RESUMEN
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.
Asunto(s)
Proteínas Asociadas a CRISPR/química , Sistemas CRISPR-Cas , Endorribonucleasas/química , Pyrococcus furiosus/genética , ARN de Archaea/química , Adenosina/metabolismo , Secuencias de Aminoácidos , Proteínas Arqueales , Secuencia de Bases , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/inmunología , Endorribonucleasas/genética , Endorribonucleasas/inmunología , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Pyrococcus furiosus/inmunología , ARN de Archaea/genética , ARN de Archaea/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Sulfolobus/genética , Sulfolobus/inmunologíaRESUMEN
The prokaryotic immune system CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) is a defense system that protects prokaryotes against foreign DNA. The short CRISPR RNAs (crRNAs) are central components of this immune system. In CRISPR-Cas systems type I and III, crRNAs are generated by the endonuclease Cas6. We developed a Cas6b-independent crRNA maturation pathway for the Haloferax type I-B system in vivo that expresses a functional crRNA, which we termed independently generated crRNA (icrRNA). The icrRNA is effective in triggering degradation of an invader plasmid carrying the matching protospacer sequence. The Cas6b-independent maturation of the icrRNA allowed mutation of the repeat sequence without interfering with signals important for Cas6b processing. We generated 23 variants of the icrRNA and analyzed them for activity in the interference reaction. icrRNAs with deletions or mutations of the 3' handle are still active in triggering an interference reaction. The complete 3' handle could be removed without loss of activity. However, manipulations of the 5' handle mostly led to loss of interference activity. Furthermore, we could show that in the presence of an icrRNA a strain without Cas6b (Δcas6b) is still active in interference.
Asunto(s)
Proteínas Arqueales/inmunología , Sistemas CRISPR-Cas/inmunología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/inmunología , Regulación de la Expresión Génica Arqueal , Haloferax volcanii/inmunología , Plásmidos/genética , ARN de Archaea/inmunología , Proteínas Arqueales/genética , Northern Blotting , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Ingeniería Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Interferencia de ARN , ARN de Archaea/genéticaRESUMEN
To fend off foreign genetic elements, prokaryotes have developed several defense systems. The most recently discovered defense system, CRISPR/Cas, is sequence-specific, adaptive and heritable. The two central components of this system are the Cas proteins and the CRISPR RNA. The latter consists of repeat sequences that are interspersed with spacer sequences. The CRISPR locus is transcribed into a precursor RNA that is subsequently processed into short crRNAs. CRISPR/Cas systems have been identified in bacteria and archaea, and data show that many variations of this system exist. We analyzed the requirements for a successful defense reaction in the halophilic archaeon Haloferax volcanii. Haloferax encodes a CRISPR/Cas system of the I-B subtype, about which very little is known. Analysis of the mature crRNAs revealed that they contain a spacer as their central element, which is preceded by an eight-nucleotide-long 5' handle that originates from the upstream repeat. The repeat sequences have the potential to fold into a minimal stem loop. Sequencing of the crRNA population indicated that not all of the spacers that are encoded by the three CRISPR loci are present in the same abundance. By challenging Haloferax with an invader plasmid, we demonstrated that the interaction of the crRNA with the invader DNA requires a 10-nucleotide-long seed sequence. In addition, we found that not all of the crRNAs from the three CRISPR loci are effective at triggering the degradation of invader plasmids. The interference does not seem to be influenced by the copy number of the invader plasmid.
Asunto(s)
Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Haloferax volcanii/genética , Plásmidos , ARN de Archaea/química , ARN de Archaea/genética , Secuencia de Bases , Proteínas Asociadas a CRISPR/inmunología , Proteínas Asociadas a CRISPR/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/inmunología , ADN/genética , Haloferax volcanii/inmunología , Secuencias Invertidas Repetidas , Datos de Secuencia Molecular , Mutagénesis , Filogenia , Procesamiento Postranscripcional del ARN , ARN de Archaea/inmunología , Alineación de Secuencia , Análisis de Secuencia de ARNRESUMEN
Effective clearance of an infection requires that the immune system rapidly detects and neutralizes invading parasites while strictly avoiding self-antigens that would result in autoimmunity. The cellular machinery and complex signaling pathways that coordinate an effective immune response have generally been considered properties of the eukaryotic immune system. However, a surprisingly sophisticated adaptive immune system that relies on small RNAs for sequence-specific targeting of foreign nucleic acids was recently discovered in bacteria and archaea. Molecular vaccination in prokaryotes is achieved by integrating short fragments of foreign nucleic acids into a repetitive locus in the host chromosome known as a CRISPR (clustered regularly interspaced short palindromic repeat). Here we review the mechanisms of CRISPR-mediated immunity and discuss the ecological and evolutionary implications of these adaptive defense systems.
Asunto(s)
Inmunidad Adaptativa/genética , Archaea/inmunología , Bacterias/inmunología , Secuencias Invertidas Repetidas/genética , ARN de Archaea/genética , ARN Bacteriano/genética , Transducción de Señal/genética , Archaea/genética , Bacterias/genética , Secuencias Invertidas Repetidas/inmunología , ARN de Archaea/inmunología , ARN Bacteriano/inmunología , Transducción de Señal/inmunologíaRESUMEN
Bacteria and archaea have evolved defense and regulatory mechanisms to cope with various environmental stressors, including virus attack. This arsenal has been expanded by the recent discovery of the versatile CRISPR-Cas system, which has two novel features. First, the host can specifically incorporate short sequences from invading genetic elements (virus or plasmid) into a region of its genome that is distinguished by clustered regularly interspaced short palindromic repeats (CRISPRs). Second, when these sequences are transcribed and precisely processed into small RNAs, they guide a multifunctional protein complex (Cas proteins) to recognize and cleave incoming foreign genetic material. This adaptive immunity system, which uses a library of small noncoding RNAs as a potent weapon against fast-evolving viruses, is also used as a regulatory system by the host. Exciting breakthroughs in understanding the mechanisms of the CRISPR-Cas system and its potential for biotechnological applications and understanding evolutionary dynamics are discussed.
Asunto(s)
Archaea/genética , Bacterias/genética , ARN de Archaea/genética , ARN Bacteriano/genética , Inmunidad Adaptativa , Archaea/química , Archaea/inmunología , Archaea/virología , Bacterias/química , Bacterias/inmunología , Bacterias/virología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Bacteriófagos/genética , Bacteriófagos/inmunología , Proteínas Asociadas a CRISPR , Biología Computacional , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/genética , Proteínas de Escherichia coli , Evolución Molecular , Regulación de la Expresión Génica Arqueal , Regulación Bacteriana de la Expresión Génica , Sitios Genéticos , Plásmidos/inmunología , Interferencia de ARN , Procesamiento Postranscripcional del ARN , ARN de Archaea/química , ARN de Archaea/inmunología , ARN Bacteriano/química , ARN Bacteriano/inmunología , ARN Pequeño no Traducido/química , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/inmunología , Transcripción Genética , Virosis/genética , Virosis/inmunología , Virosis/virologíaRESUMEN
The small CRISPR-derived RNAs of bacteria and archaea provide adaptive immunity by targeting the DNA of invading viruses and plasmids. Hale et al. (2009) now report on a new variant CRISPR/Cas complex in the archaeon Pyrococcus furiosus that uses guide RNAs to specifically target and cleave RNA not DNA.
Asunto(s)
Pyrococcus furiosus/genética , Pyrococcus furiosus/inmunología , Interferencia de ARN , ARN de Archaea/inmunología , Pyrococcus furiosus/virología , ARN de Archaea/genética , ARN Viral/inmunología , ARN Pequeño no TraducidoRESUMEN
Compelling evidence indicates that the CRISPR-Cas system protects prokaryotes from viruses and other potential genome invaders. This adaptive prokaryotic immune system arises from the clustered regularly interspaced short palindromic repeats (CRISPRs) found in prokaryotic genomes, which harbor short invader-derived sequences, and the CRISPR-associated (Cas) protein-coding genes. Here, we have identified a CRISPR-Cas effector complex that is comprised of small invader-targeting RNAs from the CRISPR loci (termed prokaryotic silencing (psi)RNAs) and the RAMP module (or Cmr) Cas proteins. The psiRNA-Cmr protein complexes cleave complementary target RNAs at a fixed distance from the 3' end of the integral psiRNAs. In Pyrococcus furiosus, psiRNAs occur in two size forms that share a common 5' sequence tag but have distinct 3' ends that direct cleavage of a given target RNA at two distinct sites. Our results indicate that prokaryotes possess a unique RNA silencing system that functions by homology-dependent cleavage of invader RNAs.
