Set-up of a fluorescent alternative splicing reporter for high throughput screenings

NICOLÁS NIETO MORENO; LUCIANA E. GIONO; MANUEL J. MUÑOZ; ALBERTO R. KORNBLIHTT

Mar del Plata, Buenos Aires

Congreso; LI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2015

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Alternative splicing (AS) is the way by which two or more different mRNAs can be produced from a single gene andis the main way of expanding protein diversity in eukaryotes. Splicing is coupled to transcription, and transcriptionkinetics as well as factors interacting with the transcriptional machinery can influence AS decisions. We have seenthat DNA damage produced by ultraviolet (UV) radiation induces a systemic transcriptional response in cells,resulting in a reduction of RNA polymerase II (RNAPII) elongation rate that leads to changes in AS patterns.Nevertheless, the factors mediating the pathway from the DNA lesions to the transcriptional machinery are stillelusive. To identify these factors, we obtained a stably-transfected HeLa Flp-In T-Rex cell line with a fluorescentreporter of AS. This reporter expresses two different fluorescent proteins depending on the inclusion or exclusion ofan exon cassette. We showed that this reporter changes its AS pattern in response to UV light and thus constitutes anappropriate tool for deciphering the pathway. With this tool, we plan to perform a non-biased high-throughputanalysis using an siRNA platform in order to identify factors involved in the UV-AS response. Additionally, this toolcould be useful for studying AS regulation at single-cell level.