Who won the 2024 Nobel Prize in Physiology or Medicine?
The Nobel Prize for Physiology or Medicine has been awarded to Victor Ambros (University of Massachusetts Medical School) and Gary Ruvkun (Harvard Medical School [HMS], both MA, USA) for their discovery of microRNA – small, non-coding RNA molecules – and its role in gene regulation.
Revealing new layers of genome regulation
Since their discovery in the 1960s, transcription factors have been seen as the drivers of gene modulation, responsible for fine tuning the expression of our genomes within individual cells to yield the specific differentiated cell types required to construct complex tissues.
However, in the late 1980s, two postdocs working together in the Robert Horvitz lab at MIT (MA, USA), Ambros and Ruvkun, made some interesting findings. The pair were investigating genes that modulate the genetic mechanisms that lead to cell differentiation, ensuring that each mechanism is stimulated at the correct time. Caenorhabditis elegans (C. elegans) worms, small simple organisms that still contain many specialized cell types, were used as their model.
The first of the mutations they were investigating had been identified by researchers in the Sydney Brenner lab at MIT in the late 70s after a mutagenesis screen revealed that worms with a mutation in the lin-4 gene completely lacked key physiological structures, while others were heavily disturbed. This suggested that the protein lin-4 was a master regulator of developmental timing. A further mutant in lin-14 was reported in a 1987 paper. Discovered previously by the Horvitz lab, the mutant was shown to have opposing developmental timing defects to those of the lin-4 mutant, which suggested that lin-4 could be a negative regulator of lin-14, a hypothesis Ambros was later able to validate.
This validation was obtained in the same year that Ambros and Ruvkun were able to clone the lin-14 gene using a classical restriction fragment length polymorphism approach, reported in 1989. Ruvkun, having started his own lab at HMS, then demonstrated that lin-14 is expressed primarily during the L1 stage of C. elegans’ development. Ruvkun identified many variants of the lin-14 gene, some of which led to expression of the protein beyond the L1 stage. These mutations were shown to be located in the 3’UTR region of the gene and left the resultant protein unaffected but prolonged its expression. This finding led Ruvkun to consider that a post-transcriptional mechanism was responsible for preventing its translation to protein and the full expression of the gene.
Meanwhile, Ambros, now running his own lab at Harvard University (MA, USA), was working on lin-4, which at the time had only one identified variant. Using restriction fragment length polymorphism and Southern blot probing, he successfully cloned lin-4 and then set out to establish the smallest amount of the gene that could be preserved to save its function, identifying a 693 bp Sal l restriction enzyme fragment. The short open reading frame of the lin-4 gene suggested that it may produce a noncoding RNA. This was confirmed when the team introduced frameshift mutations, which would typically render a protein totally functionless, and found that it had no effect on the gene’s function. Further experimentation with northern blot and RNase protection assay revealed that the gene produced two transcripts that were 61 and 22 nucleotides long.
microRNAs as a diagnostic and prognostic biomarker for psychiatric disorders
Professor Cecilia Flores and PhD student Alice Morgunova discussed their work on the molecular and cellular pathways that underlie depression in adolescence, and explained the techniques utilized to accurately profile biomarkers and determine the differences between healthy control individuals and adolescents diagnosed with depression.
A fruitful reunion
Having kept in communication and discussed their work, in June 1992 the pair met to share sequence data for their respective genes, quickly identifying partial complementarity between the lin-4 transcript and the 3’UTR of lin-14. Ambros used their sequence data for lin-4 to find other nematode worms containing the gene and identify an additional lin-4 mutant with a single nucleotide variant in the complementary sequence.
Meanwhile Ruvkun set out to confirm his suspicions of lin-14’s post-transcriptional regulation, comparing lin-14 protein versus RNA abundance in wild-type and gain-of-function mutants. The team found that protein was dramatically overexpressed while RNA remained the same, confirming that the gene’s expression was controlled at a step between transcription and translation.
By splicing the 3’UTR of lin-14 onto a reporter gene and then iteratively reducing the size of the 3’UTR section attached, Ruvkun was able to identify a 124 nucleotide-long fragment of the 3’UTR conserved in another species of worm and composed of segments partially complementary to lin-4. Further experiments from both labs revealed that lin-4 microRNA negatively regulates lin-14 by annealing to sections of the lin-14 mRNA. As they couldn’t identify lin-4 outside of the Caenorhabditis family of nematodes, the phenomenon was considered specific to these species, and the findings, released in 1993, were largely ignored.
MicroRNA receives recognition
The research remained relatively inconspicuous until the turn of the millennium when the Ruvkun lab identified a second microRNA gene, lin-7, using a genetic screen in C. elegans worms with mutations in lin-14 and egl-35 loci. They were searching for a mutation that would supress aspects of the phenotype typical to these mutations.
The same year, Ruvkun searched for the lin-7 sequence in nucleotide databases for several animals, where they found matches in fruit fly and human genomes. The presence of lin-7 microRNA was confirmed in several human tissues. Further studies revealed that lin-7 was widely conserved among several species, from crustaceans and insects to molluscs and annelids.
These discoveries led to a flurry of research into microRNAs and their potential ubiquity throughout the animal kingdom, and by the end of 2001, the evidence of microRNA’s highly conserved presence and importance in gene regulation was substantial enough to update prevailing opinions on gene regulation and the functions of RNA.
Today, over 1000 microRNA species have been identified in the human genome and 48,860 mature microRNA gene sequences have been identified in 271 organisms, while evidence has also been collected to show that microRNA can be produced outside of the animal kingdom, by viruses. 30 years on from their initial discovery of microRNA, Ambros and Ruvkun have now been awarded the Nobel Prize for Physiology and Medicine in recognition of a truly foundational piece of research on which so much of our current understanding of gene regulation relies.
