Because the molecular mechanisms responsible for splice site choice remain poorly understood, Nilsen and his colleagues decided to study how some potential splice sites are "silenced," preventing them from forming splices while causing other, weaker splice sites to make splices instead. To do this they created a synthetic pre-mRNA containing two alternative splice sites, a weak site and a much stronger site downstream of it. Only one of them can be activated to make each possible mRNA, and whichever one the genetic machinery chooses, it then deletes a section of pre-mRNA from that beginning point to a common ending point downstream of both beginning splice points.
The research team randomized all possible combinations of nucleotides in a sequence of 12 nucleotides just downstream of the stronger splice site and in 12 just upstream of it, their exact positions chosen so that they would be as close as possible to the splice site without interfering with the very nearest nucleotides, which are required to complete the splices. This work was done both in vitro and in vivo, with similar results.
In control synthetic pre-mRNA's where the weak upstream splice site was left out, splicing always took place between the preferable downstream splice site and the common splice ending point. But when the weak upstream site was present, changes in nucleotide type and order near the downstream splicing site sometimes silenced it and sometimes did not.
Using bioinformatics techniques, the researchers organized the 89 unique intronic silencer sequences they found into four distinct clusters, while the 47 unique exonic silencers formed
|Contact: Christina DeAngelis|
Case Western Reserve University