Figure 1. Identification of a ribonucleoprotein complex containing psiRNAs and Cas proteins.

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.