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Abstract
Recent evidence supports the presence of a small RNA-based genome defense system in prokaryotic organisms. This system is comprised of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and a large family of CRISPR-associated proteins. The presence of invader-derived sequences within the CRISPR loci has been shown to confer immunity against that invader to the CRISPR-containing organism. This system is predicted to depend on small RNAs that are transcribed from the CRISPR loci. This work focuses on characterization of these prokaryotic silencing (psi)RNAs and their associated proteins in the hyperthermophilic archaeon Pyrococcus furiosus. Characterization of RNA products from the CRISPR loci revealed unidirectional transcription of all of the CRISPRs in Pyrococcus furiosus originating from the conserved leader sequences. Small RNAs derived from all parts of a given CRISPR locus were found, with a greater number found of the more recently incorporated leader-proximal invader targeting sequences. Furthermore, CRISPR transcripts were shown to be processed into small RNAs that contain mostly invader-derived sequence, with an 8-nucleotide portion of the 5 repeat sequence called the psi-tag. Mature psiRNAs were found to exist in several chromatographically distinct RNA-protein complexes, one of which contained the Cas module RAMP (Cmr) proteins. The psiRNA-Cmr protein complex was found to recognize and cleave single-stranded RNAs that are complementary to the incorporated psiRNAs. Cleavage was shown to occur at a site that is 14 nucleotides from the 3 end of the psiRNA. This works presents the first evidence of small RNA-guided RNA destruction by the CRISPR/Cas system, and may represent a powerful system for sequence-specific degradation of RNAs in vivo.