NIH Research Festival
CRISPR-Cas systems are divided into Class 1, with multi-subunit effector complexes, and Class 2, with effectors comprised by a single, multidomain protein. Class 2 encompasses types II, V and VI that differ primarily by the domain architectures of the effector proteins. In all type II systems as well as subtype V-B, maturation of the precursor crRNA (pre-crRNA) requires involvement of a trans-acting CRISPR (tracr) RNA and, at least in type II, is catalyzed by RNase III. In contrast, in subtype V-A and type VI, no tracrRNA has been identified, and pre-crRNA processing is mediated by the effector proteins themselves. By comparing the available structures of Class 2 effector complexed with the crRNA, tracrRNA (where involved) and the target, we identified the nexus structure of the tracrRNA in types II-A and II-B and a similar local fold in type V-B, where it involves both tracrRNA and the repeat portion of the crRNA, and in subtype V-A, where it is formed by the repeat alone. This observation suggests an important function of the nexus structure that is common to all type II and type V systems but could be formed by convergent evolution involving tracrRNA alone, the tracrRNA-repeat hybrid or repeat alone. Using a larger set of tracrRNA-repeat pairs representative of the diversity of type II systems, we predicted the structures of each crRNA-tracrRNA co-folding and identified the nexus structure in almost all of them. The nexus is almost always located 2 to 3 nucleotides away from the tracrRNA-repeat hybrid region. We also observed that the 5’-terminal base of the repeat is always paired with the tracrRNA. We propose that the role of the nexus structure is to prevent pairing between tracrRNA and spacer whereas the pairing of the 5' base of the repeat prevents possible pairing between the repeat and the DNA target. Thus, both features appear to ensure full availability of the spacer to interact specifically and completely with the DNA target. We investigated the origins of tracrRNA by tracing the coevolution of the tracrRNA and the corresponding repeats using simulated and ancestral RNA guide, and observed a conservation of optimal hybrid energy that corresponds to partial, rather than complete, base-pairing. Finally, we analyzed the tracrRNA in microbial genomes and found that tracrRNAs located close to CRISPR arrays are expressed downstream from the same strand and downstream of the CRISPR array, and can even overlap with the array. We propose an evolutionary model in which tracrRNA emerges from the distal decaying repeat of the CRISPR array.
Scientific Focus Area: Computational Biology
This page was last updated on Friday, March 26, 2021