Transcriptome-wide Interrogation of the Functional Intronome by Spliceosome Profiling

Area of Science:

• Molecular Biology
• Genomics
• RNA Biology

Background:

• Eukaryotic transcriptome complexity necessitates understanding all intron excision sites.
• RNA sequencing (RNA-seq) is limited by low abundance of spliced transcripts due to rapid decay.
• A comprehensive view of RNA processing requires methods beyond standard RNA-seq.

Purpose of the Study:

• To introduce spliceosome profiling, a novel strategy for deep sequencing of RNAs associated with late-stage spliceosomes.
• To enable high-resolution mapping of intron ends and branchpoint identification.
• To expand the known repertoire of spliced RNA species and improve annotation accuracy.

Main Methods:

• Developed "spliceosome profiling" by deep sequencing RNAs co-purifying with late-stage spliceosomes.
• Applied the method to study intron excision in S. pombe.
• Achieved single-nucleotide resolution for intron end mapping and branchpoint identification.

Main Results:

• Identified hundreds of previously unknown introns in S. pombe.
• Corrected numerous previously misannotated introns.
• Demonstrated the power of spliceosome profiling for deep interrogation of spliceosome assembly and catalysis.

Conclusions:

• Spliceosome profiling offers unprecedented depth for analyzing RNA processing.
• The technique significantly enhances the discovery of novel introns and improves transcript annotation.
• This method is poised to revolutionize the study of nuclear RNA processing, analogous to ribosome profiling's impact on translation studies.