PDF file: EURASNET Annual Report 2008

Participant 17 – Ian C. Eperona) Work performed during the period• WP7: We have characterized the binding of GFP-tagged PTB to alpha-tropomyosin premRNA,in collaboration with Chris Smith (group 23). RNA sequences spanning the regulated exon andthe regulatory sequences in the introns were incubated in nuclear extract, and single molecule TIRFmicroscopy was done to examine the complexes. The number of fluorescent molecules bound to eachRNA molecule was examined by newly-developed statistical methods. Cross-linking was used to showthat the binding of PTB and GFP-PTB was equally probable. RNA substrates carrying mutations invarious PTB binding sites were examined also. The results showed that PTB associated as a definedcomplex, and the length of the polypyrimidine tract affected both the number of proteins bound and theaffinity of binding. Further splicing factors were cloned in expression vectors. The validity offluorescently-tagged U1A was demonstrated by showing that it formed U1 snRNPs and bound to premRNA.• WP14: Continued testing effects on recovery of SMN2 splicing in vitro and in cellsfrom patient fibroblasts when a bifunctional oligonucleotide trans-acting enhancer of splicing (TOES)is altered in respect of its tail length, site of annealing, tail sequence and chemistry; analysis of uptakeby FRET. The binding of activating proteins to the oligonucleotides has been studied, and theefficiency of different activatingsequences on SMN2 exon 7 has been shown to be correlated well with factor binding activity.b) Major cost items with justificationParticipant 1A Type of expenditure Budget total costspersonnel 15167.70consumables 6041.33training 0equipment 1255.57travel 711.34= total direct costs 23175.94+ overhead 4635.19= total eligible costs 27811.13Major cost items• ResearchPersonnelL.P. Eperon [WP 7] 439 hours 11480.99EuroG.E. Eperon [WP 7] 356.6 hours 3686.71EuroFigures include National Insurance contribution paid by university to government, and do not representamount received by employee. Number of hours worked by LPE, who was paid monthly and nothourly, is after subtraction of normal holidays.Dr Eperon was engaged part-time in PCR amplification of cDNA for splicing factors, sequencing,cloning and expression, supporting the single molecule work being done by Dr Cherny; Mr G. Eperoncontinued his in vitro splicing studies on a series of substrates containing putative hnRNP A1 highaffinity sites and to do splicing assays; he also tested the ability of PDMS to make ultra-small subfemtolitreswells; he did X-lining experiments on PTB in support of the single molecule work; hecharacterized the use of displaceable oligonucleotides to trap splicing complexes at defined states.170