Ruvkun and Ambros recognized for miRNAs

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The 2024 Nobel Prize in Physiology or Medicine was awarded to Victor Ambros and Gary Ruvkun for their discovery of microRNAs (miRNAs). Their insightful discoveries altered our understanding of how non-coding RNAs regulate gene expression.

In 1969, Britten and Davidson, extending the work of Jacob and Monod in prokaryotes, published a model of gene regulation in eukaryotic cells evoking the idea of various genetic regulatory RNAs.¹ This model was consistent with their work using quantitative RNA-DNA Cot hybridization assays showing that the complexity of the RNA transcripts was far greater than that which could be accounted for by protein coding sequences. By 1977, some of the complexity discrepancies were explained by the discovery of introns and RNA splicing,^2,3 for which Phil Sharp and Robert Roberts were awarded the 1993 Nobel Prize in Physiology or Medicine. Most small non-tRNAs detected with various techniques were thought to represent degradation products, and little was known about what has been called the dark matter of the genome. In fact, during this era, great debates about complex genomes containing junk sequences populated the literature. However, as we now know, much of the genome encodes non-coding transcripts with various functional activities that continue to be discovered.

Leading up to the discovery of miRNAs in 1992, about a decade earlier, two post-docs in the Horvitz lab, Gary Ruvkun and Victor Ambros were investigating the genetic and molecular processes involved in the development of the small worm known as Caenorhabditis elegans. This model organism was and continues to be well studied, in part because of the extensive knowledge base, toolkit, and collection of genetic mutants affecting development. Notably, C. elegans was the first animal for which cell lineage mapping from fertilization to the 959-cell adult had been determined and recognized with the 2002 Nobel Prize in Physiology or Medicine awarded to Sydney Brenner, John Sulston, and Robert Horvitz.

Ambros and Ruvkun were studying various developmental mutants, including the lin-14 locus, for which various gain-of-function and loss-of-function lin-14 alleles had opposing developmental effects.^4,5 The lin-14 gain-of-function mutations and a lin-4 loss of function mutation resulted in the same developmental phenotype. Based on genetic epistasis studies, lin-14 and lin-4 were placed into the same developmental pathway. The two, working first together as post-docs⁶ and then as independent investigators, began to uncover the molecular mechanisms that would explain the developmental genetic findings. Ruvkin’s group discovered the lin-14 gain-of-function mutant mapped to the 3′ UTR, suggesting the mutation leading to an increase in the lin-14 encoded nuclear protein was potentially regulated at the level of mRNA stability or translation.⁷ Based on the genetic studies, it was reasoned that the lin-4 locus would likely encode a protein that interacted with the 3′ UTR of the lin-14 mRNA to regulate the production of the protein product. However, everything changed in 1992, when Ambros discovered that the lin-4 locus encoded a small non-coding RNA (also known as the 22-nt miRNA). In 1993, Ambros and Ruvkun published two separate studies that in total showed that the lin-4 locus encoded a short non-coding miRNA that could form imperfect RNA duplexes, with a portion of the lin-14 3′ UTR allowing for down-regulation of the lin-14 mRNA and chimeric reporter mRNAs with a fused lin-14 3′ UTR.^8,9 Some years later, Ruvkun’s group described a second let-7 miRNA that regulated lin-41 through a comparable interaction.¹⁰ Importantly, the let-7 miRNA was found to be conserved across phylogeny, including humans. The family of let-7 miRNAs was found to play important roles in regulating cellular differentiation as well as cell growth and was soon revealed to play a role in various types of tumor suppression. Ambros, Ruvkun, and many others went on to demonstrate how a single miRNA regulated not only a single mRNA but also networks (perhaps as many as several hundred) of mRNAs (reviewed in Shang et al.¹¹).

In 1998, Fire and Mello and colleagues also working with C. elegans discovered RNAi in which short double-stranded RNAs with strand full complementarity to an mRNA resulted in cleavage and degradation of the target RNA.¹² This ultimately explained why plant molecular biologists who attempted to introduce genetic sequences to enhance pigmentation in petunias created white petals. The discovery of RNAi has led to six US Food and Drug Administration-approved therapeutics (with more on the way), the first being patisiran for hereditary transthyretin amyloidosis. In 2006, Fire and Mello were awarded the Nobel Prize in Physiology or Medicine.

While miRNAs generally result in a dampening of mRNA translation and/or shortening the half-life of a transcript, RNAi can virtually eliminate or come close to eliminating the target mRNA through a cleavage mechanism. It soon became clear that processes involved in miRNA and RNAi function entail overlapping cellular machineries, with many intervening observations indicating that miRNA-mediated gene repression is a key regulatory process at work in mammalian cells.

While our understanding of miRNA biology has greatly advanced, it is still far from complete (reviewed in Shang et al.¹¹). Nevertheless, miRNAs have become essential to our understanding of post-transcriptional gene regulation, acting within intricate regulatory networks that influence cellular processes. Research has shown that miRNA biogenesis itself is regulated by feedback loops and complex circuits, making miRNAs versatile and sensitive to disruption through mutations or other forms of dysregulation.¹¹ Such disruptions are increasingly linked to many different diseases, which has fueled interest in miRNAs as potential diagnostic and prognostic biomarkers, as well as therapeutic targets (reviewed by Seyhan, Takahashi et al., and Tian et al.^13,14,15). In the therapeutic realm, strategies like miRNA mimics and sponges—delivered either as nucleic acids or through transcriptional templates via gene therapy vectors—are actively being explored. As an example, miR-122 is only produced in hepatocytes and makes up 70% of liver miRNAs. This miRNA plays an important role in regulating metabolism and was also found to be required for hepatitis C virus replication. Thus, this miR122 became one of the earliest therapeutic targets. An antisense oligonucleotide approach was pursued through early phase 1/2 clinical trials before being supplanted by anti-HCV small-molecule therapies.

Although no miRNA-based drug has yet been approved, multiple ongoing clinical trials reflect the therapeutic potential of these molecules. The discovery of miRNAs, along with RNAi, in model organisms like C. elegans underscored the importance of fundamental basic research in revealing new gene regulatory pathways and providing insights into developmental processes and disease mechanisms. The translation of these basic biological endeavors highlights how curiosity-driven research can yield unanticipated benefits, advancing both scientific understanding and medical innovation.

Ruvkun and Ambros truly exemplify the journey of two dedicated scientists who continued their collaborative efforts from postdoctoral studies through senior faculty status. In doing so, they have not only transformed our understanding of gene regulation but also pioneered a new field. Their groundbreaking discoveries in gene silencing and regulatory RNA have laid the foundation for further research that will reshape how we understand molecular mechanisms underlying complex diseases. This paradigm-shifting work is likely to continue driving innovations in treating and diagnosing conditions like cancer, autoimmune diseases, neurological disorders, and cardiovascular diseases for years to come.

Their story is such an inspiring example of how partnerships and perseverance in research can lead to revolutionary scientific advances.