Structural Biochemistry/Alternative Splicing

Alternative Splicing in Eukaryotes Gene expression is the process that transfers genetic information from a gene made of DNA to a functional gene product made of RNA or protein. Genetic Information flows from DNA to RNA by the process of transcription, and then from RNA to protein by the process of translation. In order to ensure that the proper products are produced, gene expression is regulated at many different stages during and in between transcription and translation. In eukaryotes, the gene contains extra sequences that do not code for protein. In these organisms, transcription of DNA produces pre-mRNA, which must be spliced into mRNA that lacks these intervening sequences or introns, before translation begins. Splicing can be regulated so that different mRNAs can contain or lack regions called exons, in a process called alternative splicing. Alternative splicing allows more than one protein to be produced from a gene Douglas L. Black (2003) Mechanisms of Alternative Pre-Messeneger RNA Splicing. Annual Review of Biochemistry, 72: 291-336, and is an important regulatory step in determining which functional proteins are produced from gene expression. Splicing of pre-mRNA has been proven to be an important mechanism to controlling human development, and misregulation of splicing can lead to disease Mo Chen, James L. Manely (2009) Mechanisms of alternative splicing regulation: insights from molecular and genomic approaches. Nature, 10: 741-754. Mechanism of Alternative Splicing Regulation of Alternative Splicing by the rate of RNAPII Transcription Mechanisms that influence splice site selection are important in regulating alternative splicing, and ensure production of the proper proteins at the right time. Many genes have been proven to be spliced co-transcriptionally Pandya-Jones A., Black DL (2009) Co-Transcriptional Splicing of constitutive and alternative exons. RNA, 10: 1896-908, and the rate of RNA Polymerase II transcription has been suggested to be a regulatory mechanism of co-transcriptional Alternative splicing Kenneth James Howe, Caroline M. Kane, Manuel Ares Jr. (2003) Perturbation of transcription elongation influences the fidelity of internal exon inclusion in Saccharomyces Cerevisiae. RNA, 8: 993-1006 Cramer P., Caceres J.F., Cazalla D., Kadener S., Muro A.F., Baralle F.E., Kornblihtt A.R. Coupling of transcription with alternative splicing: RNA pol II promoters modulate SF2/ASF and 9G8 effects on an exonic splicing enhancer (1999) Molecular Cell, 4 (2), pp. 251-258.. Aebi and Weissman’s “first come, first served” model Aebi, M. and Weissman, S.M. 1987. Precision and orderliness in splicing. Trends Genet. 3: 102-107 shows how rate of transcription can influence the inclusion of internal exons. Slow recognition of the first intron or rapid recognition of the second intron creates a transcription dependent competition for 3' splice site selection. Slow transcription through the second intron could allow recognition of the first intron before the second is even synthesized, while fast transcription through the first two introns could increase the ability of the second intron to compete with the first intron for recognition. Formation of the “A Complex” (figure 2) is a step in alternative splicing which creates a commitment to splice site selection Lim SR, Hertel KJ (2004) Commitment to splice site pairing coincides with A complex formation. Mol. Cell 15: 477–483., and is completed by pairing of the U2 snRNP with the branch point Qin Li, Ji-Ann Lee, Douglas L. Black (2007) Neuronal regulation of alternative pre-mRNA splicing. Nature Reviews Neuroscience 8: 819-831. This data shows control over the rate of transcription could be a very powerful tool in regulation of 3’ splice site selection. One method eukaryotes use to regulate the rate of transcription is sequence based arrest sites located in DNA Spencer CA, Groudine M. Transcription elongation and eukaryotic gene regulation. Oncogene. 1990; 5:777–785.. Using the thermodynamic theory of DNA-dependent transcriptional arrest, Artificial Arrest Sites (ARTAR sites) have been created and shown to affect rates of RNA Polymerase II transcription Dmitry Kulish, Kevin Struhl (2001) TFIIS Enhances Transcriptional Elongation through an Artificial Arrest Site In Vivo. Mol Cell Biol. 13: 4162-4168. By placing ARTAR sites at specific positions in a gene, we have demonstrated the ability to influence 3’ splice site selection (Figure 3). When placed downstream of both 3’ splice sites, the ARTAR site will have no effect on alternative splicing. If placed in a location in between the first and second branch points, the ARTAR site can increase inclusion by decreasing 3’ splice site competition. If placed upstream of either branch point the ARTAR site decreases inclusion by increasing competition for both 3’ splice sites, although the reason for increased competition in this scenario is unknown. Sources 1. Douglas L. Black (2003) Mechanisms of Alternative Pre-Messeneger RNA Splicing. Annual Review of Biochemistry, 72: 291-336 2. Mo Chen, James L. Manely (2009) Mechanisms of alternative splicing regulation: insights from molecular and genomic approaches. Nature, 10: 741-754 3. Pandya-Jones A., Black DL (2009) Co-Transcriptional Splicing of constitutive and alternative exons. RNA, 10: 1896-908 4. Aebi, M. and Weissman, S.M. 1987. Precision and orderliness in splicing. Trends Genet. 3: 102-107 5. Kenneth James Howe, Caroline M. Kane, Manuel Ares Jr. (2003) Perturbation of transcription elongation influences the fidelity of internal exon inclusion in Saccharomyces Cerevisiae. RNA, 8: 993-1006 6. Cramer P., Caceres J.F., Cazalla D., Kadener S., Muro A.F., Baralle F.E., Kornblihtt A.R. Coupling of transcription with alternative splicing: RNA pol II promoters modulate SF2/ASF and 9G8 effects on an exonic splicing enhancer (1999) Molecular Cell, 4 (2), pp. 251-258. 7. Lim SR, Hertel KJ (2004) Commitment to splice site pairing coincides with A complex formation. Mol. Cell 15: 477–483. 8. Spencer CA, Groudine M. Transcription elongation and eukaryotic gene regulation. Oncogene. 1990; 5:777–785. 9. Dmitry Kulish, Kevin Struhl (2001) TFIIS Enhances Transcriptional Elongation through an Artificial Arrest Site In Vivo. Mol Cell Biol. 13: 4162-4168 10. Qin Li, Ji-Ann Lee, Douglas L. Black (2007) Neuronal regulation of alternative pre-mRNA splicing. Nature Reviews Neuroscience 8: 819-831 11. Dmitry Kulish, Kevin Struhl (2001) TFIIS Enhances Transcriptional Elongation through an Artificial Arrest Site In Vivo. Mol Cell Biol. 13: 4162-4168 References