Biophysical characterization of the U5-200K protein involved in Trypanosoma brucei pre-mRNA processing

• Grant number: 13/27013-5
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: March 01, 2014
• End date: December 10, 2015
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Otavio Henrique Thiemann
• Grantee: Guilherme Eduardo de Souza
• Host Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil
• Associated research grant: 08/57910-0 - National Institute of Structural Biotechnology and Medicinal Chemistry in Infectious Diseases, AP.TEM

Abstract

The Trypanosomatidae family is considered of major medical and economic importance, since the parasites of this family are capable of causing lethal diseases in their hosts. The major impact of these parasites on human health has directed investigations of peculiar molecular processes of these organisms, such as processing of pre - mRNA by Splice Leader RNA ( SL trans - splicing ) required to resolve polycistronic RNA transcripts into mature mRNAs. Being a vertebrate absent mechanism in which the cis -splicing occurs, participants proteins may be considered important parasite- specific targets. The spliceosome is a complex that interacts with nuclear SL RNA and pre -mRNA to catalyze trans-splicing SL, comprising small nuclear ribonucleoprotein particles (U1, U2, U4, U6 and U5 snRNPs) formed by protein factors and snRNAs specific members. After assembly of the pre - mRNA, the spliceosome requires rearrangements to take its catalytic conformation. The protein U5 -200K, U5 particle component T. brucei, outlines the areas helicase DEAD / Deah box and Sec63 preserved. In yeast, the Sec63 domain N - terminal cassette Brr2 is an integral part of the active site, allowing the disruption of the U4/U6 duplex during catalytic activation of the spliceosome, but the function of U5 - 200K has not yet been identified in T. brucei. This project aims to molecular cloning, heterologous expression and purification of recombinant products corresponding to the U5 - 200K protein of T. brucei and corresponding to its two conserved domains regions. If successful, crystallization trials of recombinant products could be realized. Additionally, molecular modeling of the three dimensional structure of conserved domains should contribute to the understanding of essential elements for its function. (AU)