Nonpolyadenylated RNA polymerase II termination is induced by transcript cleavage

Area of Science:

• Molecular Biology
• RNA Biology
• Gene Expression

Background:

• Eukaryotic mRNA transcription termination and 3' processing are typically linked to polyadenylation signals and transcript cleavage.
• Mechanisms for termination and processing of nonpolyadenylated RNA polymerase II transcripts remain less understood.
• U3 small nucleolar RNA (snoRNA) in Schizosaccharomyces pombe is a nonpolyadenylated transcript requiring specific regulatory mechanisms.

Purpose of the Study:

• To investigate the in vivo mechanisms of transcription termination and 3' end processing for Schizosaccharomyces pombe U3 small nucleolar RNA (snoRNA).
• To determine the role of cis-acting sequences and transcript cleavage in the maturation and termination of U3 snoRNA.
• To elucidate the underlying model for termination and processing of nonpolyadenylated RNA polymerase II transcripts.

Main Methods:

• Utilized an efficiently expressed plasmid-based expression system in Schizosaccharomyces pombe.
• Employed a termination assay linked to cell transformation and restriction fragment length polymorphism to quantify U3 snoRNA levels.
• Conducted in vivo mutation analyses and introduced endonuclease-labile structures to assess the impact on RNA processing and termination.

Main Results:

• 3' end maturation of U3 snoRNA is dependent on transcript cleavage, not external cis-acting sequences or structures.
• Cleavage can occur significantly downstream of the mature U3 snoRNA sequence, influencing both termination and processing.
• A single endonuclease-labile structure can efficiently induce both termination and maturation simultaneously, supporting a unified mechanism.

Conclusions:

• The termination and 3' end processing of Schizosaccharomyces pombe U3 snoRNA are coupled to a single transcript cleavage event.
• These findings support a "reversed torpedoes" model where cleavage facilitates access for exonucleases and protein factors.
• This model explains how a single cleavage event can drive both transcription termination and RNA maturation in nonpolyadenylated transcripts.