Mechanisms of splicing inhibition in apolipoprotein B exon 26 (ApoB ex26)
B Khoo, SA Akker & SL Chew
ApoB isoforms are components of the chylomicron, and of the atherogenic LDL and Lp(a) particles. Ex26 is exceptionally long at 7.57kb as most exons are <500bp. Ex26 is also the site of RNA editing, which generates the ApoB48 isoform instead of ApoB100. The first 3kb of ex26 contains 15 sequences matching the splice site consensus, which could be used in splicing, but are not. Splice sites matching the consensus but which are not used are called pseudosites. How the spliceosome ignores pseudosites and correctly splices such a long exon is unknown.
HYPOTHESES: [I] Ex26 contains: [a] multiple exonic splicing silencer sequences that silence the pseudosites, or [b] a single powerful silencer. [II] The correct use of the splice sites flanking ex26 is facilitated either by [a] absence of a silencer, or [b] the presence of an adjacent splicing enhancer.
EXPERIMENTAL DESIGN: A splicing reporter, DNA ligase III C-beta, was used. Initially, six 400bp fragments of the 5' 2.4kb of ex26 were positioned in place of the native silencer using PCR. Key elements were also tested in a reporter of cryptic splicing, beta-thal. Pre-mRNAs were transcribed and were assayed for splicing efficiency using HeLa nuclear extracts.
RESULTS: Silencing activity was present in all fragments assayed, even with smaller fragments of 50nt, consistent with [Ia]. This activity was not merely due to fragment length: a 338nt fragment (IGF-I ex3+4) did not silence. Enhancing activity was absent in the 5' 400nt of ex26, more consistent with [IIa], although it is possible that this fragment contains both an enhancer and a dominant silencer.
Ascertainment of the mechanisms of ex26 splicing would open up a novel therapeutic pathway towards lowering LDL and Lp(a) concentrations, by switching isoforms and reducing atherosclerosis.