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

CRAMER, P.1, PETRILLO, E. 1, NOGUÉS, G. 1, BEN-DOV, C.2, VALCÁRCEL, J. 2, FERRERO, F.V. 1, KORNBLIHTT, A.R.

Bariloche, Rio Negro, Argentina

Congreso; JOINT MEETING "Gene Expression and RNA Processing" (ICGEB) and "Cell Biology, Signaling and Alternative Splicing" (EURASNET); 2007

ICGEB-EURASNET

Use of DNA microarrays for the study of the effects of RNA polymerase II elongation rate on alternative splicing Cramer, P.1, Petrillo, E. 1, Nogués, G. 1, Ben-Dov, C.2, Valcárcel, J. 2, Ferrero, F.V. 1, Kornblihtt, A.R. 1 1Laboratorio de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, UBA 2 Centre de Regulació Genómica, Barcelona, Spain. Although some factors and mechanisms affecting alternative splicing have been described, most remain unknown to date. We have shown that the recruitment of transcription coactivators that promote initiation of transcription favor the inclusion of a model alternative exon, while transcription coactivators that increase the elongation rate do not favor its inclusion in the mature mRNA. Moreover, both the use of drugs that decrease pol II elongation rate and the use of a slow mutant version of pol II provoke an increase in the inclusion of model exons in minigenes and also in endogenous genes. As not all minigenes tested have shown this behaviour, we set out to study the effect of transcription elongation rates on alternative splicing in a large number of endogenous genes. Flavopiridol specifically blocks the transition from initiation to elongation, a step controlled by P-TEFb, which phosphorylates the carboxyterminal domain of pol II. Our model system consists of Hep3B cells that have been treated or mock-treated with 100 nM flavopiridol. After 24 h we have harvest the RNA and hybridize it to a microarray consisting 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 are represented by three to five probes. The array is printed by Agilent Technologies and utilizes the two-channel technology. The signal from the probes is compared to the signal of probes from the corresponding alternative exon, and levels of expression across the genes are determined by using 60-mer probes located in constitutive exons at 5´ and 3´ 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 being validated in Barcelona, we have validated some of the predictions from the microarray by real time RT-PCR.