Splicing Fidelity: Enforcement, Modulation and Impairment.

Introduction

In eukaryotes, the spliceosome removes non-coding introns from precursor messenger RNAs during pre-mRNA splicing. Crucially, many pre-mRNAs are spliced differently depending on cellular status or external stimuli. This “alternative splicing” reshapes the genetic information from a given mRNA to encode several protein isoforms and greatly diversifies proteomes.

Importance of Splicing Fidelity

Splicing must be extremely precise as errors produce aberrant mRNA encoding potentially toxic proteins. Splicing fidelity relies on the accuracy of the spliceosome that assembles de novo on each pre-mRNA in a cotranscriptional manner. It gets activated and selects the precise boundaries of introns to catalyze their excision.

Properties of the Spliceosome

The spliceosome is endowed with two apparently conflicting properties:

• It must be very accurate to avoid splicing errors.
• It must be tolerant to accommodate alternative splicing changes.

I aim to unveil the molecular basis of splicing fidelity’s enforcement and modulation by the spliceosome.

Challenges in Spliceosome Research

Due to its extreme complexity, the spliceosome has long been a major challenge for molecular and structural investigations. Recent progress in cryo-EM allows the structural analysis of very dynamic and low-abundance biological objects such as spliceosomes. Hence, it is now possible to explore the most elusive aspects of RNA splicing, provided that the biochemical challenges of trapping and stabilizing transient spliceosome intermediates are overcome beforehand.

Research Methodology

I will use innovative methods to capture spliceosomes from cell extracts and cryo-EM to investigate how splicing fidelity is:

1. Enforced by molecular checkpoints during spliceosome activation.
2. Maintained/modulated by other cellular processes.
3. Impaired during viral infection.

Expected Outcomes

The outcome of this research will bring us closer to answering a fundamental question of biology: how does the spliceosome give rise to the immense combinatorial space of alternative splicing without errors and allow the 20,000 human genes to yield 200,000 proteins.