Effect of aire gene mutations induced in vivo in the thymus by CRISPR-Cas9 on alternative splicing of mRNAS that encode peripheral tissue antigens in mTEC cells

• Grant number: 21/02459-7
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: May 01, 2021
• End date: January 31, 2024
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Eduardo Antônio Donadi
• Grantee: Adriana Arruda Matos
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 17/10780-4 - Effect of CRISPR-Cas9-induced mutations in the Aire gene (APS1 syndrome) on protein conformation, mTEC cell transcriptome and their interaction with thymocytes, AP.TEM

Abstract

The fellow will study the effect of specific mutations in the Aire gene associated with the APS1 Syndrome on one of the essential molecular processes of mTEC cells, the alternative processing (alternative splicing) of mRNA encoding PTAs. It has already been observed that the Aire gene controls alternative processing by studying CD80high and CD80low mTEC cells, but it is not yet known whether mutations in that gene would affect this process. The selection of mutations in the human Aire gene (SAND and PHD domains) will be made to design the components of the CRISPR-Cas9 system, following the in vivo transfections in the thymus of mice and subsequent separation of mTEC cells and characterization of the mutations in the Aire gene. Preparations of total RNA of mTEC wt cells (from unmodified thymuses) and Aire mutants (from thymuses transfected with the CRISPR-Cas9 elements) will be used in the transcriptome sequencing (RNA-Seq). The data will be analyzed using specialized bioinformatics programs, whose pipeline for analyzing groups of mRNAs according to their biological functions and also isoforms of mRNAs from alternative processing, is already in use in our laboratories. Differential expression maps of mRNAs and isoforms will be constructed with heat-map / hierarchical clustering comparing wt cells with mutant Aire cells. As the alternative processing increases the diversity of TSAs, the identification of the mRNAs that encode the respective TSAs (differentially expressed mRNAs) will allow the evaluation of the effect of Aire mutations on promiscuous gene expression and, consequently, on the representation of itself by mTEC cells, an essential process for the induction of immunological tolerance. (AU)