Virus silencer

US researchers have used Berkeley Lab's Advanced Light Source to obtain a crystal structure of the endoribonuclease enzyme known as Csy4. The enzyme is present in prokaryotes and initiates the production of small RNA molecules that target and silence invading viruses and plasmids.

Microbes, including bacteria and archaea, face an interminable attack from invading viruses and plasmid rings of nucleic acid. As such, they use various defence mechanisms, including an adaptive immune system that exploits the properties of a genetic element known as a clustered regularly interspaced short palindromic repeat, a CRISPR, for short.

CRISPRs work together with CRISPR-associated "Cas" proteins to help microbes silence those sections of an invader's genetic code critical to the invasion and so acquire immunity to future attacks. When a bacterium recognizes that it has been invaded by a virus or a plasmid, it incorporates a small piece of the foreign DNA into one of its CRISPR units as a new spacer sequence. The CRISPR unit is then transcribed as a long RNA segment called the pre-crRNA. The Csy4 enzyme cleaves this pre-crRNA within each repeat element to create crRNAs about 60 nucleotides long that will contain sequences which match portions of the foreign DNA. Cas proteins will use these matching sequences to bind the crRNA to the invading virus or plasmid and silence it.

Jennifer Doudna has now led a team of researchers, Rachel Haurwitz, Martin Jinek, Blake Wiedenheft and Kaihong Zhou, from the Lawrence Berkeley National Laboratory (Berkeley Lab) and the University of California (UC) Berkeley who hope to better understand microbial immunity. They have focused on how CRISPR's customized small RNA molecules are produced.

CRISPR is essentially a chunk of DNA, commonly found on the microbe's chromosome, that comprises repeat elements of base-pair sequences 30 to 60 bases long and separated by variable spacer sequences that are also 30-60 nucleotides in length. Microbes can contain several CRISPR loci any of which might contain between four and 100 CRISPR repeat-spacer units. About 40 percent of bacteria so far sequenced have been shown to carry CRISPR units. For archaea, that figure is closer to 90 percent. Doudna's team looked at CRISPR in the ubiquitous soil and environmental microbe, Pseudomonas aeruginosa.

The team used beamlines 8.2.1 and 8.3.1 at Berkeley Lab's Advanced Light Source to obtain a precise (1.8 Angstroms) crystal structure of the endoribonuclease Csy4, identified as the enzyme in prokaryotes that initiates the production of CRISPR-derived RNAs (crRNAs), the small RNA molecules that target and silence invading viruses and plasmids.

"Our model reveals that Csy4 and related endoribonucleases from the same CRISPR/Cas subfamily utilize an exquisite recognition mechanism to discriminate crRNAs from other cellular RNAs to ensure the selective production of crRNA for acquired immunity in bacteria," explains Doudna. "We also found functional similarities between the RNA recognition mechanisms in Cys4 and Dicer, the enzyme that plays a critical role in eukaryotic RNA interference."

With their crystal structure model of the Csy4 enzyme bound to its cognate RNA, the team has shown that Csy4 makes sequence-specific interactions in the major groove of the CRISPR RNA repeat stem-loop. Together with electrostatic contacts to the phosphate backbone, these interactions enable Csy4 to selectively bind to and cleave pre-crRNAs using phylogenetically conserved residues of the amino acids serine and histidine in the active site.

"Our model explains sequence- and structure-specific processing by a large family of CRISPR-specific endoribonucleases," Doudna explains.

The crRNAs used by the CRISPR/cas system for the targeted interference of foreign DNA join the growing ranks of small RNA molecules that mediate a variety of processes in both eukaryotes and prokaryotes. Understanding how these small RNA molecules work can improve our basic understanding of cell biology and provide important clues to the fundamental role of RNA in the evolution of life.

"By investigating how bacteria produce and use small RNAs for selective gene targeting, we hope to gain insight into the fundamental features of the pathways that have proven evolutionarily useful for genetic control, both in the bacterial world and in the world of eukaryotes," says Doudna. "Right now it looks like bacteria and eukaryotes have evolved entirely distinct pathways by which RNAs are used for gene regulation and that is pretty amazing!"

SpectroscopyNOW asked Doudna about the implications for the research in the wider life sciences. "We think the Csy4 protein looks more like a viral (phage) protein than anything encoded by cells, and hence may have originated with a virus," she told us. "One of the fascinating things about the CRISPR system is the evidence for very rapid and ongoing co-evolution of the RNAs and proteins involved, presumably in response to extensive viral challenge in natural microbial communities."

We were also curious as to whether this research thread might have an impact on medical science. "It would be exciting if a CRISPR-like system could be transferred into mammalian cells," Doudna told us, "where it might be engineered to silence the expression of deleterious host cell genes, or genes encoded by viral or bacterial pathogens. If this were possible, it could avoid complications of using the RNA interference (RNAi) pathway intrinsic to mammalian cells, which is currently the focus of many biotechnology and pharmaceutical companies."