RNA and kitchen tools – Slicers, Dicers, and CRISPRs

RNA-based regulation is all the rage in biology today.  The more familiar mechanisms involve small RNAs such as microRNAs and silencing-associated RNAs.  The biogenesis and functioning of these RNAs involves enzymes and complexes that have been termed, among other things, Dicers and Slicers.  These subcellular kitchen utensils work by processing either the small RNA precursor or the base-paired target RNA.  This mode of regulation is most often associated with eukaryotes, and indeed homologous enzymes and mechanisms are not found in prokaryotes. However, systems with remarkable functional similarity may occur in bacteria.  A recent review by Sorek et al. brings one such example into focus.

One curious feature of bacterial genome is the occurrence of arrays of direct repeats in which the repeated units are separated by so-called spacers of unique sequence unrelated to the repeat units.  The sizes of the repeat units vary from bacteria to bacteria, ranging from between 24 to 47 bp.  Likewise, the spacer sizes vary from 26-72 bp.  These arrays are flanked by an apparent leader sequence, and yet again by arrays of protein-coding (CAS) genes, the number and composition of which vary considerably from bacteria to bacteria.  The general arrangement is shown in the following figure, which is part a of Figure 1 from Sorek et al. (shown beneath the fold):

Figure 1a from Sorek et al., showing the arrangement of a generic CRISPR element.

Several aspects of this genetic unit are thought-provoking.  While the spacer sequences are not related to the repeat units, they are often similar or identical to sequences found in bacteriophages or plasmids.  Intriguingly, in one recent study, it was shown that a CRISPR element was responsible for phage resistance in Streptococcus thermophilus.  This resistance requires sequence identity between spacer element and the phage, and at least two of the CAS genes (and presumably the encoded proteins).

While the number and specific sequences of CAS genes are rather variable in different bacteria, the complement of encoded proteins possesses a core of functionalities.  Intriguingly, among these are endonucleases and exonucleases, helicases, and RNA- and DNA- binding domains.  These suggest that the action of CRISPR elements involve nucleic acid metabolism of a sort, and are consistent with the need for sequence identity in the functioning of CRISPR elements.

The leader-repeat arrangement of the CRISPR motif suggests (weakly) that of a transcription unit, and it is thus not surprising that CRISPR elements are transcribed into RNA.  Interestingly, however, while the CRISPR element is transcribed into a single apparent primary transcript, this RNA is processed into small RNA units, each of which consists of one spacer and half of each flanking repeat unit.

These various observations have yet to be pieced together into a well-defined mechanism for CPISPR action.  But one model holds that CRISPR elements act much as do small RNAs in eukaryotes.  The general idea is illustrated in the following (part c of Fig. 1 from Sorek et al.):

Figure 1c from Sorek et al., showing a proposed model for CRISPR function.

It should be emphasized that this hypothesis is relatively “young” (although a recent study lends some good experimental support to the first few steps of the model).  If confirmed, though, this model would reveal that mechanisms in which small RNAs guide an effector complex so as to silence or otherwise modulate gene expression to be remarkably ubiquitous in nature.

References:

Sorek R, Kunin V, Hugenholtz P. 2008.  CRISPR–a widespread system that provides acquired resistance against phages in bacteria and archaea.  Nat Rev Microbiol. 2008 Nat Rev Microbiol 6: 181-1866.

A study showing an involvement of CRISPR elements in phage resistance:

Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P. 2007. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709-1712.

A more recent study that confirms some of the “siRNA” model for CRISPR action (this study corroborates Barrangou et al in showing a direct involvement of CRISPR elements in phage resistance):

Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ, Snijders AP, Dickman MJ, Makarova KS, Koonin EV, van der Oost J. 2008.  Small CRISPR RNAs Guide Antiviral Defense in Prokaryotes.  Science 321, 960-964.