title: Molecular determinants for CRISPR RNA maturation in the Cas10-Csm complex and roles for non-Cas nucleases.
abstract: CRISPR–Cas (Clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) is a prokaryotic immune system that destroys foreign nucleic acids in a sequence-specific manner using Cas nucleases guided by short RNAs (crRNAs). Staphylococcus epidermidis harbours a Type III-A CRISPR–Cas system that encodes the Cas10–Csm interference complex and crRNAs that are subjected to multiple processing steps. The final step, called maturation, involves a concerted effort between Csm3, a ruler protein in Cas10–Csm that measures six-nucleotide increments, and the activity of a nuclease(s) that remains unknown. Here, we elucidate the contributions of the Cas10–Csm complex toward maturation and explore roles of non-Cas nucleases in this process. Using genetic and biochemical approaches, we show that charged residues in Csm3 facilitate its self-assembly and dictate the extent of maturation cleavage. Additionally, acidic residues in Csm5 are required for efficient maturation, but recombinant Csm5 fails to cleave crRNAs in vitro. However, we detected cellular nucleases that co-purify with Cas10–Csm, and show that Csm5 regulates their activities through distinct mechanisms. Altogether, our results support roles for non-Cas nuclease(s) during crRNA maturation and establish a link between Type III-A CRISPR–Cas immunity and central nucleic acid metabolism.
authors.affiliation: Department of Biological Sciences, University of Alabama, Tuscaloosa, AL 35487, USA.
authors.forename: Forrest C
authors.initials: FC
authors.lastname: Walker
authors.affiliation: Department of Biological Sciences, University of Alabama, Tuscaloosa, AL 35487, USA.
authors.forename: Lucy
authors.initials: L
authors.lastname: Chou-Zheng
authors.affiliation: Department of Chemistry, University of Alabama, Tuscaloosa, AL, 35487, USA.
authors.forename: Jack A
authors.initials: JA
authors.lastname: Dunkle
authors.affiliation: Department of Biological Sciences, University of Alabama, Tuscaloosa, AL 35487, USA.
authors.forename: Asma
authors.initials: A
authors.lastname: Hatoum-Aslan
mesh_terms: D064130:CRISPR-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D064113:CRISPR-Cas Systems; D011117:Polyribonucleotide Nucleotidyltransferase; D012329:RNA, Bacterial / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism*; D013212:Staphylococcus epidermidis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism
chemical_list: D064130:CRISPR-Associated Proteins; D012329:RNA, Bacterial; D011117:Polyribonucleotide Nucleotidyltransferase; D012260:Ribonucleases