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 (9–11).
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 endoribo-nuclease 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 essen-
tial for immunity only when phage mRNA
was part of the late-expressed phage genes
or when phage mRNA sequence was mu-
tated (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 at-
tempt to cleave and destroy foreign DNA.
If this fails and the infection process pro-
ceeds with the transcription of phage DNA,
the type III system goes into action, senses
the foreign mRNA, and attempts to termi-
nate the phage infection by cleaving both
the mRNA and its DNA template. During
the course of this targeting, the Cas10 pro-
tein in the type III effector RNP complex
generates a measured amount of cOA. Pos-
sibly, a small amount of cOA will not suf-
fice 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 ac-
tivate Csm6, leading to massive degrada-
tion of cellular RNA and possibly to cell
dormancy or death. This suggested mode
of action ensures that if the last line of de-
fense 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
Kazlauskiene et al. and Niewoehner et
al. report the discovery of cOA as an in-
tracellular 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 do-
mains (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 bacte-
rial and archaeal cells as part of their over-
all defense against phages. j
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1. Recognition of phage
mRNA by the CRISPR-Cas
efector RNP complex
triggers mRNA cleavage
and activates the HD and
3. Cas10 Palm domains
synthesize cOA, which
disperses through the cell.
4. Activation of Csm6
by cOA induces
of cellular RNA.
CARF domain RNase
CRISPR-Cas efector RNP complex
2. Cas10 HD domain
cleaves phage DNA.
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.