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2017 ; 45
(4
): 2112-2123
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Molecular determinants for CRISPR RNA maturation in the Cas10-Csm complex and
roles for non-Cas nucleases
#MMPMID28204542
Walker FC
; Chou-Zheng L
; Dunkle JA
; Hatoum-Aslan A
Nucleic Acids Res
2017[Feb]; 45
(4
): 2112-2123
PMID28204542
show ga
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.