Role of free DNA ends and protospacer adjacent motifs for CRISPR DNA uptake in Pyrococcus furiosus.

To acquire CRISPR-Cas immunity against invasive mobile genetic elements, prokaryotes must first integrate fragments of foreign DNA into their genomic CRISPR arrays for use in future invader silencing. Here, we found that the hyperthermophilic archaeaon, Pyrococcus furiosus, actively incorporates DNA fragments (spacers) from both plasmid (foreign) and host genome (self) sequences into its seven CRISPR loci. The majority of new spacers were derived from DNA immediately downstream from a 5'-CCN-3' protospacer adjacent motif (PAM) that is critical for invader targeting. Interestingly, spacers were preferentially acquired from genome or plasmid regions corresponding to active transposons, CRISPR loci, ribosomal RNA genes, rolling circle origins of replication, and areas where plasmids recombined with the host chromosome. A common feature of the highly sampled spacers is that they arise from DNA regions expected to undergo DNA nicking and/or double-strand breaks. Taken together with recent results from bacterial systems, our findings indicate that free DNA termini and PAMs are conserved features important for CRISPR spacer uptake in diverse prokaryotes and CRISPR-Cas systems. Moreover, lethal self-targeting by CRISPR systems may contribute to host genome stability by eliminating cells undergoing active transposon mobility or chromosomal uptake of autonomously replicating foreign mobile genetic elements.

The CRISPR-associated Csx1 protein of Pyrococcus furiosus is an adenosine-specific endoribonuclease.

Prokaryotes are frequently exposed to potentially harmful invasive nucleic acids from phages, plasmids, and transposons. One method of defense is the CRISPR-Cas adaptive immune system. Diverse CRISPR-Cas systems form distinct ribonucleoprotein effector complexes that target and cleave invasive nucleic acids to provide immunity. The Type III-B Cmr effector complex has been found to target the RNA and DNA of the invader in the various bacterial and archaeal organisms where it has been characterized. Interestingly, the gene encoding the Csx1 protein is frequently located in close proximity to the Cmr1-6 genes in many genomes, implicating a role for Csx1 in Cmr function. However, evidence suggests that Csx1 is not a stably associated component of the Cmr effector complex, but is necessary for DNA silencing by the Cmr system in Sulfolobus islandicus. To investigate the function of the Csx1 protein, we characterized the activity of recombinant Pyrococcus furiosus Csx1 against various nucleic acid substrates. We show that Csx1 is a metal-independent, endoribonuclease that acts selectively on single-stranded RNA and cleaves specifically after adenosines. The RNA cleavage activity of Csx1 is dependent upon a conserved HEPN motif located within the C-terminal domain of the protein. This motif is also key for activity in other known ribonucleases. Collectively, the findings indicate that invader silencing by Type III-B CRISPR-Cas systems relies both on RNA and DNA nuclease activities from the Cmr effector complex as well as on the affiliated, trans-acting Csx1 endoribonuclease.

Three CRISPR-Cas immune effector complexes coexist in Pyrococcus furiosus.

CRISPR-Cas immune systems function to defend prokaryotes against potentially harmful mobile genetic elements including viruses and plasmids. The multiple CRISPR-Cas systems (Types I, II, and III) each target destruction of foreign nucleic acids via structurally and functionally diverse effector complexes (crRNPs). CRISPR-Cas effector complexes are comprised of CRISPR RNAs (crRNAs) that contain sequences homologous to the invading nucleic acids and Cas proteins specific to each immune system type. We have previously characterized a crRNP in Pyrococcus furiosus (Pfu) that contains Cmr (Type III-B) Cas proteins associated with one of two size classes of crRNAs and cleaves complementary target RNAs. Here, we have isolated and characterized two additional native Pfu crRNPs containing either Csa (Type I-A) or Cst (Type I-G) Cas proteins and distinct profiles of associated crRNAs. For each complex, the Cas proteins were identified by mass spectrometry and immunoblotting and the crRNAs by RNA sequencing and Northern blot analysis. The crRNAs associated with both the Csa and Cst complexes originate from all seven Pfu CRISPR loci and contain identical 5' ends (8-nt repeat-derived 5' tag sequences) but heterogeneous 3' ends (containing variable amounts of downstream repeat sequences). These crRNA forms are distinct from Cmr-associated crRNAs, indicating different 3' end processing pathways following primary cleavage of common pre-crRNAs. Like other previously characterized Type I CRISPR-Cas effector complexes, we predict that the newly identified Pfu Csa and Cst crRNPs each function to target invading DNA, adding an additional layer of protection beyond that afforded by the previously characterized RNA targeting Cmr complex.

The three major types of CRISPR-Cas systems function independently in CRISPR RNA biogenesis in Streptococcus thermophilus.

CRISPR-Cas systems are small RNA-based immune systems that protect prokaryotes from invaders such as viruses and plasmids. We have investigated the features and biogenesis of the CRISPR (cr)RNAs in Streptococcus thermophilus (Sth) strain DGCC7710, which possesses four different CRISPR-Cas systems including representatives from the three major types of CRISPR-Cas systems. Our results indicate that the crRNAs from each CRISPR locus are specifically processed into divergent crRNA species by Cas proteins (and non-coding RNAs) associated with the respective locus. We find that the Csm Type III-A and Cse Type I-E crRNAs are specifically processed by Cas6 and Cse3 (Cas6e), respectively, and retain an 8-nucleotide CRISPR repeat sequence tag 5' of the invader-targeting sequence. The Cse Type I-E crRNAs also retain a 21-nucleotide 3' repeat tag. The crRNAs from the two Csn Type II-A systems in Sth consist of a 5'-truncated targeting sequence and a 3' tag; however, these are distinct in size between the two. Moreover, the Csn1 (Cas9) protein associated with one Csn locus functions specifically in the production of crRNAs from that locus. Our findings indicate that multiple CRISPR-Cas systems can function independently in crRNA biogenesis within a given organism - an important consideration in engineering coexisting CRISPR-Cas pathways.

Programmable plasmid interference by the CRISPR-Cas system in Thermococcus kodakarensis.

CRISPR-Cas systems are RNA-guided immune systems that protect prokaryotes against viruses and other invaders. The CRISPR locus encodes crRNAs that recognize invading nucleic acid sequences and trigger silencing by the associated Cas proteins. There are multiple CRISPR-Cas systems with distinct compositions and mechanistic processes. Thermococcus kodakarensis (Tko) is a hyperthermophilic euryarchaeon that has both a Type I-A Csa and a Type I-B Cst CRISPR-Cas system. We have analyzed the expression and composition of crRNAs from the three CRISPRs in Tko by RNA deep sequencing and northern analysis. Our results indicate that crRNAs associated with these two CRISPR-Cas systems include an 8-nucleotide conserved sequence tag at the 5' end. We challenged Tko with plasmid invaders containing sequences targeted by endogenous crRNAs and observed active CRISPR-Cas-mediated silencing. Plasmid silencing was dependent on complementarity with a crRNA as well as on a sequence element found immediately adjacent to the crRNA recognition site in the target termed the PAM (protospacer adjacent motif). Silencing occurred independently of the orientation of the target sequence in the plasmid, and appears to occur at the DNA level, presumably via DNA degradation. In addition, we have directed silencing of an invader plasmid by genetically engineering the chromosomal CRISPR locus to express customized crRNAs directed against the plasmid. Our results support CRISPR engineering as a feasible approach to develop prokaryotic strains that are resistant to infection for use in industry.

Essential features and rational design of CRISPR RNAs that function with the Cas RAMP module complex to cleave RNAs.

Small RNAs target invaders for silencing in the CRISPR-Cas pathways that protect bacteria and archaea from viruses and plasmids. The CRISPR RNAs (crRNAs) contain sequence elements acquired from invaders that guide CRISPR-associated (Cas) proteins back to the complementary invading DNA or RNA. Here, we have analyzed essential features of the crRNAs associated with the Cas RAMP module (Cmr) effector complex, which cleaves targeted RNAs. We show that Cmr crRNAs contain an 8 nucleotide 5' sequence tag (also found on crRNAs associated with other CRISPR-Cas pathways) that is critical for crRNA function and can be used to engineer crRNAs that direct cleavage of novel targets. We also present data that indicate that the Cmr complex cleaves an endogenous complementary RNA in Pyrococcus furiosus, providing direct in vivo evidence of RNA targeting by the CRISPR-Cas system. Our findings indicate that the CRISPR RNA-Cmr protein pathway may be exploited to cleave RNAs of interest.

A global view of CK2 function and regulation.

The wealth of biochemical, molecular, genetic, genomic, and bioinformatic resources available in S. cerevisiae make it an excellent system to explore the global role of CK2 in a model organism. Traditional biochemical and genetic studies have revealed that CK2 is required for cell viability, cell cycle progression, cell polarity, ion homeostasis, and other functions, and have identified a number of potential physiological substrates of the enzyme. Data mining of available bioinformatic resources indicates that (1) there are likely to be hundreds of CK2 targets in this organism, (2) the majority of predicted CK2 substrates are involved in various aspects of global gene expression, (3) CK2 is present in several nuclear protein complexes predicted to have a role in chromatin structure and remodeling, transcription, or RNA metabolism, and (4) CK2 is localized predominantly in the nucleus. These bioinformatic results suggest that the observed phenotypic consequences of CK2 depletion may lie downstream of primary defects in chromatin organization and/or global gene expression. Further progress in defining the physiological role of CK2 will almost certainly require a better understanding of the mechanism of regulation of the enzyme. Beginning with the crystal structure of the human CK2 holoenzyme, we present a molecular model of filamentous CK2 that is consistent with earlier proposals that filamentous CK2 represents an inactive form of the enzyme. The potential role of filamentous CK2 in regulation in vivo is discussed.

Genetic interactions among ZDS1,2, CDC37, and protein kinase CK2 in Saccharomyces cerevisiae.

We report here the identification of the homologous gene pair ZDS1,2 as multicopy suppressors of a temperature-sensitive allele (cka2-13(ts)) of the CKA2 gene encoding the alpha' catalytic subunit of protein kinase CK2. Overexpression of ZDS1,2 suppressed the temperature sensitivity, geldanamycin (GA) sensitivity, slow growth, and flocculation of multiple cka2 alleles and enhanced CK2 activity in vivo toward a known physiological substrate, Fpr3. Consistent with the existence of a recently described positive feedback loop between CK2 and Cdc37, overexpression of ZDS1,2 also suppressed the temperature sensitivity, abnormal morphology, and GA sensitivity of a CK2 phosphorylation-deficient mutant of CDC37, cdc37-S14A, as well as the GA sensitivity of a cdc37-1 allele. A likely basis for all of these effects is our observation that ZDS1,2 overexpression enhances Cdc37 protein levels. Activation of the positive feedback loop between CK2 and Cdc37 likely contributes to the pleiotropic nature of ZDS1,2, as both CK2 and Cdc37 regulate diverse cellular functions.

Enzymatic function of loop movement in enolase: preparation and some properties of H159N, H159A, H159F, and N207A enolases.

The hypothesis that His159 in yeast enolase moves on a polypeptide loop to protonate the phosphoryl of 2-phosphoglycerate to initiate its conversion to phosphoenolpyruvate was tested by preparing H159N, H159A, and H159F enolases. These have 0.07%-0.25% of the native activity under standard assay conditions and the pH dependence of maximum velocities of H159A and H159N mutants is markedly altered. Activation by Mg2+ is biphasic, with the smaller Mg2+ activation constant closer to that of the "catalytic" Mg2+ binding site of native enolase and the larger in the mM range in which native enolase is inhibited. A third Mg2+ may bind to the phosphoryl, functionally replacing proton donation by His159. N207A enolase lacks an intersubunit interaction that stabilizes the closed loop(s) conformation when 2-phosphoglycerate binds. It has 21% of the native activity, also exhibits biphasic Mg2+ activation, and its reaction with the aldehyde analogue of the substrate is more strongly inhibited than is its normal enzymatic reaction. Polypeptide loop(s) closure may keep a proton from His159 interacting with the substrate phosphoryl oxygen long enough to stabilize a carbanion intermediate.

A positive feedback loop between protein kinase CKII and Cdc37 promotes the activity of multiple protein kinases.

We report here the identification of CDC37, which encodes a putative Hsp90 co-chaperone, as a multicopy suppressor of a temperature-sensitive allele (cka2-13(ts)) of the CKA2 gene encoding the alpha' catalytic subunit of protein kinase CKII. Unlike wild-type cells, cka2-13 cells were sensitive to the Hsp90-specific inhibitor geldanamycin, and this sensitivity was suppressed by overexpression of either Hsp90 or Cdc37. However, only CDC37 was capable of suppressing the temperature sensitivity of a cka2-13 strain, implying that Cdc37 is the limiting component. Immunoprecipitation of metabolically labeled Cdc37 from wild-type versus cka2-13 strains revealed that Cdc37 is a physiological substrate of CKII, and Ser-14 and/or Ser-17 were identified as the most likely sites of CKII phosphorylation in vivo. A cdc37-S14,17A strain lacking these phosphorylation sites exhibited severe growth and morphological defects that were partially reversed in a cdc37-S14,17E strain. Reduced CKII activity was observed in both cdc37-S14A and cdc37-S17A mutants at 37 degrees C, and cdc37-S14A or cdc37-S14,17A overexpression was incapable of protecting cka2-13 mutants on media containing geldanamycin. Additionally, CKII activity was elevated in cells arrested at the G(1) and G(2)/M phases of the cell cycle, the same phases during which Cdc37 function is essential. Collectively, these data define a positive feedback loop between CKII and Cdc37. Additional genetic assays demonstrate that this CKII/Cdc37 interaction positively regulates the activity of multiple protein kinases in addition to CKII.

Interaction of casein kinase II with ribosomal protein L22 of Drosophila melanogaster.

The ubiquitous eukaryotic protein kinase CKII (casein kinase II) has been found to interact with a number of cellular proteins, either through the catalytic subunit or the regulatory subunit. Using the yeast two-hybrid screening method, we found that the catalytic subunit of Drosophila melanogaster CKII (DmCKII) interacts with Drosophila ribosomal protein L22 (rpL22). This interaction was also observed in vitro with a glutathione-S-transferase (GST)-rpL22 fusion protein. The predicted full-length Drosophila rpL22 protein has an N-terminal extension rich in alanine, lysine, and proline that appears to be unique to Drosophila. Deletion mapping revealed that the conserved core of rpL22 is responsible for the interaction with CKII. Moreover, purified DmCKII can phosphorylate a GST-L22 fusion protein at the C-terminal end, suggesting that this protein may be a substrate of CKII in Drosophila.