A conserved intronic U1 snRNP-binding sequence promotes trans-splicing in Drosophila

Area of Science:

• Molecular Biology
• Genetics
• RNA Processing

Background:

• Trans-splicing, which joins exons from separate transcripts, is common in eukaryotes but its mechanism in higher organisms is poorly understood.
• The trans-spliced gene mod(mdg4) in Drosophila serves as a model to investigate this process.
• Previous studies have characterized trans-splicing in simpler eukaryotes like trypanosomes and nematodes, often involving spliced leader RNA.

Purpose of the Study:

• To elucidate the mechanism of trans-splicing in the higher eukaryote Drosophila melanogaster.
• To identify and characterize the RNA elements responsible for promoting trans-splicing of the mod(mdg4) gene.
• To investigate the role of U1 small nuclear ribonucleoprotein (snRNP) in the trans-splicing process.

Main Methods:

• Investigated the mod(mdg4) gene in Drosophila.
• Utilized CRISPR/Cas9 gene editing to delete key RNA sequences (TSA and TSB) within the 5' intron.
• Analyzed the effects of these deletions on trans-splicing efficiency and organismal development.
• Performed compensatory mutations in U1 snRNA to assess its role in rescuing trans-splicing.

Main Results:

• Identified two critical RNA sequences, TSA and TSB, in the 5' intron of mod(mdg4) that promote trans-splicing.
• The TSA sequence contains a conserved 13-nt core motif essential for trans-splicing, binding U1 snRNP via base-pairing with U1 snRNA.
• TSB acts as an enhancer, and its deletion, along with TSA, leads to developmental defects in flies.
• Compensatory mutations in U1 snRNA restored trans-splicing in TSA mutants, confirming the critical role of U1 recruitment.
• TSA core-like motifs are present in other trans-spliced Drosophila genes, suggesting a conserved mechanism.

Conclusions:

• Discovered a novel mechanism for trans-splicing in higher eukaryotes mediated by RNA motifs within the 5' intron.
• These RNA motifs (TSA and TSB) are sufficient to bring separate transcripts together, facilitating chimeric mRNA formation.
• U1 snRNP recruitment, mediated by the TSA motif, is crucial for promoting trans-splicing in vivo.