A bacterial Argonaute with noncanonical guide RNA specificity

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Eukaryotic Argonaute proteins mediate gene silencing through small RNA-guided mRNA recognition and cleavage.
• Structural similarities exist between human and prokaryotic Argonaute proteins, suggesting shared functions.
• However, Argonautes within bacterial CRISPR-cas loci were hypothesized to have distinct activities.

Purpose of the Study:

• To investigate the functional and structural properties of the CRISPR-associated Marinitoga piezophila Argonaute (MpAgo) protein.
• To determine the mechanism of guide RNA interaction and target cleavage by MpAgo.
• To identify other potential Argonaute proteins with similar characteristics within bacterial CRISPR-cas systems.

Main Methods:

• Biochemical assays to assess target cleavage activity of MpAgo.
• X-ray crystallography to determine the 3D structure of the MpAgo-RNA complex at 2.0 Å resolution.
• Structure-based sequence alignment to identify related Argonaute proteins.

Main Results:

• MpAgo cleaves single-stranded target sequences.
• MpAgo uniquely utilizes 5 extbackslash'-hydroxylated guide RNAs for cleavage, unlike the 5 extbackslash'-phosphorylated guides used by other known Argonautes.
• The crystal structure revealed specific residues in the guide strand binding site that prevent 5 extbackslash' phosphate interactions.
• Identification of other MpAgo-like proteins exclusively within CRISPR-cas loci.

Conclusions:

• Marinitoga piezophila Argonaute (MpAgo) represents a distinct subclass of Argonaute proteins.
• This subclass exhibits noncanonical specificity, utilizing 5 extbackslash'-hydroxylated guide RNAs.
• The findings suggest an evolutionary divergence within Argonaute proteins, particularly in bacterial CRISPR-cas systems.