting HEK cells using lipofectamine 3000 transfection reagent per manufacturers protocol. RNA was extracted from cells that were treated with either an empty vector, a vector containing SRSF3 cDNA, a non-targeting siRNA or a SRSF3 targeting siRNA using the QIAGEN RNeasy Plus Mini Kit.
Primers were designed to flank regions of SRSF1, SRSF3, SRSF5, SRSF6, and CLK1 that were known to contain alternatively spliced regions. RT-PCR was performed with RNA extracts using the QIAGEN One-Step RT-PCR Kit.
RT-PCR products were visualized by performing gel electrophoresis using a 1% agarose gel, and imaged by UV-fluorescence.
RT-PCR products were then isolated, purified and sent to GENEWIZ for Sanger sequencing.
Overexpression and silencing of SRSF3 in samples was determined through RT-PCR using primers specific to SRSF3. SRSF3 appeared to be overexpressed in samples transfected with a vector containing an SRSF3 insert as compared to a sample containing a vector that had no insert. There was no detectable effect on the expression of SRSF3 in samples transfected with silencing RNA (Fig. 1).
The effect of overexpression of SRSF3 on the splicing patterns of three different SR proteins was tested. The splicing patterns of SRSF1, SRSF5, and SRSF6 appeared to be unchanged in response to SRSF3 overexpression (Fig. 2).
CDC2-like kinase 1 (CLK1), an enzyme that phosphorylates SR proteins, is a known target of SRSF3. Studies have shown when SRSF3 expression is decreased, there is an increased production of the CLK1 transcript that contains the exon 4 sequence . This study was able to demonstrate an increased production of the transcript excluding exon 4 when SRSF3 is overexpressed (Fig. 3).
The results of this study indicate that the splice patterns of SRSF1, SRSF5, and SRSF6 are unaffected as a result of SRSF3 overexpression. Silencing of SRSF3 may have an impact on their splice patterns, but further study needs to be performed in order to make this determination.
While overexpression of SRSF3 had no impact on the splicing of SRSF1, SRSF5 or SRSF6, expression was not successfully decreased in the SRSF3 sample transfected with silencing RNA targeting SRSF3. Due to the silencing of SRSF3 being unsuccessful, these experiments can only determine splice patterns are unaffected by the overexpression of SRSF3 specifically.
It is uncertain how the silencing of SRSF3 impacts SRSF1, SRSF5, and SRSF6. Changes in SRSF3 expression could potentially have an impact on any of the 8 other SR proteins not tested in this study. In order to gain insight into how each SRSF interacts with one another and impacts their individual splice patterns, an in-depth study assessing each splicing factor would need to be conducted.
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2. Lee, Y., & Rio, D. C. (2015). Mechanisms and Regulation of Alternative Pre-mRNA Splicing. Annual review of biochemistry, 84, 291-323.
3. Liu, B., Anderson, S. L., Qiu, J., & Rubin, B. Y. (2013). Cardiac glycosides correct aberrant splicing of IKBKAP‐encoded mRNA in familial dysautonomia derived cells by suppressing expression of SRSF3. The FEBS journal, 280(15), 3632-3646.
4. Park, E., Pan, Z., Zhang, Z., Lin, L., & Xing, Y. (2018). The Expanding Landscape of Alternative Splicing Variation in Human Populations. American journal of human genetics, 102(1), 11-26.
5. Yang, S., Jia, R., & Bian, Z. (2018). SRSF5 functions as a novel oncogenic splicing factor and is upregulated by oncogene SRSF3 in oral squamous cell carcinoma. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1865(9), 1161-1172.
6. Zhou, Z., & Fu, X. D. (2013). Regulation of splicing by SR proteins and SR protein-specific kinases. Chromosoma, 122(3), 191-207.