A molecular basis of kinetoplastids SL trans-splicing

Introduction

Kinetoplastids are unicellular eukaryotic parasites responsible for severe human pathologies, such as sleeping sickness, Chagas disease, and leishmaniasis. Kinetoplastids have diverged early during evolution and harbor many intriguing cellular and molecular peculiarities.

Genome Characteristics

Among these peculiarities is their remarkably streamlined nuclear genome characterized by a high gene density and significantly divergent and specialized gene expression systems. A main example of such divergence is the polycistronic transcription of all their genes, producing long messenger RNA precursors (pre-mRNAs) containing tens to hundreds of coding sequences.

Trans-splicing Mechanism

These pre-mRNAs are dissected into monocistronic mRNAs by SL trans-splicing, a process during which a spliced leader (SL) RNA is intermolecularly fused to the 5’ end of all mRNAs by the trans-spliceosome. Although this machinery is essential for gene expression, the lack of structural information and the high divergence of its RNA and protein elements have hindered a mechanistic understanding of how kinetoplastids employ SL trans-splicing to generate their entire mRNA transcriptome.

Research Objectives

Here, we will use innovative approaches to provide the structural and mechanistic basis of SL trans-splicing in kinetoplastids by using cutting-edge structural biology techniques and state-of-the-art genetic and in silico tools.

1. By combining genome editing,
2. Purification of endogenous trans-splicing machineries,
3. High-resolution cryo-electron microscopy,
4. AI-based interactome approaches, and
5. Novel in vivo assays,

we will reveal the mechanism of SL snRNP biogenesis, the molecular basis for SL snRNP intermolecular recognition and activation by the trans-spliceosome, and the central mechanism of trans-splicing orchestrated by the highly divergent kinetoplastids trans-spliceosome.

Expected Outcomes

The outcome of this research will transform our understanding of RNA trans-splicing in eukaryotes and will pave the way for developing new drugs that specifically target this unique and essential pathway.