Sequence-specific Endoribonuclease Research Articles

Using nucleolytic toxins as restriction enzymes enablesnew RNA applications.

Over the past five decades, DNA restriction enzymes have revolutionized biotechnology. While these enzymes are widely used in DNA research and DNA engineering, the emerging field of RNA and mRNA therapeutics requires sequence-specific RNA endoribonucleases. Here, we describe EcoToxN1, a member of the type III toxin-antitoxin family of sequence-specific RNA endoribonucleases, and its use in RNA and mRNA analysis. This enzyme recognizes a specific pentamer in a single-stranded RNA and cleaves the RNA within this sequence. The enzyme is neither dependent on annealing of guide RNA or DNA oligos to the template nor does it require magnesium. Furthermore, it performs over a wide range of temperatures. With its unique functions and characteristics, EcoToxN1 can be classified as an RNA restriction enzyme. EcoToxN1 enables new workflows in RNA analysis and biomanufacturing, meeting the demand for faster, cheaper, and more robust analysis methods.

MazEF Homologs in Symbiobacterium thermophilum Exhibit Cross-Neutralization with Non-Cognate MazEFs.

Toxin-antitoxin systems are preserved by nearly every prokaryote. The type II toxin MazF acts as a sequence-specific endoribonuclease, cleaving ribonucleotides at specific sequences that vary from three to seven bases, as has been reported in different host organisms to date. The present study characterized the MazEF module (MazEF-sth) conserved in the Symbiobacterium thermophilum IAM14863 strain, a Gram-negative syntrophic bacterium that can be supported by co-culture with multiple bacteria, including Bacillus subtilis. Based on a method combining massive parallel sequencing and the fluorometric assay, MazF-sth was determined to cleave ribonucleotides at the UACAUA motif, which is markedly similar to the motifs recognized by MazF from B. subtilis (MazF-bs), and by several MazFs from Gram-positive bacteria. MazF-sth, with mutations at conserved amino acid residues Arg29 and Thr52, lost most ribonuclease activity, indicating that these residues that are crucial for MazF-bs also play significant roles in MazF-sth catalysis. Further, cross-neutralization between MazF-sth and the non-cognate MazE-bs was discovered, and herein, the neutralization mechanism is discussed based on a protein-structure simulation via AlphaFold2 and multiple sequence alignment. The conflict between the high homology shared by these MazF amino acid sequences and the few genetic correlations among their host organisms may provide evidence of horizontal gene transfer.

Arg-73 of the RNA endonuclease MazF in Salmonella enterica subsp. arizonae contributes to guanine and uracil recognition in the cleavage sequence

The sequence-specific endoribonuclease MazF is widely conserved among prokaryotes. Approximately 20 different MazF cleavage sequences have been discovered, varying from three to seven nucleotides in length. Although MazFs from various prokaryotes were found, the cleavage sequences of most MazFs are unknown. Here, we characterized the conserved MazF of Salmonella enterica subsp. arizonae (MazF-SEA). Using massive parallel sequencing and fluorometric assays, we revealed that MazF-SEA preferentially cleaves the sequences UˆACG and UˆACU (ˆ represents cleavage sites). In addition, we predicted the 3D structure of MazF-SEA using AlphaFold2 and aligned it with the crystal structure of RNA-bound Bacillus subtilis MazF to evaluate RNA interactions. We found Arg-73 of MazF-SEA interacts with RNAs containing G and U at the third position from the cleavage sites (UˆACG and UˆACU). We then obtained the mutated MazF-SEA R73L protein to evaluate the significance of Arg-73 interaction with RNAs containing G and U at this position. We also used fluorometric and kinetic assays and showed the enzymatic activity of MazF-SEA R73L for the sequence UACG and UACU was significantly decreased. These results suggest Arg-73 is essential for recognizing G and U at the third position from the cleavage sites. This is the first study to our knowledge to identify a single residue responsible for RNA recognition by MazF. Owing to its high specificity and ribosome-independence, MazF is useful for RNA cleavage in vitro. These results will likely contribute to increasing the diversity of MazF specificity and to furthering the application of MazF in RNA engineering.

Induction of E. coli-derived endonuclease MazF suppresses HIV-1 production and causes apoptosis in latently infected cells

The current antiretroviral therapy cannot cure the patients infected with human immunodeficiency virus type 1 (HIV-1) due to the existence of latently infected cells capable of virus production from harboring proviral DNA. MazF is an ACA nucleotide sequence-specific endoribonuclease derived from Escherichia coli. The conditional expression of MazF by binding of HIV-1 Tat to the promoter region of a MazF-expression vector has previously been shown to selectively inhibit HIV-1 replication in acutely infected cells. The expression of MazF significantly suppressed tumor necrosis factor (TNF)-α-induced HIV-1 production and viral RNA expression in the HIV-1 latently infected cell line OM-10.1 transduced with the MazF-expression vector (OM-10.1/MFR). Moreover, the viability of OM-10.1/MFR cells decreased with increasing concentrations of TNF-α, whereas such decrease was not observed for HL-60 cells transduced with the MazF-expression vector (HL-60/MFR), the uninfected parental cell line of OM-10.1. TNF-α increased the expression of cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase in OM-10.1/MFR cells, indicating that the cell death was caused by the induction of apoptosis. TNF-α-induced expression of MazF mRNA was detected in OM-10.1/MFR but not HL-60/MFR cells, suggesting that TNF-α-induced apoptosis of latently infected cells was due to the expression of MazF. Thus, the anti-HIV-1 gene therapy using the MazF-expression vector may have potential for the cure of HIV-1 infection in combination with suitable latency reversing agents through reducing the size of latently infected cells without viral reactivation.

A CUGGU/UUGGU-specific MazF homologue from Methanohalobium evestigatum

MazF is a sequence-specific endoribonuclease or mRNA interferase, which cleaves RNA at a specific sequence. Since the expression of a specific gene or a group of specific genes can be regulated by MazF, expanding the repertoire of recognition sequences by MazF mRNA interferases is highly desirable for biotechnological and medical applications. Here, we identified a gene for a MazF homologue (MazFme) from Methanohalobium evestigatum, an extremely halophilic archaeon. In order to suppress the toxicity of MazFme to the E. coli cells, the C-terminal half of the cognate antitoxin MazEme was fused to the N-terminal end of MazFme. Since the fusion of the C-terminal half of MazEme to MazFme was able to neutralize MazFme toxicity, the MazEme-MazFme fusion protein was expressed in a large amount without any toxic effects. After purification of the MazEme, the free MazFme RNA cleavage specificity was determined by primer extension and synthetic ribonucleotides, revealing that MazFme is a CUGGU/UUGGU-specific endoribonuclease.

Identification of MazF Homologue in Legionella pneumophila Which Cleaves RNA at the AACU Sequence

MazF is a sequence-specific endoribonuclease that is widely conserved in bacteria and archaea. Here, we found an MazF homologue (MazF-lp; LPO-p0114) in Legionella pneumophila. The mazF-lp gene overlaps 14 base pairs with the upstream gene mazE-lp (MazE-lp; LPO-p0115). The induction of mazF-lp caused cell growth arrest, while mazE-lp co-induction recovered cell growth in Escherichia coli. In vivo and in vitro primer extension experiments showed that MazF-lp is a sequence-specific endoribonuclease cleaving RNA at AACU. The endoribonuclease activity of purified MazF-lp was inhibited by purified MazE-lp. We found that MazE-lp and the MazEF-lp complex specifically bind to the palindromic sequence present in the 5′-untranslated region of the mazEF-lp operon. MazE-lp and MazEF-lp both likely function as a repressor for the mazEF-lp operon and for other genes, including icmR, whose gene product functions as a secretion chaperone for the IcmQ pore-forming protein, by specifically binding to the palindromic sequence in 5′-UTR of these genes.

Deinococcus radiodurans Toxin-Antitoxin MazEF-dr Mediates Cell Death in Response to DNA Damage Stress.

Here we identified a functional MazEF-dr system in the exceptionally stress-resistant bacterium D. radiodurans. We showed that overexpression of the toxin MazF-dr inhibited the growth of Escherichia coli. The toxic effect of MazF-dr was due to its sequence-specific endoribonuclease activity on RNAs containing a consensus 5′ACA3′, and it could be neutralized by MazE-dr. The MazF-dr showed a special cleavage preference for the nucleotide present before the ACA sequence with the order by U>A>G>C. MazEF-dr mediated the death of D. radiodurans cells under sub-lethal dose of stresses. The characteristics of programmed cell death (PCD) including membrane blebbing, loss of membrane integrity and cytoplasm condensation occurred in a fraction of the wild-type population at sub-lethal concentration of the DNA damaging agent mitomycin C (MMC); however, a MazEF-dr mutation relieved the cell death, suggesting that MazEF-dr mediated cell death through its endoribonuclease activity in response to DNA damage stress. The MazEF-dr-mediated cell death of a fraction of the population might serve as a survival strategy for the remaining population of D. radiodurans under DNA damage stress.

Toxins of Prokaryotic Toxin-Antitoxin Systems with Sequence-Specific Endoribonuclease Activity

Protein translation is the most common target of toxin-antitoxin system (TA) toxins. Sequence-specific endoribonucleases digest RNA in a sequence-specific manner, thereby blocking translation. While past studies mainly focused on the digestion of mRNA, recent analysis revealed that toxins can also digest tRNA, rRNA and tmRNA. Purified toxins can digest single-stranded portions of RNA containing recognition sequences in the absence of ribosome in vitro. However, increasing evidence suggests that in vivo digestion may occur in association with ribosomes. Despite the prevalence of recognition sequences in many mRNA, preferential digestion seems to occur at specific positions within mRNA and also in certain reading frames. In this review, a variety of tools utilized to study the nuclease activities of toxins over the past 15 years will be reviewed. A recent adaptation of an RNA-seq-based technique to analyze entire sets of cellular RNA will be introduced with an emphasis on its strength in identifying novel targets and redefining recognition sequences. The differences in biochemical properties and postulated physiological roles will also be discussed.

Sequence-specific endoribonucleases

Ribonucleases are nucleolytic enzymes that commonly occur in living organisms and act by cleaving RNA molecules. These enzymes are involved in basic cellular processes, including the RNA maturation that accompanies the formation of functional RNAs, as well as RNA degradation that enables removal of defective or dangerous molecules or ones that have already fulfilled their cellular functions. RNA degradation is also one of the main processes that determine the amount of transcripts in the cell and thus it makes an important element of the gene expression regulation system. Ribonucleases can catalyse reactions involving RNA molecules containing specific sequences, structures or sequences within a specific structure, they can also cut RNAs non-specifically. In this article, we discuss ribonucleases cleaving the phosphodiester bond inside RNA molecules within or close to particular sequences. We also present examples of protein engineering of ribonucleases towards the development of molecular tools for sequence-specific cleavage of RNA.

Characterization of MazF-Mediated Sequence-Specific RNA Cleavage in Pseudomonas putida Using Massive Parallel Sequencing.

Under environmental stress, microbes are known to alter their translation patterns using sequence-specific endoribonucleases that we call RNA interferases. However, there has been limited insight regarding which RNAs are specifically cleaved by these RNA interferases, hence their physiological functions remain unknown. In the current study, we developed a novel method to effectively identify cleavage specificities with massive parallel sequencing. This approach uses artificially designed RNAs composed of diverse sequences, which do not form extensive secondary structures, and it correctly identified the cleavage sequence of a well-characterized Escherichia coli RNA interferase, MazF, as ACA. In addition, we also determined that an uncharacterized MazF homologue isolated from Pseudomonas putida specifically recognizes the unique triplet, UAC. Using a real-time fluorescence resonance energy transfer assay, the UAC triplet was further proved to be essential for cleavage in P. putida MazF. These results highlight an effective method to determine cleavage specificity of RNA interferases.

Characterization of RNA Helicase CshA and Its Role in Protecting mRNAs and Small RNAs of Staphylococcus aureus Strain Newman.

The toxin MazFsa in Staphylococcus aureus is a sequence-specific endoribonuclease that cleaves the majority of the mRNAs in vivo but spares many essential mRNAs (e.g., secY mRNA) and, surprisingly, an mRNA encoding a regulatory protein (i.e., sarA mRNA). We hypothesize that some mRNAs may be protected by RNA-binding protein(s) from degradation by MazFsa. Using heparin-Sepharose-enriched fractions that hybridized to sarA mRNA on Northwestern blots, we identified among multiple proteins the DEAD box RNA helicase CshA (NWMN_1985 or SA1885) by mass spectroscopy. Purified CshA exhibits typical RNA helicase activities, as exemplified by RNA-dependent ATPase activity and unwinding of the DNA-RNA duplex. A severe growth defect was observed in the cshA mutant compared with the parent when grown at 25°C but not at 37°C. Activation of MazFsa in the cshA mutant resulted in lower CFU per milliliter accompanied by a precipitous drop in viability (∼40%) compared to those of the parent and complemented strains. NanoString analysis reveals diminished expression of a small number of mRNAs and 22 small RNAs (sRNAs) in the cshA mutant versus the parent upon MazFsa induction, thus implying protection of these RNAs by CshA. In the case of the sRNA teg049 within the sarA locus, we showed that the protective effect was likely due to transcript stability as revealed by reduced half-life in the cshA mutant versus the parent. Accordingly, CshA likely stabilizes selective mRNAs and sRNAs in vivo and as a result enhances S. aureus survival upon MazFsa induction during stress.

Detection of endogenous MazF enzymatic activity in Staphylococcus aureus

The mazEFSa toxin–antitoxin (TA) system is ubiquitous in clinical isolates of Staphylococcus aureus, yet its physiological role is unclear. MazFSa is a sequence-specific endoribonuclease that inhibits the growth of S. aureus and Escherichia coli on ectopic overexpression. MazFSa preferentially cleaves RNA at UACAU sites, which are overrepresented in genes encoding pathogenicity factors. The exploitation of the inherent toxicity of MazFSa by artificial toxin activation has been proposed as an antibacterial strategy; however, enzymatic activity of endogenous MazFSa has never been detected, and tools for such analyses are lacking. Here we detail methods for detection of the ribonuclease activity of MazFSa, including a continuous fluorometric assay and a gel-based cleavage assay. Importantly, these methods allowed for the first detection of endogenous MazFSa enzymatic activity in S. aureus lysate. These robust and sensitive assays provide a toolkit for the identification, analysis, and validation of stressors that induce MazF enzymatic activity and should assist in the discovery of artificial activators of the mazEFSa TA system.

A regulatory role for Staphylococcus aureus toxin–antitoxin system PemIKSa

Toxin-antitoxin systems were shown to be involved in plasmid maintenance when they were initially discovered, but other roles have been demonstrated since. Here we identify and characterize a novel toxin-antitoxin system (pemIKSa) located on Staphylococcus aureus plasmid pCH91. The toxin (PemKSa) is a sequence-specific endoribonuclease recognizing the tetrad sequence U↓AUU, and the antitoxin (PemISa) inhibits toxin activity by physical interaction. Although the toxin-antitoxin system is responsible for stable plasmid maintenance our data suggest the participation of pemIKSa in global regulation of staphylococcal virulence by alteration of the translation of large pools of genes. We propose a common mechanism of reversible activation of toxin-antitoxin systems based on antitoxin transcript resistance to toxin cleavage. Elucidation of this mechanism is particularly interesting because reversible activation is a prerequisite for the proposed general regulatory role of toxin-antitoxin systems.

Sustained Inhibition of HIV-1 Replication by Conditional Expression of theE. coli-Derived Endoribonuclease MazF in CD4+T cells

Gene therapy using a Tat-dependent expression system of MazF, an ACA nucleotide sequence-specific endoribonuclease derived from Escherichia coli, in a retroviral vector appears to be an alternative approach to the treatment of human immunodeficiency virus type 1 (HIV-1) infection. MazF can cleave HIV-1 RNA, since it has more than 240 ACA sequences. Significant inhibition of viral replication, irrespective of HIV-1 strains, was observed in CD4(+) T cells that had been transduced with the MazF-expressing retroviral vector (MazF-T cells). The growth and viability of MazF-T cells were not affected by HIV-1 infection. Interestingly, the infectivity of HIV-1 produced from MazF-T cells was found to be lower than that from control CD4(+) T cells. A long-term culture experiment with HIV-1-infected cells revealed that viral replication was always lower in MazF-T cells than in CD4(+) T cells transduced with or without a control vector for more than 200 days. MazF was expressed and mainly localized in the cytoplasm of the infected cells. Unlike in CD4(+) T cells, the expression level of Tat gradually decreased rather than increased in MazF-T cells after HIV-1 infection. As a consequence, the expression level of MazF appeared to be well regulated and sustained during HIV-1 infection in MazF-T cells. Furthermore, the levels of cellular mRNA were not affected by HIV-1 infection. Thus, the Tat-dependent MazF expression system has great potential for inhibition of HIV-1 replication in vivo without apparent toxicity and may be able to avoid the emergence of resistant strains.

Growth and Translation Inhibition through Sequence-specific RNA Binding by Mycobacterium tuberculosis VapC Toxin

The Mycobacterium tuberculosis genome harbors an unusually large number of toxin-antitoxin (TA) modules. Curiously, over half of these are VapBC (virulence-associated protein) family members. Nonetheless, the cellular target, precise mode of action, and physiological role of the VapC toxins in this important pathogen remain unclear. To better understand the function of this toxin family, we studied the features and biochemical properties of a prototype M. tuberculosis VapBC TA system, vapBC-mt4 (Rv0596c-Rv0595c). VapC-mt4 expression resulted in growth arrest, a hallmark of all TA toxins, in Escherichia coli, Mycobacterium smegmatis, and M. tuberculosis. Its expression led to translation inhibition accompanied by a gradual decrease in the steady-state levels of several mRNAs. VapC-mt4 exhibited sequence-specific endoribonuclease activity on mRNA templates at ACGC and AC(A/U)GC sequences. However, the cleavage activity of VapC-mt4 was comparatively weak relative to the TA toxin MazF-mt1 (Rv2801c). Unlike other TA toxins, translation inhibition and growth arrest preceded mRNA cleavage, suggesting that the RNA binding property of VapC-mt4, not RNA cleavage, initiates toxicity. In support of this hypothesis, expression of VapC-mt4 led to an increase in the recovery of total RNA with time in contrast to TA toxins that inhibit translation via direct mRNA cleavage. Additionally, VapC-mt4 exhibited stable, sequence-specific RNA binding in an electrophoretic mobility shift assay. Finally, VapC-mt4 inhibited protein synthesis in a cell-free system without cleaving the corresponding mRNA. Therefore, the activity of VapC-mt4 is mechanistically distinct from other TA toxins because it appears to primarily inhibit translation through selective, stable binding to RNA.

The Escherichia coli Extracellular Death Factor EDF Induces the Endoribonucleolytic Activities of the Toxins MazF and ChpBK

Escherichia coli (E. coli) mazEF is a toxin-antitoxin (TA) stress-induced module that mediates cell death requiring the quorum-sensing pentapeptide NNWNN designated EDF (extracellular death factor). E. coli toxin MazF is a sequence-specific endoribonuclease cleaving single-stranded mRNAs at ACA sequences. E. coli ChpBK, a toxin homologous to MazF, is a sequence-specific endoribonuclease cleaving single-stranded mRNAs at ACA, ACG, and ACU sequences. Here we report that, in vitro, the signaling molecule EDF significantly amplifies the endoribonucleolytic activities of both MazF and ChpBK. EDF also overcomes the inhibitory activity of the antitoxins MazE over the toxin MazF and ChpBI over ChpBK. EDF sequence is important for both functions. Moreover, direct sequence-specific binding of EDF to MazF has been confirmed. Peptide-protein modeling revealed parallel contacts between EDF-MazF and MazE-MazF. These findings are intriguing, since most known quorum-sensing molecules monitor gene expression on the transcriptional level, while EDF monitors posttranscriptionally.

Inhibitory Mechanism of Escherichia coli RelE-RelB Toxin-Antitoxin Module Involves a Helix Displacement Near an mRNA Interferase Active Site

In Escherichia coli, RelE toxin participates in growth arrest and cell death by inducing mRNA degradation at the ribosomal A-site under stress conditions. The NMR structures of a mutant of E. coli RelE toxin, RelE(R81A/R83A), with reduced toxicity and its complex with an inhibitory peptide from RelB antitoxin, RelB(C) (Lys(47)-Leu(79)), have been determined. In the free RelE(R81A/R83A) structure, helix alpha4 at the C terminus adopts a closed conformation contacting with the beta-sheet core and adjacent loops. In the RelE(R81A/R83A)-RelB(C) complex, helix alpha3(*) of RelB(C) displaces alpha4 of RelE(R81A/R83A) from the binding site on the beta-sheet core. This helix replacement results in neutralization of a conserved positively charged cluster of RelE by acidic residues from alpha3(*) of RelB. The released helix alpha4 becomes unfolded, adopting an open conformation with increased mobility. The displacement of alpha4 disrupts the geometry of critical residues, including Arg(81) and Tyr(87), in a putative active site of RelE toxin. Our structures indicate that RelB counteracts the toxic activity of RelE by displacing alpha4 helix from the catalytically competent position found in the free RelE structure.

Staphylococcus aureus MazF Specifically Cleaves a Pentad Sequence, UACAU, Which Is Unusually Abundant in the mRNA for Pathogenic Adhesive Factor SraP

Escherichia coli mRNA interferases, such as MazF and ChpBK, are sequence-specific endoribonucleases encoded by toxin-antitoxin (TA) systems present in its genome. A MazF homologue in Staphylococcus aureus (MazF(Sa)) has been shown to inhibit cell growth when induced in E. coli. Here, we determined the cleavage site for MazF(Sa) with the use of phage MS2 RNA as a substrate and CspA, an RNA chaperone, which prevents the formation of secondary structures in the RNA substrate. MazF(Sa) specifically cleaves the RNA at a pentad sequence, U downward arrow ACAU. Bioinformatics analysis revealed that this pentad sequence is significantly abundant in several genes, including the sraP gene in the S. aureus N315 strain. This gene encodes a serine-rich protein, which is known to play an important role in adhesion of the pathogen to human tissues and thus in endovascular infection. We demonstrated that the sraP mRNA became extremely unstable in comparison with the ompA mRNA only when MazF(Sa) was induced in E. coli. Further bioinformatics analysis indicated that the pentad sequence is also significantly abundant in the mRNAs for all the pathogenic factors in S. aureus. This observation suggests a possible regulatory relationship between the MazEF(Sa) TA module and the pathogenicity in S. aureus.

The mRNA interferases, MazF‐mt3 and MazF‐mt7 from Mycobacterium tuberculosis target unique pentad sequences in single‐stranded RNA

mRNA interferases are sequence-specific endoribonucleases encoded by toxin-antitoxin (TA) systems in bacterial genomes. Previously, we demonstrated that Mycobacterium tuberculosis contains at least seven genes encoding MazF homologues (MazF-mt1 to -mt7) and determined cleavage specificities for MazF-mt1 and MazF-mt6. Here we have developed a new general method for the determination of recognition sequences longer than three bases for mRNA interferases with the use of phage MS2 RNA as a substrate and CspA, an RNA chaperone, which prevents the formation of secondary structures in the RNA substrate. Using this method, we determined that MazF-mt3 cleaves RNA at UU CCU or CU CCU and MazF-mt7 at U CGCU ( indicates the cleavage site). As pentad sequence recognition is more specific than those of previously characterized mRNA interferases, bioinformatics analysis was carried out to identify M. tuberculosis mRNAs that may be resistant to MazF-mt3 and MazF-mt7 cleavage. The pentad sequence was found to be significantly underrepresented in several genes, including members of the PE and PPE families, large families of proteins that play a role in tuberculosis immunity and pathogenesis. These data suggest that MazF-mt3 and MazF-mt7 or other mRNA interferases that target longer RNA sequences may alter protein expression through differential mRNA degradation, a regulatory mechanism that may allow adaptation to environmental conditions, including those encountered by pathogens such as M. tuberculosis during infection.

A Novel Family of Sequence-specific Endoribonucleases Associated with the Clustered Regularly Interspaced Short Palindromic Repeats

Clustered regularly interspaced short palindromic repeats (CRISPRs) together with the associated CAS proteins protect microbial cells from invasion by foreign genetic elements using presently unknown molecular mechanisms. All CRISPR systems contain proteins of the CAS2 family, suggesting that these uncharacterized proteins play a central role in this process. Here we show that the CAS2 proteins represent a novel family of endoribonucleases. Six purified CAS2 proteins from diverse organisms cleaved single-stranded RNAs preferentially within U-rich regions. A representative CAS2 enzyme, SSO1404 from Sulfolobus solfataricus, cleaved the phosphodiester linkage on the 3'-side and generated 5'-phosphate- and 3'-hydroxyl-terminated oligonucleotides. The crystal structure of SSO1404 was solved at 1.6A resolution revealing the first ribonuclease with a ferredoxin-like fold. Mutagenesis of SSO1404 identified six residues (Tyr-9, Asp-10, Arg-17, Arg-19, Arg-31, and Phe-37) that are important for enzymatic activity and suggested that Asp-10 might be the principal catalytic residue. Thus, CAS2 proteins are sequence-specific endoribonucleases, and we propose that their role in the CRISPR-mediated anti-phage defense might involve degradation of phage or cellular mRNAs.