What do variegate petunias and blood cholesterol-lowering biologics have in common? Scientists used both to discover RNA interference (RNAi), a mechanism for using RNA to regulate gene expression. Today, RNAi is a powerful technology for developing effective therapeutics for a range of conditions, including neurological and cardiovascular diseases.
Download this milestone article from Drug Discovery News to learn how scientists discovered the mechanism of RNAi and developed this technology into a robust therapeutic platform.
BY DANIELLE GERHARD, PHD
In the early 1990s, a number of unexpected and perplexing results emerged from the corners of plant and worm research. Little did scientists know at the time that they were writing the first chapters of a story that would culminate in a paradigm shift for understanding gene regulation.
1990
The paradox of purple petunias
Interest in modifying plants for agricultural and ornamental purposes piqued in the 1980s with the advent of transgenic technologies. At the time, Richard Jorgensen was a plant geneticist at DNA Plant Technology Corporation who wondered if he could increase the vibrancy of purple petunias. To achieve this, Jorgensen and colleagues overexpressed the gene responsible for the purple pigment (1). Unexpectedly, this produced a mix of completely white and purple-and-white flowers. Upon closer examination of the completely white flowers, the researchers discovered a significant reduction in messenger RNA (mRNA) for both the introduced transgene and the endogenous gene for the purple pigment. These findings suggested that introducing the extra pigment gene caused coordinated suppression, or co-suppression, of both the extra gene and the endogenous gene, thereby producing white flowers. An explanation for this phenomenon evaded the researchers. Nevertheless, Jorgensen published these findings instead of sticking them in the file drawer as a failed experiment. For the time, the case of the purple petunias remained a mystery.
With the hopes of producing a vibrant, purple petunia, scientists overexpressed the transgene responsible for purple pigment. Instead, the plants yielded purple-and-white speckled flowers as a result of co-suppression.
1993
Just a weird worm thing
While plant biologists dove into horticulture, worm researchers tinkered with genes driving worm development. In the 1980s, Gary Ruvkun, a molecular biologist now at Harvard University, joined the lab of the famous worm biologist H. Robert Horvitz at the Massachusetts Institute of Technology (MIT) as a postdoctoral researcher. There, he befriended another postdoctoral researcher in the lab, Victor Ambros, a developmental biologist currently at the University of Massachusetts Chan Medical School. In the larval stages of worm development, one gene in particular, lin-4, turns o! another gene, lin-14, to trigger the switch to the next larval stage, and mutations in either gene disrupt the developmental cadence (2). At the time, mapping of the C. elegans genome was still underway, and the products of these genes were unknown. Ambros and Ruvkun decided to divide and conquer: Ambros focused his research on lin-4 while Ruvkun pursued lin-14.
Victor Ambros and Rosalind Lee from the University of Massachusetts Chan Medical School discovered the first microRNA (top). Gary Ruvkun from Harvard University discovered the second microRNA (bottom).
After several years, Ambros and his team successfully cloned the lin-4 gene. Interestingly, they found that lin-4 did not encode a protein, but instead produced two small transcripts, one of which was only 22 nucleotides long (3). Across campus, Ruvkun’s team investigated how mutations in lin-14 mRNA affect protein products (4). In a late-night phone call, Ruvkun and Ambros compared their sequences and observed complementarity between the lin-14 mRNA and the short lin-4 transcript. Specifically, the tiny lin-4 transcript paired to several sites in the 3’ untranslated region of the lin-14 mRNA, somehow causing less protein to be made from the lin- 14 mRNA. The pair published their findings in the same issue of Cell in 1993. “At the time, there was no framework for thinking about a very short antisense transcript,” said Ambros. The function of these tiny RNAs remained a mystery for nearly a decade. “There was always a view that worms were quirky and weird,” said Ruvkun. This sentiment led many to cast aside these findings as just some weird worm developmental event.
1998
Twitching worms
In the 1990s, scientists thought they knew the basic principles of gene regulation, but a number of anomalous findings popped up that were difficult to explain, including Jorgensen’s puzzling petunias. Around this time, worm researchers had observed that experimentally introducing RNA that was antisense to a specific mRNA led to repression of the gene it encoded. However, in 1996, researchers at Cornell University found that injecting either antisense or sense RNA suppressed gene expression (5). These findings caught the eye of Andrew Fire, now at Stanford University, who knew that the experimental procedure used to make the RNAs could inadvertently produce some doublestranded products consisting of the sense and antisense RNAs. He wondered if these double-stranded RNAs (dsRNAs) played a role in gene regulation, so he contacted Craig Mello, a biologist at the University of Massachusetts Chan Medical School. To test this hypothesis, Fire and Mello injected worms with either sense, antisense, or double-stranded RNAs designed to target the mRNA encoding a muscle protein. Knowing the potential for this technique to randomly produce aberrant RNA products, the researchers made sure to purify the RNA before injections. Surprisingly, either the sense or the antisense RNA alone had only a modest effect on the worms, but the doublestranded RNA caused a substantial loss of the muscle protein, causing the worms to twitch (6). With the loss of the worm’s smooth moves came the discovery of RNA interference (RNAi), or the regulation of genes by double-stranded RNA, a finding that won Fire and Mello the 2006 Nobel Prize in Physiology and Medicine. They uncovered the causative agent for RNAi, but the molecular mechanisms underlying this phenomenon were a mystery. “This one discovery opened the floodgates,” said Phillip Zamore, a molecular biologist at the University of Massachusetts Chan Medical School. “Their paper pretty much set a roadmap for 20 years of an entire field.”
Many early studies on microRNAs and RNAi were done in worms like Caenorhabditis elegans.
1999
Tiny RNAs are a tomato thing too
Shortly after the discovery of RNAi in worms, researchers observed RNAi in insects and protozoa. Plant biologists identified a similar phenomenon of gene silencing as a defense mechanism to reduce exogenous and viral mRNAs, but how this occurred remained unclear. In 1999, David Baulcombe, a plant scientist at the Sainsbury Laboratory in the United Kingdom, and his student Andrew Hamilton published a paper in Science showing that in response to the introduction of certain transgenes, tomato plants created short antisense RNAs of only 25 nucleotides in length, later named small interfering RNAs (siRNAs), that were complimentary to the mRNA of the transgene (7). In this paper, Baulcombe and Hamilton reported that RNAi also occurred in plants and suggested that the 25-nucleotide RNAs they found might play a role. This paper was the first to suggest a link between RNAi and the short RNAs Ambros observed in 1993. Individual pieces to a puzzle were appearing one by one, but the connecting pieces, or the molecular mechanisms driving RNAi, were still missing.
2000-2001
Slicing and dicing dsRNA
The 1998 Fire and Mello paper made such a strong impression on Zamore, then a postdoctoral researcher at MIT in the lab of molecular biologist David Bartel, that he redirected his future research plans towards RNAi. He published a paper in 1999 introducing the first cell-free system for biochemically studying RNAi in flies, which he wrote in collaboration with another postdoctoral researcher, Thomas Tuschl, who is now a biochemist at Rockefeller University (8). This system allowed the researchers to demonstrate that cells process each strand of dsRNA into short RNA segments that are 21-23 nucleotides long (9), thus linking the findings from Fire and Mello with those of Hamilton and Baulcombe. Soon after, Gregory Hannon’s research team at Cold Spring Harbor Laboratory added more pieces to the puzzle, demonstrating in flies that the enzyme Dicer chops dsRNA into the 21-22 nucleotide segments (10), which then instruct a multiprotein complex called RNA-induced silencing complex (RISC) to destroy a specific mRNA (11).
microRNAs converge on RNAi machinery
Around the same time, Ruvkun discovered a second short, 21-nucleotide RNA involved in orchestrating worm development, let-7 (12). While at home working on a grant submission, Ruvkin decided to compare the worm let-7 sequence to the human genome. At the time, only approximately one-third of the human genome had been sequenced, but updates were coming in daily. To his surprise, he found a complete match, suggesting that this wasn’t just some weird worm thing, but something that was conserved in humans too (13). This prompted him to request RNA from researchers located all over the world who worked on different organisms. “A postdoc in my lab ran these northern blots on zoos of RNA, proving that these tiny RNAs were universal to animals,” said Ruvkun. “When I saw the paper, it just changed my life,” said Ambros. “I can remember to this day the moment I opened up Nature and saw that. Holy cow!” He thought that if there were two of these tiny RNAs, there must be others. Indeed, a trio of papers published in Science in 2001 from the labs of Tuschl, Bartel, and Ambros revealed dozens of tiny endogenous RNAs, which they called microRNAs (14,15,16). They noted that many of these newly identified RNAs were highly conserved and expressed not only during early development, but also later in life. “This is when many biologists started to get excited about microRNAs,” said Bartel. “It was clear that humans had more than a hundred different microRNAs, and that any gene that you might be interested in — not just genes involved in developmental transitions — might be regulated by a microRNA.”
For RNAi to occur, the enzyme Dicer cleaves dsRNA into siRNA, which serves as a template to direct the RNAinduced silencing complex (RISC) to find and destroy the complementary mRNA transcript.
2018
The first RNAi-based therapeutic
In the meantime, Tuschl studied the features of siRNAs produced in the fly cell-free system. Using these insights, he chemically synthesized siRNAs and used them to achieve gene-specific knockdown in mammalian cells (17). These experiments provided a powerful new research tool that transformed the study of mammalian gene function. As studies continued to accumulate, a link between siRNAs and microRNA became increasing apparent. Synthetic dsRNA is processed into 21-23-nucleotide segments of siRNAs, about the same length as microRNAs. “It just couldn’t be coincidence,” said Ambros.
In the early 2000s, Phillip Zamore from the University of Massachusetts Medical School (left) and David Bartel from Massachusetts Institute of Technology (right) helped found the RNAibased therapeutics company Alnylam Pharmaceuticals, which developed the first FDA-approved RNAi drug.
Sure enough, researchers quickly found that Dicer also snips long pre-microRNAs to produce 21-nucleotide microRNAs (18), which then direct the RISC complex to hunt down its target mRNA (19). Although they originate from different sources, siRNAs and microRNAs converge mechanistically to drive gene suppression. “Once they get into RISC, the cell has no way of distinguishing the chemically synthesized siRNA from a microRNA,” said Bartel. The widespread utility of siRNA as a research tool suggested that they might also be useful in the clinic to treat diseases. In 2002, several scientists, including Zamore, Tuschl, and Bartel, joined forces with investors to found the RNAi-based therapeutics company Alnylam Pharmaceuticals. In 2018, the FDA approved the first RNAi drug, patisiran, to treat a rare and fatal neurodegenerative disease. Now, Alnylam Pharmaceuticals has several FDA-approved RNAi drugs with more in the pipeline. One promising drug, inclisiran, blocks the gene PCSK9 to lower cholesterol concentrations in the blood. “This drug shows the promise of a true platform drug,” said Zamore, meaning that once drug delivery is optimized to target a specific cell type, like liver cells, scientists can slightly modify the drug to target any number of other genes to treat a variety of diseases. RNAi drugs give new hope to patients su!ering from debilitating diseases for which there are no effective treatments.
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