Long-read sequencing of nascent RNA reveals coupling among RNA processing events

Area of Science:

• Molecular Biology
• RNA Biology
• Gene Regulation

Background:

• Pre-mRNA splicing by the spliceosome is a crucial step in gene expression.
• Splicing occurs cotranscriptionally, but the order and regulation of splicing events on nascent transcripts are poorly understood.
• Existing methods primarily analyze mature mRNA, limiting insights into splicing intermediates.

Purpose of the Study:

• To investigate the order of intron removal during cotranscriptional splicing.
• To explore the regulatory potential of nascent transcript remodeling by splicing.
• To understand the interplay between splicing, 3' end formation, and mRNA stability.

Main Methods:

• Utilized long-read RNA sequencing to analyze nascent, multi-intron transcripts in Schizosaccharomyces pombe.
• Employed a temperature-sensitive mutation in prp2 to mildly inhibit splicing.
• Investigated the impact of splicing status on transcriptional read-through and cotranscriptional degradation by the nuclear exosome.

Main Results:

• Most multi-intron transcripts were fully spliced, supporting rapid cotranscriptional splicing.
• A significant proportion of transcripts were either fully spliced or fully unspliced, indicating intron splicing interdependence.
• Inhibition of splicing led to an increase in fully unspliced transcripts, which were prone to read-through and degradation.
• Caffeine treatment reduced mRNA levels in genes with unspliced nascent transcripts, highlighting regulatory significance.

Conclusions:

• Splicing of individual introns within a nascent transcript is interdependent.
• The splicing status of introns influences 3' end formation and mRNA half-life.
• Crosstalk exists between spliceosomes and the polyadenylation machinery during transcription elongation.