RNAi from Petunias to Worms

OK a breif history of RNA interference.

1990 Rich Jorgensen at the University of Arizona wanted to make petunias a deeper purple. His group tried expressing extra copies of the same gene and ... he got white flowers. The very gene he wanted to overexpress got turned off. This effect was named "cosuppression".

Cossupression was then seen in other plants and fungi. Plant virologists also found that plants expressing viral genes, or simply small bits of viral RNA that did not encode for any protein, developed resistance to the virus from which the genes originally came from.

1993 Victor Ambrose's group (at MGH) found that the expression of a small RNA (lin-4) in C. Elegans (worms) inhibited the expression of a larger gene (lin-14). At the time the Ambrose lab believed that the small RNA maybe inhibiting the larger transcript by base pairing with it. The idea was that the double stranded RNA (dsRNA) became inaccessible to the ribosome and was degraded. No RNA = no protein.

1995 Kemphues' group tries to silence polarity genes (in this case par1) in worms. The idea is to form dsRNA between par1 transcript and RNA introduced by the experimenter (i.e. exogenously or from "outside"). When they injected "antisense" RNA to base pair with the par1 mRNA, the expression of par1 protein went down as expected. Surprisingly when they injected a sense stranded RNA as a control ... that worked too!

Why?

1998 Fire (Carnegie, now Stanford) and Mello (U Mass) demonstrate that double stranded RNA was extremely efficient at turning off gene expression when injected into the gut of worms. It didn't take much dsRNA, and the silencing was transmittable to the next generation. It's as if giving worms just a few dsRNAs turned on some mechanism that allowed specific gene silencing. This gene silencing could spread to the whole organism and also over generations.

In retrospect it turned out that Kemphues' sense RNA was contaminated with a bit of antisense RNA and that his antisense RNA was contaminated with sense RNA. Jorgensen's results were caused by leaky transcription of the antisense RNA. His sense + antisense formed some dsRNA which then became processed for RNAi. In both cases the RNA species formed were siRNA (silencer RNA). If organisms are in contact with double stranded RNA they chop it up via a protein called dicer and then use the bits (21 to 23 nucleotides long) to recognize any single stranded RNA that may have been produced from the double stranded RNA via a protein complex called RISC (RNA Interference Silencing Complex). The targetted single stranded RNAs are then destroyed. This is thought to have been evolved as a defence against viruses that have a double stranded RNA genome and produced mRNA transcripts that encode proteins that are dangerous. Chop the genome and get rid of any mRNAs.

In the Ambrose case, eukaryotes produced their own short-hairpin RNAs. These RNAs have short double stranded bits that can get processed into miRNAs (micro RNAs). miRNAs are almost complementary to regions at the end of mRNAs and are loaded onto thne RISC complex. The miRNAs are used to turn off the translation of the target mRNAs by chopping OR by storing the RNAs in P-bodies. (Click here to see the latest on siRNAs and miRNAs.)

We probably make hundreds of types of miRNAs. Small RNAs can also be used to turn off large portions of the genome near centromeres via the RITS complex. Slightly longer RNAs (piRNAs) are playing some role in germ cell development. And many more species of RNAs keep appearing. Only 10% of the RNA in a typical cell is the conventional type of RNA (i.e. mRNA). It looks as if there was a whole RNA zoo there all along and RNAi was the entry ticket. That's why RNAi won this year's Nobel Prize.