Structural Biochemistry/Eukaryotic 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, 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.



Mechanism of Alternative Splicing
Alternative splicing is a 2 step process carried out by the splicesome that ligates the 5’ splice site of an upstream exon to the 3’ splice site of a downstream exon. The splicesome is primarily composed of 5 small nuclear ribonuceloproteins (snRNP’s). snRNP’s carry out splicing through the recognition of 5’, 3’, and a branch point sequences in an intron.

Steps of Alternative Splicing:

1.) U1 snRNP recognizes GU of 5’ splice site, while U2 snRNP recognizes branch point bulged Adenosine.

2.) The A-complex is formed when U1 and U2 position the branch point near the 5’ splice site.

3.) The B-complex is formed when the tri-snRNP U4-U5-U6 bind to the A complex.

4.) The activated B-complex is formed when U1 and U4 leave, forming the catalytic splicesome.

5.) The C-complex is formed after the 1st step of splicing when the branch site adenosine attacks the 5’ splice site, creating an intron lariat.

6.) The 2nd step of splicing is carried out by the C-complex when the 5’ splice site attacks the 3’ splice site, resulting in the ligation of both exons.

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, and the rate of RNA Polymerase II transcription has been suggested to be a regulatory mechanism of co-transcriptional Alternative splicing. Aebi and Weissman’s “first come, first served” model 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, and is completed by pairing of the U2 snRNP with the branch point. 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. 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. 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.