Functions of prokaryotic Argonautes (pAgo) have long remained elusive. Recently, Argonautes of the bacteria Rhodobacter sphaeroides and Thermus thermophilus were demonstrated to be involved in host defense. The Argonaute of the archaeon Pyrococcus furiosus (PfAgo) belongs to a different branch in the phylogenetic tree, which is most closely related to that of RNA interference-mediating eukaryotic Argonautes. Here we describe a functional and mechanistic characterization of PfAgo. Like the bacterial counterparts, archaeal PfAgo contributes to host defense by interfering with the uptake of plasmid DNA. PfAgo utilizes small 5′-phosphorylated DNA guides to cleave both single stranded and double stranded DNA targets, and does not utilize RNA as guide or target. Thus, with respect to function and specificity, the archaeal PfAgo resembles bacterial Argonautes much more than eukaryotic Argonautes. These findings demonstrate that the role of Argonautes is conserved through the bacterial and archaeal domains of life and suggests that eukaryotic Argonautes are derived from DNA-guided DNA-interfering host defense systems.
To achieve adaptive and heritable immunity against viruses and other mobile genetic elements, CRISPR-Cas systems must capture and store short DNA fragments (spacers) from these foreign elements into host genomic CRISPR arrays. This process is catalyzed by conserved Cas1/Cas2 integration complexes, but the specific roles of another highly conserved protein linked to spacer acquisition, the Cas4 nuclease, are just now emerging. Here, we show that two Cas4 nucleases (Cas4-1 and Cas4-2) play critical roles in CRISPR spacer acquisition in Pyrococcus furiosus. The nuclease activities of both Cas4 proteins are required to process protospacers to the correct size. Cas4-1 specifies the upstream PAM (protospacer adjacent motif), while Cas4-2 specifies the conserved downstream motif. Both Cas4 proteins ensure CRISPR spacer integration in a defined orientation leading to CRISPR immunity. Collectively, these findings provide in vivo evidence for critical roles of Cas4 nucleases in protospacer generation and functional spacer integration at CRISPR arrays.
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 exon junction complex (EJC) is highly conserved in many organisms and is involved in various steps of mRNA metabolism. During the course of investigating the role of EJC in the germ line sex determination of the nematode Caenorhabditis elegans, we found that depletion of one of the three core subunits (Y14, MAG-1, and eukaryotic translation initiation factor 4III [eIF4AIII]) or one auxiliary subunit (UAP56) of EJC resulted in the cytoplasmic leakage of unspliced RNAs from almost all of the C. elegans protein-coding genes examined thus far. This leakage was also observed with the depletion of several splicing factors, including SF3b, IBP160, and PRP19, all of which genetically interacted with Y14. We also found that Y14 physically interacts with both pre-mRNA and spliceosomal U snRNAs, especially U2 snRNA, and that the interaction was abolished when both IBP160 and PRP19 were depleted. Our results strongly suggest that a specific set of EJC subunits is recruited onto introns and interacts with components of the spliceosome, including U2 snRNP, to provide a critical signal for the surveillance and nuclear retention of unspliced RNAs in C. elegans.T he complete removal of introns from pre-mRNA before mRNA export to the cytoplasm is critical to ensure the proper expression of the protein product in higher eukaryotes. Premature export of intron-containing RNAs gives rise to abnormal protein products that are very harmful to various cell functions, although most of such abnormal RNAs are eliminated by mRNA quality control systems, including nonsense-mediated mRNA decay (NMD) (1-4). Thus, cells must have mechanisms to retain partially spliced pre-mRNA in the nucleus. Indeed, most intron-containing RNAs, except in the case of alternative splicing, are normally localized in the nucleus. One mechanism is spliceosome formation around introns, which is confined to the nucleus because most splicing factors are localized in the nucleus. In this mechanism, the spliceosome itself or one or more spliceosomeassociated factors anchor intron-containing RNAs in the nucleus (5-7). Another mechanism for the nuclear retention of introncontaining RNAs is the splicing-dependent recruitment of mRNA export-related factors. The transcription and export (TREX) complex and exon junction complex (EJC) form on the 5= region of the nascent transcript (8, 9) and 20 to 24 nucleotides upstream from splice junctions (10, 11), respectively. The TREX complex contains the proteins UAP56 and Aly/REF (12, 13), both of which are also subunits of EJC (14-16), suggesting the tight coupling of splicing with mRNA export and the involvement of TREX and EJC for preventing the premature export of unspliced RNAs. Both spliceosome-dependent and export-related-factor-coupled mechanisms for the nuclear retention of intron-containing RNAs may operate concurrently, but the molecular details remain to be elucidated.EJC consists of four core subunits (Y14, Mago-nashi, eukaryotic translation initiation factor 4III [eIF4AIII], and Barentz/ MLN51) and several auxiliary...
CRISPR-Cas adaptive immune systems are used by prokaryotes to defend against invaders like viruses and other mobile genetic elements. Immune memories are stored in the form of ‘spacers’ which are short DNA sequences that are captured from invaders and added to the CRISPR array during a process called ‘adaptation’. Spacers are transcribed and the resulting CRISPR (cr)RNAs assemble with different Cas proteins to form effector complexes that recognize matching nucleic acid and destroy it (‘interference’). Adaptation can be ‘naïve’, i.e. independent of any existing spacer matches, or it can be ‘primed’, i.e. spurred by the crRNA-mediated detection of a complete or partial match to an invader sequence. Here we show that primed adaptation occurs in Pyrococcus furiosus. Although P. furiosus has three distinct CRISPR-Cas interference systems (I-B, I-A and III-B), only the I-B system and Cas3 were necessary for priming. Cas4, which is important for selection and processing of new spacers in naïve adaptation, was also essential for priming. Loss of either the I-B effector proteins or Cas3 reduced naïve adaptation. However, when Cas3 and all crRNP genes were deleted, uptake of correctly processed spacers was observed, indicating that none of these interference proteins are necessary for naïve adaptation.
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