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Cellular investigation of protein kinases involved with splicing, by minigene system

Grant number: 19/11320-2
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): November 01, 2019
Effective date (End): October 31, 2020
Field of knowledge:Biological Sciences - Genetics - Human and Medical Genetics
Principal Investigator:Katlin Brauer Massirer
Grantee:Gabriel Vieira Valderrama
Home Institution: Centro de Biologia Molecular e Engenharia Genética (CBMEG). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
Associated research grant:14/50897-0 - Open-acess Medicinal Chemistry Centre (OpenMedChem), AP.TEM

Abstract

Splicing is a process that occurs after gene transcription, and that involves the removal of introns from messenger RNAs, allowing the union of the exons encoding proteins. This process is dependent on the complex ribonucleoproteico, denominated spliceosomo, which is composed of several proteins who still need more exhaustive studies. One of the classes of proteins regulators of this complex is the class of protein kinases, which act by phosphorylation of target proteins (substrates), usually to activate them. Among the 500human kinases, there is a set of eight kinases that regulate some splicing factors, thus influencing the combinations of exons that generate isoforms of mature mRNAs. In this context, the changed state of splieceosome phosphorylation or the occurrence of events such as mutation or inhibition of these kinases may result in unbalance of isoforms leading to expression (such as truncated isoforms, or which are oncogenic when expressed in tissue other than expected). In view of the foregoing, we propose to evaluate if there is a relation of a new group of five protein kinases with splicing and we propose to mount an essay that can be expanded as general screening regulate splicing. Initially we will perform ectopic expression of these kinases inHEK293 cell system in combination with a published reporter gene system. This system consists of a minigene that emits chemiluminosity in the presence of substrate of the luciferase enzyme only when the minigene undergoes splicing. As long-term objective, we can expand the evaluation to a greater number of little studied proteins, by means of alteration of the minigene replacing with new ones regulatory sequences, and thus we can contribute to the understanding of waterfalls of phosphorylation regulating splicing.