Controlling Alternative Splicing via Cell Signaling Pathways
On November 18, 2014, Dr. Kristen W. Lynch’s seminar topic was about how the cell signaling pathways can control the process of alternative splicing. Dr. Lynch’s started the seminar by mentioning that about 95% of human genes undergo alternative splicing. Those genes that undergo alternative splicing further change the Open Reading Frame (ORF). ORF is a reading frame of nucleotides that begins with start codon and ends with stop codon, which can translate into polypeptide chains. Therefore, Lynch’s gave the example of how the alternative splicing of CD45 gene alters in the production of different mRNAs that further produces different proteins (polypeptide chains). Thus, the difference in the production of functional protein (tyrosine phosphatase protein) has played the role in the regulation of development and maturation of T-cells, a lymphocyte cell. The difference in the production of tyrosine phophatase protein is due to the variation in alternative splicing of exon 4, 5 and 6 of CD45 gene in T-cells. Multiple signaling pathways that are induced by antigen signaling in turn regulate the alternative splicing. Some of the multiple signaling pathways that Dr. Lynch had mentioned are RAS (G-protein), Glycogen Synthase Kinase-3 (GSK-3) and c-Jun (c-Jun N-terminal Kinase or JNK) signaling. However, Dr. Lynch had illustrated the GSK-3 and JNK signaling pathway. In GSK-3 signaling, PSF is phosphorylated. PSF usually binds to the Exon splicing silencer (ESS) and plays the role in skipping of those exons or silencing of the expression of that particular exon during alternative splicing. Under the resting conditions of T-cells, the PSF is phosphorylated at Threonine 687. The phosphorylated PSF will undergo the conformation change. Thus, the altered conformation of PSF will be recognized by TRAP-150 protein and the PSF-TRAP150 will interact to each other. The interaction of PSF-TRAP150 to each other will prevent the PSF from binding to the ESS. The hn RNP L is already bounded to the ESS for the silencing of those exons. However, the phosphorylated PSF will not be able to bind to the ESS. Hence, the exons are partially repressed during the resting conditions of T-cells (as shown in the following figure 1).

Figure 1: It shows the interaction of PSF with the TRAP-150 that prevents the binding of the PSF to the Exon Splicing Silencer (ESS). hnRNP L ( repressor) is already bounded to the ESS but the phosphorylated PSF cannot bind to the ESS (Lynch, K., and Mena, M. L, 2013). On the other hand, when the foreign antigen activates the T-cell then the T-cell further deactivates the GSK-3. The inactive GSK-3 would not be able to phosphorylated the PSF. The unphosphorylated PSF cannot interact with TRAP-150. Thus, PSF is free to bind to the ESS along with repressor hnRNP L and hnRNP LL. L and LL had competed with splicing factors (activator of splicing) for binding to the Enhancer element. The binding of PSF to the ESS had stabilized the binding of L and LL to the enhancer sequence of CD45 pre-mRNA. Hence, the exon 4 and 5 of pre-mRNA of CD45 is fully repressed as shown in the figure 2.

Figure 2: It shows the free unbound PSF that can easily bind to the Exon Splicing Silencer along with L and LL. As a result, exon 4 and 5 of CD45 are fully repressed (Lynch, K., and Mena, M. L, 2013).
The second signaling pathway that Dr. Lynch had mentioned was about the JNK (or c-Jun N-terminal Kinase) signaling. Dr. Lynch talked about how the activation of c-Jun regulates the transcriptional activity of T-cells. The summary of regulation of transcription through JNK or Jun signaling pathway is shown in the following figure 3.

Figure 3: It shows the signaling pathway of Jun or JNK that regulates the transcriptional activity (I did not use any other source to draw this signaling pathway diagram).
TAK1 is a protein kinase that plays the role in the regulation of transcription. The encounter of foreign antigen by T-cells stimulates the TAK1 kinase. TAK 1 kinase will in turn activates the MKK7 (Mitogen activated protein kinases-7), which is very important component for the JNK signaling transduction pathway. The MKK7 is activated by phosphorylation, which results into the skipping of exon 2 in MKK7 mRNAs. The disruption in skipping of exon 2 of MKK7 reduces the JNK activity. In contrast, when the splice sites are blocked with the help of antisense morpholino oligos or AMO (that blocks the splicing sites in pre-mRNA to modify the splicing events), it induces the skipping of exon 2 in MKK7. The skipped exon 2 of activated MKK7 produces the phosphorylated MKK7 protein kinase. The phosphorylated MKK7 kinase further phosphorylates the threonine and tyrosine residues that are located (within the Threonine-Proline-Tyrosine motif) in kinase subdomain VIII of JNK. The phosphorylated JNK will activate the jun gene. The activated jun gene will produce the c-jun, a protein in human that combines with the other such as c-Fos to form a transcription factor. The increasing amount of transcription factor will induce the differentiation and the activation of T-cells in response to the attacked of foreign antigens. Therefore, Dr. Lynch had revealed that how the alternative splicing, which is regulated by the cell signaling pathway i.e GSK-3 and JNK regulates the development and maturation of T-cells.

Works Cited:
1.Lynch, K., & Metta, L.M. (2013). Alternative Splicing. Encyclopedia of Biological Chemistry, vol. 1, 75-80.
2. Shankarling, G., Cole, B. S., Mallory, M. J., & Lynch, K. W. (2014). Transcriptome-wide RNA interaction profiling reveals physical and functional targets of hnRNP L in human T cells. Molecular and cellular biology, 34(1), 71-83.
3. Tournier, C., Dong, C., Turner, T. K., Jones, S. N., Flavell, R. A., & Davis, R. J. (2001). MKK7 is an essential component of the JNK signal transduction pathway activated by proinflammatory cytokines. Genes & development, 15(11), 1419-1426.
4. http://www.gene-tools.com/