Functional SELEX method

To identify exonic splicing enhancer (ESE) motifs by functional in vivo or in vitro SELEX (systematic evolution of ligands by exponential enrichment), a minigene is used that harbors ESE sequences that are required for the efficient splicing of its pre-mRNA. As shown in the accompanying figure, the natural enhancer (green box) is replaced by random sequences (blue) from an oligonucleotide library (a). The resulting pool of minigenes is then transfected into cultured cells, or is transcribed in vitro, to generate a pool of pre-mRNAs (b). Following in vivo or in vitro splicing (c), the pool of spliced mRNAs is amplified by RT-PCR and gel-purified (d). This pool of enhancer-enriched sequences is then used to reconstruct new minigene templates by overlap-extension PCR (e), to use in a new enrichment cycle. The iteration of this entire procedure yields a limited number of "winners" - sequences that possess good splicing enhancer activity.
To identify ESEs that are recognized by individual SR proteins, the splicing step was carried out in S100 extract complemented with one of four different SR proteins (SF2/ASF, SC35, SRp40 and SRp55). Transcripts were derived from an IgM-derived minigene in which the natural enhancer was substituted with a pool of 20-nucleotide random sequences. After a few cycles of enrichment, spliced products were sequenced and aligned to derive a consensus motif. The frequencies of the individual nucleotides at each position were then used to calculate a score matrix (shown below), which can be used to predict the location of SR-protein-specific putative ESEs in exonic sequences.

The current weighted matrix values (release 2.0) and the consensus motifs obtained with these four SR proteins are shown below; the height of each letter reflects the frequency of each nucleotide at a given position, after adjusting for background nucleotide composition. At each position, the nucleotides are shown from top to bottom in order of decreasing frequency; orange letters indicate above-background frequencies. (The pictogram representation method was described by Burge and colleagues (Burge, C.B.,Tuschl, T., Sharp, P.A. in The RNA world II, 525-560, CSHL press, 1999).

SF2/ASF	SC35	SRp40	SRp55
[1] [2] [3] [4] [5] [6] [7] A -1.14 0.62 -1.58 1.32 -1.58 -1.58 0.62 C 1.37 -1.1 0.73 0.33 0.94 -1.58 -1.58 G -0.21 0.17 0.48 -1.58 0.33 0.99 -0.11 T -1.58 -0.5 -1.58 -1.13 -1.58 -1.13 0.27	[1] [2] [3] [4] [5] [6] [7] [8] A -0.88 0.09 -0.06 -1.58 0.09 -0.41 -0.06 0.23 C -1.16 -1.58 0.95 1.11 0.56 0.86 0.32 -1.58 G 0.87 0.45 -1.36 -1.58 -0.33 -0.05 -1.36 0.68 T -1.18 -0.2 0.38 0.88 -0.2 -0.86 0.96 -1.58	[1] [2] [3] [4] [5] [6] [7] A -0.13 -1.58 1.28 -0.33 0.97 -0.13 -1.58 C 0.56 0.68 -1.12 1.24 -0.77 0.13 -0.05 G -1.58 -0.14 -1.33 -0.48 -1.58 0.44 0.8 T 0.92 0.37 0.23 -1.14 0.72 -1.58 -1.58	[1] [2] [3] [4] [5] [6] A -0.66 0.11 -0.66 0.11 -1.58 0.61 C 0.39 -1.58 1.48 -1.58 -1.58 0.98 G -1.58 0.72 -1.58 0.72 0.21 -0.79 T 1.22 -1.58 -0.07 -1.58 1.02 -1.58

The above material is Copyrighted. ALL RIGHTS RESERVED.