A)The indicated 5’ end-labeled (*) substrates were incubated in the presence (+) or absence (−) of the psiRNP (Figure 1C). The substrates were full-length 7.01 target RNA (F.L.), and the indicated truncations as diagramed in the lower panel. As in Figure 3, the locations of observed cleavages at sites 1 (green) and 2 (blue) are indicated on the full-length target and truncated RNAs (lower panel) and the corresponding cleavage products are indicated with green and blue arrows in the upper panels. Question mark in the lower panel indicates cleavage that could not be assessed. B) Model for cleavage at two sites directed by two psiRNAs. The 45-nucleotide psiRNA species guides cleavage at site 1 and the 39-nucleotide psiRNA guides cleavage at site 2 on each of the substrate RNAs as indicated. In both cases, cleavage occurs 14 nucleotides from the 3’ end of the psiRNA. Observed products are shown in green (site 1) and blue (site 2) and correspond to products in Figure 3 and Figure 4A.

A) 5' end-labeled 7.01 target RNA was incubated in the absence of added psiRNAs or proteins (−), in the presence of synthetic psiRNAs (+) or purified recombinant P. furiosus Cmr proteins (R) or in the presence of purified native psiRNPs (N) as indicated. The synthetic psiRNAs were 45- and 39-nucleotide forms of psiRNA 7.01. The six added recombinant Cmr proteins were P. furiosus Cmr1-1, Cmr2, Cmr3, Cmr4, Cmr5 and Cmr6. Products were resolved by denaturing gel electrophoresis. The products corresponding to cleavage at site 1 and site 2 (see Figure 3) are indicated by green and blue arrows, respectively. The sizes of RNA markers (M) are indicated. B) The 7.01 target RNA (Target) was incubated with the synthetic 7.01 psiRNAs (both 45-and 39-nucleotide species) in the absence (+ psiRNAs) and presence of the purified recombinant P. furiosus Cmr proteins (all), and also with combinations of proteins lacking individual Cmr proteins as indicated (e.g. - Cmr6). C) Cleavage activity of the recombinant psiRNP (R) reconstituted with either the individual 7.01 psiRNA species (45- or 39-nt) or both. Cleavage by the native psiRNP (N) is included for comparison.

Based on the results of this study, a psiRNA with a conserved 5’ sequence element derived from the CRISPR repeat (psi-tag) and a region of invader-targeting sequence assembles with six Cas module-RAMP proteins (Cmr1-6). The assembled psiRNP interacts with an invader RNA (e.g., viral mRNA) through base pairing between the psiRNA and invader RNA, positioning the region of the RNA-RNA duplex 14 nucleotides from the 3’ end of the psiRNA in proximity to the active site (star) of the enzyme. In P. furiosus, there are two prominent size forms of each psiRNA with different 3’ ends that guide cleavage of viral mRNAs at two distinct sites. There are also two Cmr1 proteins in P. furiosus that are both found in purified preparations and likely function redundantly.

The indicated 5’ end-labeled substrates were incubated in the presence (+) or absence (−) of the native psiRNPs (Figure 1C). Products were resolved by denaturing gel electrophoresis. The primary cleavage products are indicated by green and blue arrows (upper panel), and the corresponding sites of cleavage are indicated with green (site 1) and blue (site 2) vertical lines in the substrate sequences shown in the lower panel. The sizes of RNA markers (M) are indicated. “Target” substrates (panels 1, 3, 4, 5, 7) contain regions of perfect complementarity to the guide sequence of the indicated P. furiosus psiRNA. Grey bars demarcate the guide sequences in the lower panel. “+ ext” substrates (panels 1, 2, 3, 4, 7) contain 5’ and 3’ polyA extensions. In panel 4, a synthetic psiRNA (sequence shown in grey) was pre-annealed to the 7.01 target RNA + ext. Panel 2 shows a reverse target sequence substrate and panel 6 shows an antisense (AS) target substrate. Panel 3 shows a DNA substrate; all other substrates are RNA. Panel 8 shows unrelated RNA sR2.

A) psiRNP purification scheme. Letters indicate the location of corresponding data within Figure 1. B) psiRNA (top panel) and total RNA (bottom panel) profiles across the initial Q-sepharose anion exchange fractions and an unfractionated sample (total). Northern analysis (top panel) was performed for P. furiosus psiRNA 7.01. The positions of the mature psiRNAs and 1X intermediate RNA (Hale et al., 2008) are indicated. The lower panel shows all RNAs detected by SYBR Gold staining. The peak fraction is indicated by an arrow in each panel. C) psiRNA (Northern analysis of psiRNA 7.01, left panel) and total RNA (SYBR staining, right panel) profiles across the S-sepharose cation exchange fractions and starting material (load). The peak fraction is indicated by an arrow in each panel. D) Native gel Northern analysis of the psiRNP. The peak S-sepharose fraction (arrow, C) was fractionated by native gel electrophoresis and analyzed by Northern blotting for psiRNA 7.01. RNA extracted from the same fraction was co-analyzed. The position of the psiRNP is indicated. E) Cas proteins identified by tandem mass spectrometry. The isolated psiRNP (D) was subject to in-gel trypsin digestion and tandem mass spectrometry. Sequence coverage and the number of unique peptides for Cas proteins identified with 99% confidence are shown. P. furiosus cas gene names are as given (Haft et al., 2005) and proposed functions are as predicted (Haft et al., 2005; Makarova et al., 2006). F) Genome organization of predicted P. furiosus cas genes. Operon organization and COG assignments were adapted from NCBI database. Core cas genes (cas) and Cas module-RAMP (cmr), Cas subtype Apern (csa) and Cas subtype Tneap (cst) genes are indicated. Proteins identified by mass spectrometry are indicated in black.

A) Sequence analysis of RNAs associated with the complex. RNA species present in the S-sepharose fraction (visualized by SYBR Gold staining) are shown in left panel. RNAs in the upper and lower bands were isolated, cloned, and sequenced. Graphs show the percentage of sequenced RNAs with 5’ ends located at specific positions within the repeat sequence (black), and with indicated numbers of guide sequence nucleotides downstream of the repeat sequence (orange). The average guide sequence is 37 nucleotides in P. furiosus. A consensus for each psiRNA species is diagrammed under each graph. The 8-nucleotide repeat sequence found at the 5’ end of the majority of the psiRNAs is indicated as the psi-tag. B) Model for biogenesis of the two psiRNA species in P. furiosus. CRISPR locus transcripts containing alternating repeat (R, black segments) and guide (G, colored segments) elements are cleaved at a specific site within the repeat by the Cas6 endoribonuclease (Carte et al., 2008), ultimately producing 1X intermediate RNAs that contain a full invader-targeting sequence flanked on both sides by segments of the repeat. The mature RNAs retain the 5’ end repeat sequence (psi-tag). Uncharacterized 3’ end processing of the 1X intermediate by endo- and/or exo-nucleases forms the two major mature psiRNAs: a 45-nucleotide species that contains the 8-nucleotide psi-tag and a full guide sequence, and a 39-nucleotide species that contains a shorter 31-nucleotide guide sequence. C) Deep sequencing of small RNAs from P. furiosus confirms the presence of the psi-tag. The 5’ ends of the sequenced psiRNAs are graphed as in A. The number of total clones analyzed (n) is indicated in the graphs of panels A and C.