en
Master
English

Structural and functional studies of RNA Editing TUTase 1 from Trypanosoma brucei

ContributorsLong, Marius
Master program titleMaster en biologie
Defense date2015
Abstract

The kinetoplastid clade regroups many species including the Trypanosomatids that are human parasites. The mitochondrial genome of these organisms is organized into a network of circular DNA called kinetoplast. The kinetoplast encodes for pre-messenger RNAs (pre-mRNAs), pre-ribosomal RNAs (pre-rRNAs) and guide RNAs (gRNAs). The RNA Editing Core Complex (RECC) is responsible for the RNA editing reaction on mRNAs which can be either uridine insertion or uridine deletion depending on the information provided by the particular gRNA base-pairing with the pre-mRNA. The editing of the transcripts by the RECC is an essential physiological process for the survival of the organism. The prerequisite for the editing process is the proper pre-processing of gRNA and the mRNA, which involves uridylation of the gRNA 3' end. The processing of the transcripts is done by RET1/DSS1 (RDS) complex (unpublished data from Aphasizhev group). RET1 is a polyuridylating enzyme that also participates in another complex involved in the processing and the production of translationally competent mRNAs. The later complex consists of primarily two enzymes, the RET1 and the kinetoplast polyadenylation polymerase (KPAP1) as well as a heterodimeric complex of two pentatricopeptide repeat (PPR) proteins called kinetoplast polyadenylation/uridylation factors 1 and 2 (KPAF1 & KPAF 2) complex. This large complex generates long A/U tails that are essential for the mRNA to be translated by the ribosome. Since RET1 is an essential protein and participates in two majors pathways of gRNA and mRNA maturation, we decided to characterize it structurally to further understand how this protein can interact with its various substrates, how it can be recruited by multiple proteins to act in these two major pathways, and finally, to generate a tool (the atomic model) to help design new inhibitors potentially interesting to fight this life threatening parasitic infection. During my time in the laboratory, we have been able to express, purify and crystallize a truncated version of RET1 from Trypanosoma brucei in its apo and in complex with the UTP analogue UMPNPP to a resolution of 1.6 and 1.8 angstroms respectively. We further solve the structure of an inactive RET1 mutant with the UTP molecule in the active site. These three atomic structures gave us multiple insights into the uridylation mechanism. RET1 being an essential protein in the life cycle of the 5 parasite, we further use those high resolution structures to design new drugs to fight the African sleeping sickness. We are collaborating with the group of R. Amaro, UCSD, which are performing molecular simulations using our high-resolution structures to identify new compounds potentially able to inhibit the activity of this protein.

eng
Citation (ISO format)
LONG, Marius. Structural and functional studies of RNA Editing TUTase 1 from Trypanosoma brucei. 2015.
Main files (1)
Master thesis
accessLevelRestricted
Identifiers
  • PID : unige:75417
164views
6downloads

Technical informations

Creation09/22/2015 2:01:00 PM
First validation09/22/2015 2:01:00 PM
Update time03/14/2023 11:38:15 PM
Status update03/14/2023 11:38:15 PM
Last indexation01/29/2024 8:32:12 PM
All rights reserved by Archive ouverte UNIGE and the University of GenevaunigeBlack