A bacterial Argonaute with noncanonical guide RNA specificity

Emine Kaya; Kevin W Doxzen; Kilian R Knoll; Ross C Wilson; Steven C Strutt; Philip J Kranzusch; Jennifer A Doudna

doi:10.1073/pnas.1524385113

A bacterial Argonaute with noncanonical guide RNA specificity

Proc Natl Acad Sci U S A. 2016 Apr 12;113(15):4057-62. doi: 10.1073/pnas.1524385113. Epub 2016 Mar 30.

Authors

Emine Kaya¹, Kevin W Doxzen², Kilian R Knoll³, Ross C Wilson³, Steven C Strutt³, Philip J Kranzusch⁴, Jennifer A Doudna⁵

Affiliations

¹ Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720;
² Biophysics Graduate Group, University of California, Berkeley, CA 94720;
³ Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720;
⁴ Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; Howard Hughes Medical Institute, University of California, Berkeley, CA 94720;
⁵ Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; Biophysics Graduate Group, University of California, Berkeley, CA 94720; Howard Hughes Medical Institute, University of California, Berkeley, CA 94720; Center for RNA Systems Biology, University of California, Berkeley, CA 94720; Innovative Genomics Initiative, University of California, Berkeley, CA 94720; Department of Chemistry, University of California, Berkeley, CA 94720 [email protected].

Abstract

Eukaryotic Argonaute proteins induce gene silencing by small RNA-guided recognition and cleavage of mRNA targets. Although structural similarities between human and prokaryotic Argonautes are consistent with shared mechanistic properties, sequence and structure-based alignments suggested that Argonautes encoded within CRISPR-cas [clustered regularly interspaced short palindromic repeats (CRISPR)-associated] bacterial immunity operons have divergent activities. We show here that the CRISPR-associated Marinitoga piezophila Argonaute (MpAgo) protein cleaves single-stranded target sequences using 5'-hydroxylated guide RNAs rather than the 5'-phosphorylated guides used by all known Argonautes. The 2.0-Å resolution crystal structure of an MpAgo-RNA complex reveals a guide strand binding site comprising residues that block 5' phosphate interactions. Using structure-based sequence alignment, we were able to identify other putative MpAgo-like proteins, all of which are encoded within CRISPR-cas loci. Taken together, our data suggest the evolution of an Argonaute subclass with noncanonical specificity for a 5'-hydroxylated guide.

Keywords: Argonaute; RNA interference; small noncoding RNA.

Publication types

Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Argonaute Proteins / chemistry
Argonaute Proteins / genetics
Argonaute Proteins / metabolism*
Bacterial Proteins / chemistry
Bacterial Proteins / genetics
Bacterial Proteins / metabolism*
Clustered Regularly Interspaced Short Palindromic Repeats
Genes, Bacterial
Models, Molecular
RNA, Guide, CRISPR-Cas Systems / metabolism*

Substances

Argonaute Proteins
Bacterial Proteins
RNA, Guide, CRISPR-Cas Systems

Associated data

PDB/5I4A

Grants and funding

R01 GM073794/GM/NIGMS NIH HHS/United States