You'd Prefer An Argonaute

RNA Journal Club 12/3/09

Posted in RNA Journal Club, RNAJC w/ review by YPAA on December 3, 2009

RNA-Guided RNA Cleavage by a CRISPR RNA-Cas Protein Complex

Caryn R. Hale, Peng Zhao, Sara Olson, Michael O. Duff, Brenton R. Graveley, Lance Wells, Rebecca M. Terns and Michael P. Terns

Cell 139 (5): 945-56, 25 November 2009.
doi:10.1016/j.cell.2009.07.040

This week’s ace summary and analysis by Robin Friedman:

The CRISPR (clustered regularly interspaced short palindromic repeats) system is a set of DNA sequences and associated genes involved in prokaryotic immune defense. Since it was discovered that CRISPR loci generate small (usually 25-60 nt) RNAs that often match phage or plasmid sequence, it has been tempting to make an obvious analogy with eukaryotic RNA interference, which can also be used to protect against viruses. However, what little was known about CRISPR mechanism pointed to a DNA-dependent mechanism of invader recognition and defense. One complicating factor is that there are at least nine distinct subtypes of the CRISPR system based on different sets of Cas (CRISPR-associated) genes. In this paper Hale et al. examine a subtype, Cmr, that had not previously been studied.

The authors first showed that in P. furiosis, there are two main species of CRISPR RNA product (termed psiRNAs), 39 and 45 nucleotides long. They purified protein complexes containing these psiRNAs and subjected them to mass spectrometry, finding seven members of the CRISPR-associated Cmr family. Sequencing of the psiRNAs revealed that each species had an 8-nucleotide “psi-tag”, consisting of the 3’ end of the constant repeat sequence, followed by unique guide sequence.

To test the mechanism of the psiRNA-Cmr complex, the authors used several synthetic constructs with sequence similarity to P. furiosis psiRNAs. The complementary RNA sequence was specifically cleaved at two spots, but the sense RNA sequence, unrelated RNA sequences, and a complementary DNA sequence were not cleaved. Truncations of these synthetic complementary RNAs showed that cleavage occurs in the same location, suggesting that the Cmr complex cleaves 14 nucleotides from the 3’ end of the psiRNA. Finally, the authors reconstituted the Cmr complex in vitro with recombinant proteins and synthetic psiRNA and recapitulated the cleavage behavior of the native complex. Only one of the six included Cmr proteins were dispensable for cleavage.

This is one of the simplest Cell papers I have seen. However, its few results are thoroughly proven. There are many questions left to answer about CRISPR function both in this model system and in others, but the scope of this paper is merely to show that the CRISPR system can function through RNA cleavage. This paper finally provides evidence strengthening the appealing analogy between CRISPRs and eukaryotic RNAi, which is sure to stimulate more interest in the system.

Advertisements

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s

%d bloggers like this: