The Mis-splicing of REST
We discussed that there are many potential applications for our proposed system. After confiding with academic and industrial experts; however, we decided the best application for our system would be in the realm of therapeutics. We sought a disease in which aberrant splicing patterns could contribute. After long discussions and thorough research, we finally decided on a disease and cell type to apply our system.
We have selected misregulation of the transcription factor REST in certain forms of aggressive cancer as a disease state that could potentially be treated with our system for alternative splicing regulation. REST protein, encoded by the REST gene, is a tumor suppressor protein that binds to more than 2000 known target oncogenes. (1) It represses transcription by binding a DNA sequence element called the neuron-restrictive silencer element, known as RE1, recruiting co-repressors as well as chromatin modifying enzymes that change the topology of the oncogenes, all in all down-regulating the expression of these oncogenes.
In a normal splicing event, exon 4 in the pre-mature mRNA is skipped and the mature mRNA is accordingly translated. A certain balance of isoforms from the REST gene is required for functional suppression function.
Loss of REST protein has been linked to forms of small cell lung cancer (SCLC), colon cancer, and recently researchers at Wisconsin University have discovered a subset of breast cancers which display a highly aggressive disease course in which the tumor suppressor gene REST is lost. (2) In this subset, REST gene is mis-spliced, leading to the inclusion of the normally skipped exon. This exon includes a premature stop codon, which, upon its identification by the translation machinery, results in the production of a truncated REST protein called REST4.
This truncated protein has only 5 out of 8 the zinc fingers, resulting in an incomplete DNA domain necessary for oncogene binding. The new protein also lacks a C terminus for recruiting the corepressors and chromatin modifying enzymes, leading to the protein’s loss of function as a tumor suppressor. (3)
REST4 isoform producing splicing event is caused by abnormal levels of a trans splicing factor called SR100. SR100 gene also has an RE1 site to which the REST protein would bind and suppress the SR100 gene.(4) Accordingly, REST protein can regulate its own levels to restore the normal endogenous balance of isoforms. In fact, it has been shown that successful introduction of REST protein into small cell lung cancer cell line via protein augmentation has restored REST levels on a longer term compared to the time frame associated with the usual administration period.(5)
We ran preliminary experiments on the H82 Small Cell Lung Cancer cell line. To see the our results click here
References(1) (2) (3) Wagoner, Matthew P. et al. “The Transcription Factor REST Is Lost in Aggressive Breast Cancer.” Ed. Jason D. Lieb. PLoS Genetics 6.6 (2010): e1000979. PMC. Web. 1 Nov. 2017.
(4)(5) Shimojo, "The small cell lung cancer-specific isoform of RE1-silencing transcription factor (REST) is regulated by neural-specific Ser/Arg repeat-related protein of 100 kDa (nSR100)." MCR (2013): PMC. Web. 1 Nov. 2017.