A stem/loop in U6 RNA defines a conformational switch required for pre-mRNA splicing

Area of Science:

• Molecular Biology
• RNA Biology
• Genetics

Background:

• U6 small nuclear RNA (snRNA) is a vital component of the spliceosome, responsible for pre-mRNA splicing.
• Its exact role in catalysis remains unclear, though it's hypothesized to be directly involved.

Purpose of the Study:

• To investigate the structural dynamics of U6 RNA and their impact on spliceosome assembly.
• To provide biochemical and genetic evidence for a specific RNA structure's role in U4/U6 small nuclear ribonucleoprotein (snRNP) biogenesis.

Main Methods:

• Biochemical assays to analyze RNA structures.
• Genetic analysis in Saccharomyces cerevisiae using site-directed mutagenesis.
• Assessment of growth phenotypes and RNA accumulation under varying temperature conditions.

Main Results:

• Identified a mutually exclusive intramolecular stem/loop structure in the 3' half of U6 RNA.
• Mutations stabilizing this stem caused cold-sensitive growth and blocked U4/U6 snRNP assembly in yeast.
• Overexpression of U4 snRNA and mutations disrupting the U6 3' stem suppressed the cold-sensitive growth defect.

Conclusions:

• A conformational switch, involving the melting of the U6 RNA 3' stem, is essential for U4/U6 snRNP assembly.
• This conformational change is likely required to activate U6 RNA for its catalytic role in splicing post-spliceosome assembly.