Functional characterization of linear and circular wRNAs isoforms of halophilic archaea using genetics and microscopy techniques

• Grant number: 24/13402-4
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• Start date: December 15, 2024
• End date: December 14, 2025
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Tie Koide
• Grantee: Beatriz Adas Picinato
• Supervisor: Alexandre Wilson Bisson Filho
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Institution abroad: Brandeis University, United States
• Associated to the scholarship: 22/00308-4 - Identification and comparison of circular RNAs in archaeal transcriptome data, BP.DD

Abstract

Circular RNAs (circRNAs) are RNA molecules with 5' and 3' ends covalently ligated. Their functions range from acting as genetic regulators to producing proteins, and they are often expressed in a tissue and condition-specific manner. Next-generation sequencing with prior RNA treatment with RNase R (Circ-Seq) has been widely used to identify circRNAs, mainly in model eukaryotes. However, we know little about circRNAs in prokaryotes. To date, only a few Circ-Seq studies have been done in archaea. Using our Halobacterium salinarum Circ-Seq data and computational pipeline, we have identified circRNAs primarily associated with ribosomal RNAs, transfer RNAs, and insertion sequences (IS) of the IS200/IS605 family and their associated non-coding RNAs, wRNAs. Transposases of the IS200/IS605 family (IscB and TnpB) recently were implicated as the ancestral for the Cas9 and Cas12 proteins and can act as RNA guided-endonucleases with the wRNA as the guide. However, even with recent evidence that TnpB and the wRNA can help maintain transposon copy numbers in the genome, their function in the cell is unclear. IS200/IS605 transposons have lower mobility than other classes of IS, and there is a stark difference in wRNA and tnpB mRNA and protein levels. Evidence shows that the wRNAs could interact with the ribosome or RNA degradation machinery. Besides, we have identified circular isoforms of wRNAs, with unexplored biogenesis pathways and functions, in different halophilic archaea species. In this project, we aim to understand the functions of wRNAs and their circular counterparts in the cell using genetics and microscopy techniques in halophilic archaea. Using both H. salinarum and Haloferax volcanii as models, we will (1) construct different deletion and overexpression strains to perturb the TnpB/wRNA system, (2) test the different strains phenotypes, and (3) image the cells to track the different isoforms of the wRNA and their interaction partners.