revisions to the “central dogma” of molecular biology over the last 10 years, scientists have discovered an entirely new category of non-coding RNA genes whose existence had never even been suspected; they are distinguished by their small sizes relative to traditional RNA genes (i.e. 21 to 25 bp versus 96 bp) and are therefore commonly referred to as “microRNAs” genomic DNA pre-mRNA with introns mRNA2 no introns mRNA1 no introns protein isoform1 protein isoform2 non-coding RNA genes

The Nobel Prize in Physiology or Medicine 2006 Andrew Z. Fire Stanford University School of Medicine b Craig C. Mello University of Massachusetts Medical School b for their discovery of RNA interference gene silencing by double-stranded RNA

microRNA biogenesis and action gene silencing is mediated by short double-stranded RNAs (dsRNAs) Saumet A, Lecellier CH Retrovirology 3: 3 microRNA-mediated gene silencing is observed in plants, animals, and many fungi; it plays essential roles in development and virus resistance; it has already provided powerful tools for studying gene function and spawned new therapeutic industries

biologists working with transgenic plants had long observed a bewildering array of unanticipated gene silencing phenomena; in 1998, Fire and Mello compared the silencing activity of single- stranded RNAs (ssRNAs) (sense or antisense) with double-stranded (dsRNAs) hybrids; marginal silencing was achieved by injecting C. elegans with ssRNAs, but potent and specific silencing was achieved by injecting a sense-antisense mixture; in other words it is the dsRNAs that matter

functional genomics of C. elegans chromosome 1 by RNA interference Fraser AG, …, Ahringer J Nature 408: (notice how few genes have an identifiable phenotype) ingestion of bacteria expressing dsRNAs is as effective for gene silencing as the direct injection of dsRNAs C. elegans scientists can silence nearly any desired gene just by feeding the worm with one of these custom- modified bacteria they sell on the Internet

evolutionarily conserved genes are more likely to have a phenotype percentages for all genes (blue bars) or all genes with RNAi phenotype (red bars) that have matches in S. cerevisiae (SC), D. melanogaster (DM), human (HS), all three combined (ALL), or have no matches in any organism (NO M)

commercial libraries are widely available for most popular organisms

RNA interference identifies over 250 host factors required by HIV-1 Brass AL, …, Elledge SJ Science 319:

genetic defect in CCR5 chemokine receptor provides immunity against HIV-1 and is/was a model for development of drug therapeutics susceptible normal receptor human cell HIV virus  DISEASED PLUS drug susceptible PLUS drug normal receptor DRUG human cell HIV virus  HEALTHY non-susceptible mutant receptor human cell HIV virus  HEALTHY

RNAi exists because eukaryotic cells make microRNAs of their own Griffiths-Jones S, et al Nucleic Acids Res 36: D154-D158

the miR-124 regulatory network gene batteries to global regulators of transcription and alternative splicing active interactions are solid black; inactive interactions are solid gray; weak interactions are thinner lines; gene battery expression inputs to cellular transcriptome are dashed lines; non-neuronal elements have dark blue coloring if present or light blue if absent; neuronal specific elements are likewise colored in shades of reds Makeyev EV, Maniatis T Science 319:

Amaral PP, …, Mattick JS The eukaryotic genome as an RNA machine. Science 319: 1787 how many other RNA mechanisms are remaining to be discovered?!?