Use of DNA microarrays for the study of the effects of RNA polymerase II elongation rate on alternative splicing

PAULA CRAMER; EZEQUIEL PETRILLO; GUADALUPE NOGUÉS; CLAUDIA BEN-DOV; JUAN VALCÁRCEL; MARÍA F. FERRERO; ALBERTO RODOLFO KORNBLIHTT

San Carlos de Bariloche, Neuquén, Argentina

Congreso; Gene Expression and RNA Processing Meeting and Cell Biology, Signaling and Alternative Splicing; 2007

Although some factors and mechanisms affecting alternative splicing have beendescribed, most remain unknown to date. We have shown that the recruitment oftranscription coactivators that promote initiation of transcription favor the inclusion of amodel alternative exon, while transcription coactivators that increase the elongation ratedo not favor its inclusion in the mature mRNA. Moreover, both the use of drugs thatdecrease pol II elongation rate and the use of a slow mutant version of pol II provoke anincrease in the inclusion of model exons in minigenes and also in endogenous genes. Asnot all minigenes tested have shown this behaviour, we set out to study the effect oftranscription elongation rates on alternative splicing in a large number of endogenousgenes.Flavopiridol specifically blocks the transition from initiation to elongation, a stepcontrolled by P-TEFb, which phosphorylates the carboxyterminal domain of pol II. Ourmodel system consists of Hep3B cells that have been treated or mock-treated with 100nM flavopiridol. After 24 h we have harvest the RNA and hybridize it to a microarrayconsisting of 25-mer exon probes for constituve exons plus exon-exon junction probes,spanning all the annotated splicing variants expressed from 600 selected human genes.Each junction is represented by three sets of staggered probes, while exons arerepresented by three to five probes. The array is printed by Agilent Technologies andutilizes the two-channel technology. The signal from the probes is compared to thesignal of probes from the corresponding alternative exon, and levels of expression acrossthe genes are determined by using 60-mer probes located in constitutive exons at 5' and3' of the transcripts.So far we have found changes in almost 200 splicing events (i.e., exon skipping,alternative splice donors or acceptors). While the array platform and design are beingvalidated in Barcelona, we have validated some of the predictions from the microarrayby real time RT-PCR.