Although it likely arose as a defense mechanism against viruses and transposable elements, RNA interference, as we have seen, has become thoroughly integrated into many aspects of normal cell biology, ranging from the control of gene expression to the structure of chromosomes. It has also been developed by scientists into a powerful experimental tool that allows almost any gene to be inactivated by evoking an RNAi response to it. This technique, which can be readily carried out in cultured cells and, in many cases, whole animals and plants, has made possible new genetic approaches in cell and molecular biology. We shall discuss it in detail in the following chapter where we cover modern genetic methods used to study cells (see pp. 499–501). RNAi also has potential in treating human disease. Since many human disorders result from the misexpression of genes, the ability to turn these genes off by experimentally introducing complementary siRNA molecules holds great medical promise. Although the mechanism of RNA interference was discovered a few decades ago, we are still being surprised by its mechanistic details and by its broad biological implications.
Bacteria Use Small noncoding rnAs to protect themselves from viruses
Bacteria make up the vast majority of the Earth’s biomass and, not surprisingly, viruses that infect bacteria greatly outnumber plant and animal viruses. These viruses generally have DNA genomes. A recent discovery revealed that many species of bacteria (and almost all species of archaebacteria) use a repository of small noncoding RNA molecules to seek out and destroy the DNA of the invading viruses. Many features of this defense mechanism, known as the CRISPR system, resemble those we saw above for miRNAs and siRNAs, but there are two important differences. First, when bacteria and archaea are first infected by a virus, they have a mechanism that causes short fragments of that viral DNA to become integrated into their genomes. These serve as “vaccinations,” in the sense that they become the templates for producing small noncoding RNAs known as crRNAs (CRISPR RNAs) that will thereafter destroy the virus should it reinfect the descendants of the original cell. This aspect of the CRISPR system is similar in principle to adaptive immunity in mammals, in that the cell carries a record of past exposures that is used to protect against future exposures. The second distinguishing feature of the CRISPR system is that these crRNAs then become associated with special proteins that allow them to seek out and destroy double-stranded DNA molecules, rather than single-stranded RNA molecules.
Although many details of CRISPR-mediated immunity remain to be discovered, we can outline the general process in three steps (Figure 7–78). In the first, viral DNA sequences are integrated into special regions of the bacterial genome known as CRISPR (clustered regularly interspersed short palindromic repeat) loci, named for the peculiar structure that first drew the attention of scientists. In its simplest form, a CRISPR locus consists of several hundred repeats of a host DNA sequence interspersed with a large collection of sequences (typically 25–70 nucleotide pairs each) that has been derived from prior exposures to viruses and other foreign DNA. The newest viral sequence is always integrated at the 5ʹend of the CRISPR locus, the end that is transcribed first. Each locus, therefore, carries a temporal record of prior infections. Many bacterial and archaeal species carry several large CRISPR loci in their genomes and are thus immune to a wide range of viruses.
In the second step, the CRISPR locus is transcribed to produce a long RNA molecule, which is then processed into the much shorter (approximately 30 nucleotides) crRNAs. In the third step, crRNAs complexed with Cas (CRISPR-associated) proteins seek out complementary viral DNA sequences and direct their destruction by nucleases. Although structurally dissimilar, Cas proteins are analogous to the Argonaute and Piwi proteins discussed above: they hold small single-stranded RNAs in an extended configuration that is optimized, in this case, for seeking and forming complementary base pairs with DNA.