Kevin Folta and Mark Settles present seminal RNAi research: Napoli et al.'s 1990 work on chalcone synthase suppression in petunia, and Fire et al.'s 1998 C. elegans experiments showing how double-stranded RNA triggers sequence-specific gene silencing that spreads through cells and is heritable.
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Thank you for watching Transformative Advances in Molecular Biology, a retrospective look at critical events in the history of the discipline. The presentations in this series were prepared by graduate students in a journal colloquium at the University of Florida supervised by Mark Settles and Kevin Folta. Today we review 2 papers considered to be the seminal studies that uncovered the phenomena of RNAi. What many people do not realize is that RNAi, or what was referred to as co-suppression, was first discovered in plants in 1990 by Richard Jorgensen's group. 8 years later, genetic interference by double-stranded RNA was described in C. elegans, for which the authors of the paper, Andrew Fire, Craig Mello, and colleagues, were honored with the Nobel Prize in Physiology or Medicine in 2006. We now discuss these 2 studies in detail, Highlighting the results of each paper and the experimental approaches taken to elucidate this fundamental process in biology. This splendid array of colors found in nature's flowers are in large part due to the class of secondary metabolites called flavonoids. In this study by Napoli et al., their objective was to enhance the color of petunia flowers by overexpressing the gene at the beginning of the flavonoid pathway, chalcone synthase. Chalcone synthase catalyzes the condensation of 3 malonyl-CoA units with a coumaroyl-CoA to yield chalcone, which is the gateway step to all the major flavonoid classes shown here. Each class predominates in particular plant lineages, and it is generally accepted that the anthocyanin class are what gives flowers their color. The wild-type petunia flowers used in this study were a violet variety shown here. Unexpectedly, when they overexpressed chalcone synthase, they did not recover any plants with a more enhanced violet hue, but instead plants with a variegated color pattern as shown here, and in some cases flowers that were completely white. When primary petunia transformants were outcrossed to different wild-type varieties like V26, R20, R1, and M27, the novel flower color phenotype illustrated by W for white sectors displayed segregation ratios consistent with a dominant single locus transgene, with either complete, partial, or no penetrance. For example, the segregation of the white sector phenotype of transformant 218.38 suggests complete penetrance in all crosses, as the 1:1 ratios indicate. On the other hand, the 218.18 transformant exhibited very low penetrance in each cross. The authors posited that this variability in inheritance could be due to environmental factors, the genotype of the outcross parent, or position effect of the transgene itself. Using RNase protection assays, both the endogenous and introduced chalcone synthase transcript levels were measured in wild-type and transgenic petunia flowers. Both endogenous and introduced chalcone synthase transcripts peak when petunia corollas reach approximately 40 mm in length, and decline in abundance thereafter. Strikingly, the levels of endogenous chalcone synthase were approximately 50-fold lower than found in wild type. It was observed that after many months, transgenic plants exhibiting the white-sectored flower phenotype eventually gave rise to branches with solid white-violet colors. These revertants were assayed for chalcone synthase gene expression, again by RNase protection. Interestingly, the violet revertent flowers had both endogenous and introduced chalcone synthase levels that were 30 to 50-fold higher than found in white flowers, suggesting that both chalcone synthase genes were coordinately and reversibly suppressed in white flowers. Given that chalcone synthase is expressed throughout the plant, the authors next examined whether chalcone synthase transcripts behave the same way in leaf tissue as they do in flower tissue. Indeed, low levels of endogenous chalcone synthase transcript were detected in both wild-type and violet-revertant leaf tissue, while in white-sectored petunia plants, levels of leaf chalcone synthase transcripts were undetectable. These results indicate that the co-suppression effect appears to occur throughout the plant.
The next paper that we are going to discuss is the Nobel Prize winner, for year 2006 by Fire and his group, published in Nature 1998. It was already known that RNA has the ability to cause interference to functional genes, but Fire and his group want to know if RNA interference is specific and the structures of a gene that are required for RNA interference to happen by using the model organism C. elegans. An interesting point on RNA interference is that the effect is heritable into the next generation. Prior to the paper, there are 2 papers by Izett and Winthrop in 1998 and Fire et al. in 1991 demonstrated the after- What are the effects of injecting antisense DNA? To show the specificity of RNA interference, the UNG22 gene is used. The UNG22 is an important gene that codes for myofilament protein. In the absence of the UNG22 gene, severe twitching muscle structural damage, and impaired movement are observed in C. elegans. This experiment demonstrated that antisense with sense strand are able to produce observable phenotype in C. elegans compared to either only sense or antisense strand. Another important point is The key point in this slide is that severe twitching is observed only if the double-stranded RNA is homologous to the target gene. Introns do not cause any effects in C. elegans. To further illustrate at the cellular level, Fire and his group used a stable transgenic Drosophila elegans strain PD4251 that produces GFP in all body muscles. The purpose of the GFP is to check for identical expression patterns in the progenies in comparison to the parents. For easy identification of different cell compartments, the transgenic strain PD4251 Also have a combination of mitochondrial and nuclear localization to observe whether these cells express the original GFP constructs. From the figure, it is clear that the expression levels are decreased when compared to the control. The same expression is observed in parental strain, but it's not shown in this figure. Another similar expression is observed in situ using the target transcript Max3 that is found abundantly in the gonads. There are no observable endogenous mRNA when the gonads were injected with dsRNA from Max3. On the other hand, injected purified mRNA— Sorry, can you repeat that? antisense of MaxIII showed some mRNA expression. In conclusion, Fire and his group clearly demonstrated that dsRNA is able to eliminate endogenous mRNA transcripts. Only promoters and introns are unable to play a role in RNAi. RNA interference is able to persist into the next generation and also has the ability to cross cellular boundaries.
Thank you for watching, and please check out the other exciting topics in the series.
