Intron effects on eukaryotic gene expression and a study of protein-protein interactions between human second step splicing factors

Many eukaryotic genes are interrupted by non-coding intervening sequences called introns. An obligatory step in expression of these genes involves removal of introns from the precursor mRNAs by pre-mRNA splicing. Initially thought to be impediments to gene expression, introns and splicing have now been shown to be important for efficient expression of genes in diverse organisms. In this study, I report an analysis of intron effects on multiple steps of gene expression. I found that introns enhance gene expression mainly by increasing mRNA abundance and the amount of proteins produced per mRNA, without significantly influencing the cytoplasmic localization of spliced mRNAs. Efficient translation of spliced mRNAs correlated with their enhanced polysome association. Using Xenopus oocytes as an experimental system, I was able to attribute part of the splicing effects to the exon junction complex (EJC), a subset of proteins that are deposited on mRNAs specifically as a consequence of splicing. Furthermore, both increased protein expression and enhanced polysome association could be reproduced by tethering specific EJC proteins to an intronless reporter mRNA. These results demonstrated an EJC-mediated coupling between pre-mRNA splicing and mRNA translation.; In a separate study, I performed an analysis of protein-protein interactions between factors required specifically for the second step of pre-mRNA splicing. Using yeast-two hybrid analysis and in vitro GST-pulldown experiments, I found that the human second step factor hPrp18 interacts with several proteins previously known to play roles in 3′ splice site recognition. hPrp18 interacted with the second step factors hSlu7 and hPrp16. An interaction between hPrp18 and hSlu7 was expected from previous studies showing that the yeast proteins interact, whereas the interaction of hPrp18 with hPrp16 is probably not conserved in yeast. More surprisingly, I found that hPrp18 interacted physically with U2AF35 , a protein thought to be important for recognition of the 3 ′ splice site only prior to the first step of splicing. These studies confirmed a predicted protein contact and revealed the existence of novel protein-protein interactions that may contribute to mammalian 3 ′ splice site recognition.