PerspectiveMicrobiology

Intracellular signaling in CRISPR-Cas defense

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Science  11 Aug 2017:
Vol. 357, Issue 6351, pp. 550-551
DOI: 10.1126/science.aao2210

The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein) system is known to protect bacteria against foreign invading DNA, usually from phages (viruses that infect bacteria) or plasmids (circular DNA found in the cytoplasm of bacteria). Since the first demonstration of CRISPR-Cas functionality a decade ago (1), mechanistic understanding of CRISPR-Cas has not only enabled genome editing but also revolutionized our appreciation of bacterial defense against their viruses. CRISPR-Cas systems show a high degree of sophistication in providing immunity against phages, including elaborate mechanisms to accurately identify the invading DNA, safety checks to prevent self-targeting (2), and high diversity of target destruction mechanisms among different types of CRISPR-Cas systems (3). Kazlauskiene et al. (4), on page 605 of this issue, and a study by Niewoehner et al. (5) report the discovery of an unexpected aspect of CRISPR-Cas immunity: intracellular signaling.

Kazlauskiene et al. and Niewoehner et al. show that in type III CRISPR-Cas systems, identification of phage nucleic acids by the CRISPR-Cas effector complex leads to the generation of a small molecule called cyclic-oligoadenylate (cOA). This molecule then activates a CRISPR-associated ribonuclease (RNase), the function of which was previously unclear. Both papers demonstrate that the RNase, once activated, cleaves cellular RNA nonspecifically, probably leading to dormancy or death of the infected bacterium.

CRISPR-Cas systems typically encode an array of phage- or plasmid-derived pieces of DNA (known as the CRISPR array) that forms the bacterial immune memory. The CRISPR array is transcribed and processed into short RNA sequences, CRISPR-RNAs (crRNAs), that bind Cas proteins to form the effector RNA-protein (RNP) complex. The effector complex recognizes the nucleic acids of the foreign genetic material via base-pairing with the crRNA, eventually leading to cleavage of the foreign genetic material by endonuclease domains of one or more of the Cas proteins.

CRISPR-Cas systems are grouped into six types, each of which uses a different set of Cas proteins (6). Whereas most CRISPR-Cas types target foreign DNA, the kind of nucleic acids targeted by type III systems was subject for debate. Early on, it was reported that type III systems target DNA (7); but seemingly contradictory reports indicated that RNA was targeted (8). Eventually, it was realized that the type III effector RNP complex base-pairs with messenger RNA (mRNA) derived from the transcription of the invading DNA. Upon binding, the effector complex cleaves the mRNA and also cleaves the DNA from which it is transcribed (911).

The multiprotein effector RNP complex of type III CRISPR-Cas systems includes a large protein called Cas10. Cas10 typically encodes an HD nuclease domain, which degrades the foreign DNA, and two Palm (polymerase/nucleotide cyclase-like) domains that had no known roles in the activity of CRISPR-Cas until now. Kazlauskiene et al. and Niewoehner et al. discovered that the Cas10 Palm domains are responsible for synthesizing cOA, which is a short, cyclic oligomer composed of multiple adenosine monophosphate (AMP) molecules that are derived from adenosine triphosphate (ATP). The production of cOA by Cas10 is triggered by base-pairing between the effector complex and the foreign mRNA. Once produced, cOA molecules probably disperse through the cell and activate another Cas protein, Csm6, which is a single-strand endoribonuclease that nonspecifically cleaves cellular RNA, likely degrading both bacterial and phage mRNAs.

The exact role of Csm6 in CRISPR-Cas immunity was unclear. This protein is not associated with the CRISPR-Cas effector RNP complex and was shown to be essential for immunity only when phage mRNA was part of the late-expressed phage genes or when phage mRNA sequence was mutated (12). The two studies now offer a plausible unified model for the mode of action of type III systems. Other types of CRISPR-Cas system, such as type I or type II, form the “first line of defense” and attempt to cleave and destroy foreign DNA. If this fails and the infection process proceeds with the transcription of phage DNA, the type III system goes into action, senses the foreign mRNA, and attempts to terminate the phage infection by cleaving both the mRNA and its DNA template. During the course of this targeting, the Cas10 protein in the type III effector RNP complex generates a measured amount of cOA. Possibly, a small amount of cOA will not suffice to induce fully fledged RNase activity of Csm6 in a manner substantial enough to damage the cell; but if multiple type III complexes identify phage mRNAs, the cumulative amount of cOA will fully activate Csm6, leading to massive degradation of cellular RNA and possibly to cell dormancy or death. This suggested mode of action ensures that if the last line of defense has failed and transcription of phage RNA is sensed from multiple loci (meaning that multiple phage infections co-occur, or that phage DNA has been replicated), then the cell commits “suicide” to prevent production of new phage particles and protect nearby bacteria from the spread of the infection.

CRISPR-Cas intracellular signaling

Binding of the type III CRISPR-Cas effector RNP complex to transcribed phage mRNA initiates production of cOA molecules that activate the Csm6 RNase, which degrades phage and cellular mRNA.

GRAPHIC: K. SUTLIFF/SCIENCE

Kazlauskiene et al. and Niewoehner et al. report the discovery of cOA as an intracellular signaling molecule involved in antiphage immune defense. This molecule binds Csm6 proteins at the CARF (CRISPR-associated Rossmann fold) domain, and this binding allosterically triggers RNase activity of Csm6. Interestingly, additional Cas proteins are also known to have CARF domains, and even non-CRISPR proteins associated with immunity against foreign DNA were reported to encode CARF domains (13). It is therefore plausible that this immunity-associated intracellular signaling represents just one aspect of a larger network of signaling, to be revealed by future studies, that takes place in bacterial and archaeal cells as part of their overall defense against phages.

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