Structural and mechanistic insight into stem-loop RNA processing by yeast Pichia stipitis Dicer

Area of Science:

• Biochemistry
• Molecular Biology
• Yeast Genetics

Background:

• Dicer enzymes are crucial for processing double-stranded RNA into small functional RNAs.
• Species-specific variations in Dicer domain architecture present intriguing functional adaptations.
• Budding yeast Pichia stipitis harbors a unique RNase III protein with Dicer-like activity.

Purpose of the Study:

• To characterize the structure and function of a novel non-canonical Dicer protein (PsDCR1) from Pichia stipitis.
• To elucidate the structural basis for the catalytic activity and substrate specificity of PsDCR1.
• To investigate the potential for an RNA-guided gene silencing pathway in budding yeast mediated by PsDCR1.

Main Methods:

• X-ray crystallography to determine the structure of the catalytic core of PsDCR1.
• Biochemical assays to assess RNA cleavage activity and substrate preference.
• RNA sequencing and site-directed mutagenesis to identify key RNA structural determinants for cleavage.

Main Results:

• The catalytically active region of PsDCR1 comprises a homodimeric RNase III domain and dsRNA-binding domain 1 (dsRBD1) in a unique diagonal orientation.
• A C-terminal dsRNA-binding domain, absent in related proteins like Rnt1p, enhances RNA cleavage.
• PsDCR1 cleavage is dictated by sequence motifs in the lower stem of hairpin RNAs, differing from Rnt1p's apical loop dependence.

Conclusions:

• PsDCR1 represents a novel, catalytically active Dicer homolog with distinct structural and functional properties.
• The unique domain arrangement and substrate recognition of PsDCR1 highlight evolutionary divergence in RNA processing pathways.
• Findings suggest the existence of a stem-loop RNA-guided gene silencing mechanism in budding yeast.