The protein coding information in our genome is located on genes which are very often interrupted by non-coding regions called introns. For proper gene expression, introns must be removed accurately and the remaining protein coding parts, the exons, must be rejoined. This reaction, termed splicing, is carried out by an enormous macromolecular machine called the spliceosome, and is one of the most crucial steps in gene expression. Two different intron types have been identified in eukaryotes, each removed by their own dedicated spliceosome; the U2-type (or major) introns, which constitute the majority of introns, and the U12-type (or minor) introns, of which ca. 700-800 have been identified in the human genome. The presence of a second type of intron and spliceosome has always been enigmatic. However, studies investigating U12-type intron removal have provided us with an important clue; it appears that U12-type introns are spliced less efficiently than U2-type introns. This suggests that their removal could be rate-limiting for the expression of the genes that harbor these introns, and it also offers the intriguing possibility that the activity of the minor spliceosome could be altered in response to changing cellular conditions. These implications could offer a valuable explanation for the extraordinary conservation of the U12-type introns and the components that catalyze their excision. There is currently not much known about the regulation of the minor spliceosome and this study aimed to address this issue. I have investigated the characteristics of a negative feedback loop that regulates the expression level of two essential and unique protein components of the minor spliceosome, the U11-48K and the U11/U12-65K proteins. In the genes that encode these proteins, an ultraconserved sequence element can be found which consists of a tandem repeat of U12-type 5ʹ splice sites. We uncovered that binding of U11/U12 di-snRNPs on these elements leads to alternative splicing where an mRNA isoform is produced that is targeted for degradation or nuclear retention. The presence of such enhancer elements is conserved from plants to animals, highlighting an extreme selection pressure for this regulatory mechanism. I further investigated the role of the U11-35K protein, another protein uniquely associated with the minor spliceosome, in alternative splicing, and the functional requirements for enhancer binding. Furthermore, I uncovered the molecular mechanism by which the level of translational-competent U11/U12-65K mRNA is downregulated through U11/U12 di-snRNP enhancer binding.
|Status||Publicerad - 29 jan. 2016|
|MoE-publikationstyp||G5 Doktorsavhandling (artikel)|
- 1182 Biokemi, cell- och molekylärbiologi