Endocrine Abstracts (2003) 5 P165

A unique sequence element that silences the growth hormone receptor (GHR) pseudoexon

SA Akker, B Khoo, LA Metherell, AJL Clark & SL Chew

Department of Endocrinology, St Bartholomew's Hospital, London, UK.

Pseudoexons resemble true exons by current bioinformatic criteria and may outnumber true exons by 10:1. They are, however, never spliced into mature mRNA. A point mutation in the GHR gene results in abnormal splicing of a pseudoexon, leading to Laron syndrome. The GHR pseudoexon lies between exons six and seven and the point mutation is adjacent to the pseudoexon 5' splice site.
Our studies aimed to define the elements that normally prevent splicing of this pseudoexon.
A systematic mutagenesis of the GHR pseudoexon, in a reporter construct, was performed. Mutated pre-mRNAs were transcribed and incubated in Hela nuclear extracts. The efficiency of splicing to the pseudo-exon was quantified.
Our preliminary data showed that deletion of a twenty nucleotide element (construct del78-98) in the pseudoexon increased pseudoexon splicing by 2.5-fold. This element was a sequence-specific silencer that only interacted with the wild-type pseudoexon 5' splice site. Mutation of the surrounding intronic sequences had no effect. Deletion of other elements showed weak splicing effects; del58-78 and del38-58 had weak silencing effects, del38-58 had a mutant specific silencing effect and del3-18 had a weak enhancing effect.
This is the first demonstration of a silencer element that acts specifically with one type of 5' splice site sequence. The silencing element is not active when a 5' splice site is inserted that increases the match with the consensus. These data show the coordinated action of a silencer and its nearby 5' splice site. Possible models explaining pseudoexon de-repression include a change in binding of silencing factor(s) at the silencer element, or a conformational change between factor(s) at the element and the spliceosome.

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