Target RNA capture and cleavage by the Cmr type III-B CRISPR–Cas effector complex

CRISPR (clustered regularly interspaced short palindromic repeat)–Cas (CRISPR-associated) systems confer bacteria and archaea with the ability to acquire immunity to viruses and plasmids (Terns and Terns 2011; Westra et al. 2012; Sorek et al. 2013; Barrangou and Marraffini 2014; van der Oost et al. 2014). Upon infection, short fragments of invading DNA are captured and incorporated into discrete host genome loci termed CRISPRs, which consist of multiple copies of a short repeat sequence separated by the short invader-derived sequences (Bolotin et al. 2005; Mojica et al. 2005; Pourcel et al. 2005). CRISPR transcripts are processed into small CRISPR RNAs (crRNAs) that contain an invader-derived guide sequence and sequence from the flanking repeats (Brouns et al. 2008; Hale et al. 2008, 2009). crRNAs are loaded into effector complexes that can recognize and silence corresponding RNAs or DNAs. Modules of Cas proteins function in invader sequence acquisition and crRNA biogenesis as well as target destruction (Haft et al. 2005; Makarova et al. 2011a). Bioinformatic analyses have identified three broad types of CRISPR–Cas systems, each with distinct types of crRNA-containing effector complexes (Makarova et al. 2011b). In the prototypic type I system found in Escherichia coli, multiple Cas proteins form a crRNA-containing complex that recognizes complementary DNA targets and recruits the Cas3 nuclease for target destruction (Brouns et al. 2008; Jore et al. 2011; Sinkunas et al. 2011, 2013; Wiedenheft et al. 2011a,b; Sashital et al. 2012; Westra et al. 2012; Mulepati and Bailey 2013; Hochstrasser et al. 2014; Huo et al. 2014; Jackson et al. 2014; Mulepati et al. 2014; Zhao et al. 2014). The prototypic type II effector complex also cleaves DNA targets but consists of a single protein (Cas9) associated with two essential RNAs (a crRNA guide and a tracrRNA cofactor) (Garneau et al. 2010; Deltcheva et al. 2011; Gasiunas et al. 2012; Jinek et al. 2012). Type III effector complexes (Hale et al. 2009, 2012; Spilman et al. 2013; Zhang et al. 2012; Staals et al. 2013) include multiple Cas proteins. The type III-B or Cmr complexes, characterized in Pyrococcus furiosus (Hale et al. 2009, 2012; Spilman et al. 2013), Sulfolobus solfataricus (Zhang et al. 2012), and Thermus thermophilus (Staals et al. 2013), cleave RNA targets. The type III-A or Csm subtype system targets DNA (Marraffini and Sontheimer 2008; Millen et al. 2012). The type I and type III effector complexes appear to be distantly related: The protein subunits of both complexes are members of common superfamilies that are found in strikingly similar arrangements within the complexes (Spilman et al. 2013). The functions of the individual proteins in multisubunit CRISPR–Cas complexes are largely unknown. Type III-B Cmr complexes from P. furiosus consist of the six Cmr proteins, Cmr1–6, and a crRNA comprised of 8 nucleotides (nt) of CRISPR sequence at the 5′ end (the signature 5′ tag) and typically either 37 or 31 nt of invader-derived guide sequence (45- and 39-nt crRNAs) (Hale et al. 2008, 2009, 2012). (The Cmr complex from T. thermophilus has a similar composition [Staals et al. 2013].) Cmr1, Cmr4, and Cmr6 are members of the Cas7 superfamily of proteins found in both type I and type III systems (Makarova et al. 2011a). Cmr3 is a member of the Cas5 protein superfamily, also found in both type I and type III systems (Makarova et al. 2011a). All four of these Cmr proteins (Cmr1, Cmr3, Cmr4, and Cmr6) contain a distinct ferredoxin fold or RNA recognition motif (RRM) found in numerous Cas proteins that are referred to as repeat-associated mysterious proteins (RAMPs) (Makarova et al. 2011b). Some RAMPs are nucleases (e.g., Cas6) (Brouns et al. 2008; Carte et al. 2008). Cmr2 is a member of the Cas10 “large subunit” Cas protein superfamily (Hale et al. 2009; Makarova et al. 2011a). Cmr2 contains an adenylyl cyclase-like domain that forms a nucleotide and metal-binding pocket (Cocozaki et al. 2012). Cmr2 also contains a predicted N-terminal HD nuclease domain, although this domain is dispensable for target RNA cleavage by the Cmr complex (Zhu and Ye 2012; Shao et al. 2013; Staals et al. 2013). Finally, Cmr5 is a member of the “small subunit” Cas protein superfamily and is primarily α-helical in structure (Sakamoto et al. 2009; Park et al. 2013; Reeks et al. 2013). The functions of the individual Cmr proteins are currently unknown. The mechanisms involved in target RNA cleavage by Cmr complexes are a topic of ongoing investigation. Native Cmr complexes associate with multiple size forms of crRNAs and cleave complementary target RNAs at multiple sites in the region recognized by the crRNA. For example, native P. furiosus Cmr complexes contain two major crRNA species and cleave complementary target RNAs primarily at two sites (Hale et al. 2009). However, we found that Cmr complexes reconstituted with single crRNA species generated a single cleavage product at the site located 14 nt from the 3′ end of the respective crRNA species, leading to the model that each Cmr complex cleaves the target RNA a fixed distance from the 3′ end of the crRNA (Hale et al. 2009). T. thermophilus Cmr complexes contain at least three distinct crRNA species and cleave target RNAs at up to five sites (Staals et al. 2013). Studies with reconstituted T. thermophilus complexes suggest that the complex cleaves at multiple sites measured from the 5′ end of the crRNA (Staals et al. 2013). Moreover, the molecular basis of the nucleolytic activity of the Cmr complex is unknown. We determined the basic organization and structure of the P. furiosus Cmr complex by a combination of RNA–protein cross-linking and cryo-electron microscopy (cryo-EM) of assembled Cmr complexes (Fig. 1A; Spilman et al. 2013). Our analysis revealed that Cmr4–Cmr5 units comprise the “backbone” of the particle and follow a helical path along the guide region of the crRNA. Cmr2 and Cmr3 form a “foot” at the 5′ end of the crRNA, and Cmr1 and Cmr6 form a “cap” at the 3′ end (Fig. 1A; Spilman et al. 2013). The EM structure of the T. thermophilus Cmr particle has a very similar shape, and Staals et al. (2013) developed a very similar model of the organization of the proteins in the T. thermophilus complex, while that of the Cmr complex variant characterized from S. solfataricus differs significantly (Zhang et al. 2012). Native mass spectrometric analysis of the T. thermophilus complex determined that the Cmr complex includes four Cmr4 and three Cmr5 subunits (Staals et al. 2013). Figure 1. Requirements for target RNA cleavage and crRNA binding by the Cmr complex. (A) Model of the organization of the Cmr protein subunits relative to the crRNA within the Cmr complex (Spilman et al. 2013). (B) Cmr protein requirements for target RNA cleavage. ... In this study, we investigated the processes of Cmr complex assembly, target RNA capture, and target RNA cleavage and the roles of the Cmr proteins in these processes.