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

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

Mar del Plata

Congreso; SAIB 51; 2015

Alternative splicing (AS) is the mean by which two or more different mRNAs can be produced from a single gene and is the main way of expanding protein diversity in eukaryotes. Splicing is coupled to transcrip1on, and transcrip1on kine1cs as well as factors interac1ng with the transcrip1onal machinery can influence AS decisions. We have seen that DNA damage produced by ultraviolet (UV) radia1on induces a systemic transcrip1onal response in cells, resul1ng in a reduc1on of RNA polymerase II elonga1on rate that leads to changes in AS paFerns. Nevertheless, the factors media1ng the pathway from the DNA lesions to the transcrip1onal machinery are s1ll elusive.We obtained a stably-transfected HeLa Flp-In T-Rex cell line with a fluorescent reporter of AS. This reporter expresses two different fluorescent proteins depending on whether an alterna1ve exon is included or skipped. We showed that this reporter changes its AS paFern in response to UV light, and thus cons1tutes an appropriate tool for deciphering this pathway. With this tool, we plan to perform a non-biased high-throughput analysis with an siRNA plaWorm in order to iden1fy factors involved in the UV-AS response. Addi1onally, this tool can be useful for studying AS regula1on at single-cell level.