Visualizing trans-splicing molecular machines across scales

Introduction

Trans-splicing is an essential mRNA processing step for a significant portion of living organisms. In trans-splicing, exons from two pre-mRNA precursors merge into a single mRNA, while cis-splicing rearranges exons within the mRNA.

Current Understanding

Despite recent technical advancements in cryo-electron microscopy (cryo-EM) that have movie-like resolved different stages of cis-splicing, the trans-splicing mechanism is still a black box. Input and output are defined, but the single steps of how the trans-spliceosome assembles and remodels to initiate the splicing cycle remain unclear. This limitation is partially due to the absence of molecular structures resolving trans-splicing complexes.

Project Overview

In TRANSPLIC, I will pioneer the assembly of trans-splicing complexes on pre-mRNA scaffolds to reveal the particular states unique to trans-spliceosomes. I will determine the molecular structures of trans-spliceosomes and uncover their behavior in the cellular context.

Methodology

Targeted functional assays will disclose the order of events leading to trans-splicing. The protist Trypanosoma brucei (Tb) will serve as an accessible model organism because it uses trans-splicing as an obligatory and abundant mRNA processing step.

I will apply an ambitious approach that integrates:

1. In vitro and cell lysate-based methods
2. State-of-the-art cryo-EM
3. Cryo-electron tomography
4. Proteomics
5. Artificial intelligence-based computational modeling

Complementary Studies

I will complement the study through targeted functional experiments, leading to a complete understanding of the spatial-temporal resolution of trans-splicing in trypanosomes, with wider relevance to other organisms, including humans.

Future Implications

The targeted fusion of gene sequences through trans-splicing bears potential as a molecular tool for transcriptome editing in the future.