Degree Granting Department
Denise R. Cooper, Ph.D.
Niketa A. Patel, Ph.D.
PKCbeta, PKCdelta, Splicing, SRp40, SC35
Alternative splicing is a major contributing factor to protein diversity. The human genome project validated that there are about 25,000 genes, but over 100,000 proteins. Genes contain numerous exons that are specifically regulated; thus, elucidating alternative splicing mechanisms of pre-mRNAs is an immense undertaking. Two basic tools are used to study this mammoth task. For in vitro studies, usually in vitro transcription followed by splicing assays is used. For in vivo studies, the main technique is the construction of minigenes. These techniques enable one to study the mechanisms/conditions that explain an exon's inclusion or exclusion in the mature mRNA. In this project, studies were focused in the protein kinase C (PKC) family, and specifically in certain exons of PKCbeta and PKCdelta genes. The PKCbeta gene codes two proteins: PKCbetaI and PKCbetaII. The pre-mRNA of PKCbeta contains 18 exons.
If exon-17 (exon-betaII) is included in the spliced transcript, protein PKCbetaII is expressed. If exon-betaII is skipped (excluded), protein PKCbetaI is expressed. Previous and current results indicate that insulin treatment not only favors exon-betaII inclusion, but also regulates 3?-UTR size in mature mRNA. We identified up to five different PKCbetaII mRNAs that differ only in the 3?-UTR size (all five mature transcripts express the same PKCbetaII protein). PKCbetaII is required for glucose uptake in skeletal muscle and adipocytes. Additional studies revealed that inclusion of exon-betaII involves SRp40 in its phosphorylated form. Small interfering RNA (siRNA) knockdown of Akt2 and Clk1 further revealed that SRp40 was phosphorylated by Akt2/Clk1, and that Akt2 phosphorylates Clk1. The knockdown of Clk1 or SRp40 down-regulated glucose uptake. The PKCdelta gene contains 18 exons.
The standard size of exon-10 is 97-bases, and when included in the mature transcript, PKCdeltaI is expressed. Exon-10 has an additional 5?-splice site that extends the exon size to 190-bases. When this extended size is included in the mature transcript, PKCdeltaVIII is expressed which has a profound effect in the fate of the cell by blocking apoptosis. Our results reveal that all-trans retinoic acid regulates the inclusion of exon-10 extended size. Moreover, the inclusion involves SC35 (SRp30b). Finally, we elucidated the position of the SC35 cis-element.
Scholar Commons Citation
Apostolatos, Hercules S., "Splicing systems for studying signaling to the spliceosome" (2010). Graduate Theses and Dissertations.