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
• 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.

SL Trans-Splicing Process

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.

Challenges in Understanding SL Trans-Splicing

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:

1. Cutting-edge structural biology techniques
2. State-of-the-art genetic and in silico tools

Methodology

By combining:

• Genome editing
• Purification of endogenous trans-splicing machineries
• High-resolution cryo-electron microscopy
• AI-based interactome approaches
• 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
• 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.