Structural assessment of the core domains of Leishmania major telomerase RNA

• Grant number: 22/05039-1
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• Start date: November 01, 2022
• End date: April 30, 2023
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Maria Isabel Nogueira Cano
• Grantee: Beatriz Cristina Dias de Oliveira
• Supervisor: Kausik Chakrabarti
• Host Institution: Instituto de Biociências (IBB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil
• Institution abroad: University of North Carolina at Charlotte (UNCC), United States
• Associated to the scholarship: 19/25985-6 - Studies about the effect of knocking out and overexpressing the Telomerase RNA component in L. major development and survival, BP.DD

Abstract

Leishmaniases are neglected tropical diseases caused by different species of Leishmania, a protozoa parasite belonging to the Trypanosomatidae family. Every year, about 1 million people worldwide are threatened by the disease. Besides its high incidence rate, there is still no efficient prevention and treat-ment. Therefore, a deeper understanding of parasite biology will help develop new therapies to treat the disease. Telomeres are key to maintaining genome stability and integrity and, thus, are considered potential targets for drug design. Leishmania spp telomeres, similarly to other eukaryotes, are nucleoprotein structures formed by repetitive DNA in double and single-stranded forms, with the single strand protruding towards the end of the chromosome (known as the 3' G-overhang). Telomeres are maintained by telomerase, a ribonucleoprotein complex minimally composed of a protein subunit, the Telomerase Reverse Transcriptase (TERT), and a noncoding RNA the telomerase RNA (TER). The Leishmania TER (LeishTER), like Trypanosoma brucei TER (TbTER), is one of the largest TER described so far, making it difficult to characterize its structure due to its large size and sequence diversity. Current information about LeishTER's putative architecture and secondary structure is mainly based on bioinformatics analysis. At the University of North Carolina, Charlotte, USA, Dr. Chakrabarti's group and colleagues developed a new approach to characterize unusual long RNA structures at single-nucleotide resolution, such as TbTER. The methodology combines in-cell chemical probing (in vivo and ex vivo) using DMS (dimethyl sulfate) and NAI (2-methylnicotinic acid imidazolide) with targeted high-throughput RNA sequencing and mutational mapping. We propose extending our ongoing collaboration with Dr. Elton Vasconcelos to improve the in silico structural analysis and collaborating with Dr. Chakrabarti's group to apply this methodology to elucidate the LeishTER structural domains. Due to the known structural similarities between TbTER and LeishTER, we intend to use this approach in the pro-cyclic and metacyclic promastigotes (in vivo and ex vivo) and compare the results with that obtained through bioinformatics. Furthermore, the information about LeishTER structure obtained through RNA chemical probing combined with bioinformatic analysis would be a great addition to the project's second part, which consists in uncovering novel species-specific RNA-protein interactions in Leishmania. Discovering the LeishTER binding partners is crucial to understanding its role inside the cell, since there are important protein partners associated with TER that are involved with the biogenesis and assembly of the telomerase complex. Thus, we intend to obtain information about proteins interacting with LeishTER domains (e.g., template region, TBE, Helix IV, pseudoknot) using RNA-centric approaches (i.e. affinity purification and MS2-RNA affinity tag) followed by mass spectrometry analysis. (AU)