PMC

An overview of the process leading to the isolation of B52-dependent pre-mRNA splicing substrates. The number of candidates in each step is indicated.

Identification of a B52-binding site on a sequence segment selected by the genomic SELEX. (A) BBS-like elements found in two segments by the multiple alignment program ClustalW () (http://www. ebi.ac.uk/clustalw) are shown as are their mutated derivatives. Underlined letters in the sequence of #1-3 and #1-6 indicate deviations from the BBS consensus. Lower case letters in the ‘Mut’ constructs indicate sequence differences from the original isolates. (B) A mobility shift assay with B52 shows the BBS-like element of segment #1-3 is critical for B52 binding.

B52-binding activity of the RNA transcripts derived from exonic as well as intronic regions as demonstrated by electrophoretic mobility shift assay. The binding results of in vitro transcripts of 21 genomic sequence segments selected in genomic SELEX and identified as parts of intron- containing genes are shown. Segments in those genes confirmed to have splicing defects in the B52-null mutant are indicated by circles around their names. The positive control (P) is BBS #8, an aptamer isolated in a previous SELEX experiment. The negative control (N) is MGM #1, an RNA that binds to the nitrocellulose filter used as partitioning matrix ().

Splicing deficiency of four B52-binding pre-mRNAs in B52 deletion flies revealed by RT–PCR assay. (A) Schematic diagrams (not to scale) of the four pre-mRNAs. Exons are represented by boxes, and introns by lines. Asterisks indicate the location of segments containing B52-binding sites. The location of primer sets used in RT–PCR and the predicted lengths of RT–PCR products from wild type are also indicated. All primers reside completely within exon sequences. (B) RT–PCR products visualized on an agarose gel. The identities of the lettered bands are indicated in (A). (C) Real-time PCR assays of specific RNAs in the B52 mutant and wild-type larvae. The fold reductions in the RNAs’ abundance in B52-null mutant larvae are plotted with standard deviations (N = 3). The reduction represents the ratio of the RNA level in wild-type to the B52-null mutants using β1-tubulin mRNA levels as a standard. β1-Tubulin mRNA levels are unaffected in the B52 mutant ().