Darnell made his first major finding 40 years ago, soon after researchers studying bacteria uncovered a phenomenon that every modern biology student knows: The nucleic acid messenger RNA (mRNA) serves as a short-lived copy of its close relative, DNA, allowing the information contained in the cell’s genes to be translated into proteins. In the genetic regions that encode proteins, each trio of DNA building blocks (deoxyribonucleotides) corresponds to a trio of RNA building blocks (ribonucleotides), which in turn guides a single amino acid into the growing protein.
As scientists were marveling at the elegant simplicity of this bacterial system, Darnell was tracking RNA inside mammalian cells. Soon he made a surprising finding: Very long RNAs gradually disappear from the nucleus, where they are made, and shorter RNAs show up in the cytoplasm. The nucleotide composition of the large and the smaller molecules resemble each other and are characteristic of RNAs known to compose ribosomes, the protein-making factories of the cell. Together, the results indicated that mammalian cells pare down their original RNAs; they release several mature forms of ribosomal RNA (rRNA) from a giant precursor molecule. Darnell coined the term “RNA processing” to describe this phenomenon in a 1963 paper that reports this work.
When Darnell analyzed the largest and most fleeting RNA in the nucleus, he discovered that its composition does not resemble that of rRNA, but instead corresponds to that of total mammalian DNA and mRNA. He called this RNA heterogeneous nuclear RNA (hnRNA) and speculated that hnRNA is the nuclear precursor to mRNA just as pre-rRNA is the precursor to rRNA. Perhaps the mRNA lay in the middle or at the ends of the hnRNA molecule, awaiting liberation.
He and Mary Edmonds, of the University of Pittsburgh, independently found that some hnRNAs and almost all mRNAs carry a string of adenine nucleotides — a poly(A) — at one end. Furthermore, Darnell showed that poly(A) appears first in hnRNA and later in cytoplasmic mRNA, supporting the notion that the first molecule gives rise to the second one. Subsequently, scientists discovered a distinguishing chemical structure called a cap on the other end of both mRNA and hnRNA. Darnell found that many hnRNA molecules and all mRNA molecules carry one poly(A) tail and one cap. These observations posed an enigma: How could a long hnRNA with a tail and cap give rise to a short mRNA with the same characteristic tags at its ends? In retrospect, the answer is obvious, but at the time, the dogma that DNA sequence always corresponded exactly to mRNA sequence — and that genes were thus uninterrupted — forced scientists to surmount a tremendous intellectual barrier before stumbling upon the solution in 1977. Two teams — led by Phillip Sharp of MIT and Richard Roberts of Cold Spring Harbor Laboratory — vaulted this hurdle when they used electron microscopy to look at DNA bound to the mRNA it encodes. Large regions of the DNA looped out, and the groups proposed that internal spans of sequence — now called introns — were removed as hnRNA was transformed into mRNA. With this discovery, the field of mRNA splicing burst onto the scientific scene.
Knowing how mRNA was formed, Darnell next decided to back up a step and explore how cells regulate its production — or transcription — from DNA, and how they activate particular sets of genes to accomplish specialized physiological tasks. By the early 1980s, he had discovered that a liver cell could remain a liver cell only when it resided in its natural milieu. When he separated cells from each other, they stopped manufacturing liver-specific mRNAs within a few hours, although they continued to produce basic housekeeping mRNAs. Without constant signals from their normal habitat, liver cells lost the mRNA profile that helps give them their identity.
These observations made Darnell wonder how cues from outside the cell could spark specific gene activity. He suspected that he could analyze this issue by studying how proteins called interferons (IFNs) instigate human cells to guard themselves against viruses. These proteins — subdivided into groups called alpha, beta, and gamma — belong to a hormone family called cytokines that rev up antimicrobial defenses. Immediately after binding to the outside of a cell, IFNs spur the manufacture of proteins that quash viral duplication and render the infected cells more susceptible to attack by the immune system.
To get a handle on the cellular players that turn on genes in response to IFN, Darnell decided to work backwards, reasoning that the genes triggered by the cytokine would lead him to their molecular inducers. To find IFN-prodded genes, he identified mRNAs that accumulate in response to IFN-beta. His and another research group showed that the mRNA hike didn’t depend on new protein synthesis, implying that the cell possessed the IFN-responsive gene activator — or transcription factor — in a dormant state before stimulation.
After isolating a short stretch of DNA that lies next to IFN-alpha-induced genes, he sought cellular machinery that could sit down on this sequence and drive transcription of adjacent test genes. This strategy produced three nuclear factors that bound to that particular piece of DNA and not others; one of them appeared specifically in IFN-treated cells. This IFN-responsive transcription factor was what he sought; it hangs out in the cytoplasm, waiting for IFN to jolt it into action. Two thousand liters of IFN-treated cell cultures later, Darnell’s students and postdoctoral fellows had purified this factor, which consisted of four proteins. These were among the first purified proteins that respond to signals from outside the cell by binding to DNA and stimulating genes. Darnell subsequently named two of them STAT1 and STAT2, echoing the “STAT” call of a hospital loudspeaker — in part because they trigger genes quickly.
Once he had isolated the proteins that provoke the genes, he wondered what the cell uses to sense IFN outside the cell and goad the Stats into action. He soon discovered that when IFN attaches to its receptor on the surface of cells, STAT1 and STAT2 acquire a chemical group — phosphate — known to animate proteins. Other teams identified the enzymes that tack on these phosphates as Janus kinases, or JAKs, which cozy up to molecules that span the cell’s membrane. When IFN binds on the outside, these molecules prompt the JAKs to modify Stats. A new signaling system — the Jak-Stat pathway — was born, and provided the first detailed account of a pathway that leads from a tickle at the cell’s surface to changes in gene activity inside its nucleus. The findings elucidated how cells can dramatically switch physiological directions in response to environmental signals.
Since its discovery, the mammalian Stat family has expanded to seven members, and its relatives have popped up in a wide range of organisms, including worms, slime molds, and flies. IFN-alpha represents just one chemical prod for these transcription factors and the physiological consequences of Stat activation extend beyond the anti-viral response. STAT3, for example, participates in animal cell growth regulation, inflammation, resistance to cell death, and early embryonic development in response to a variety of stimuli — and drugs that act on this Stat might hold therapeutic value. Darnell showed that a hyperactive version of STAT3 spurs rampant cellular reproduction in culture and that an inhibitory version blocks this unrestrained growth. These results and others, together with the observation that more than 50 percent of human cancers contain constantly active STAT3, suggest that the protein normally prevents deranged cancer cells from committing suicide. Thus, a pharmaceutical agent that thwarts STAT3 might repel cancer. Other Stats, too, participate in medically relevant pathways. A drug that incites STAT5 (recombinant erythropoietin) is already on the market to combat anemia.
In addition to discovering the fields of RNA processing and Stat signaling, Darnell has co-written two textbooks — General Virology and Molecular Cell Biology—that have instructed countless students. He has personally influenced a tremendous number of trainees over the course of his career, having mentored more than two dozen PhD students and 100 postdoctoral fellows in his lab. These individuals include an exceptional number of creative scientists, including Sheldon Penman, David Baltimore, Jonathan Warner, Ronald Evans, Joseph Nevins, and Jeffrey Friedman.
Darnell has also played an indispensable role in building a strong independent junior faculty at The Rockefeller University. A long-held and cherished tradition of The Rockefeller University ordained that early career investigators should work in senior faculty members’ labs. In 1985, Darnell co-chaired a university committee charged with finding strong young candidates, and hiring them as independent scientists. The first five researchers refused the university’s offers because they didn’t believe that they’d be allowed the autonomy they desired. Now, owing in large part to Darnell’s effective leadership, Rockefeller competes for junior faculty with other top-notch institutions. The program has engaged several dozen young scientists in the last decade, 20 of whom have earned tenure. Scientists at other institutions have hailed this innovation, saying that it has greatly broadened the research fabric of the university.
For almost half a century, Darnell has had a major impact on science worldwide. He has uncovered major themes in RNA’s function as an informational molecule, and provided a molecular explanation for how the cell heeds signals from its environment. Throughout his career, he has devoted himself to nurturing young scientists, assuring his legacy through their achievements as well as his own.
by Evelyn Strauss
Key publications of Willem Kolff
Kolff, W.J., Berk, H.T.J., ter Welle, Z.M., van der Ley, A.J.W., van Dijk, E.C., and van Noordwijk, J. (1943). de Kunstmatige nier:een dialysator met groot oppervlak. Geneesk. Gids. 21, 3–12.
Kolff, W.J., Berk, H.T.J., ter Welle, Z.M., van der Ley, A.J.W., van Dijk, E.C., and van Noordwijk, J. (1942). de Kunstmatige nier:eendialysator met groot oppervlak. Ned. Tijdschr. V. Geeneskunde. 87, 1684–1687.
Kolff, W.J. and Terk, H.T.J. (1944). The artificial kidney: a dialyzer with a great area. Acta Med. Scand. 117, 121.
Kolff, W.J. and Watschinger, B. (1956). Further development of a coil kidney. J. Lab. Clin. Med. 47, 969–977.
Kolff, W.J., Effler, D.B., Groves, L.K., Peereboom, G., Moraca, P., Aoyama, S., and Sones, F.M. (1956). Disposable membrane oxygenator (heart-lung machine) and its use in experimental surgery. Elective Cardiac Arrest in open-heart surgery: report on three cases. Cleveland Clinic Quart. 23, 69–114.
Kolff, W.J. (1983). Artificial organs — forty years and beyond. Trans. ASAIO. 24, 6–24.
Key publications of Belding Scribner
Scribner, B.H., Caner, J.E.Z., Buri, R., and Quinton, W. (1960). The technique of continuous hemodialysis. Trans. ASAIO. 6, 88–103.
Quinton, W., Dillard, D., and Scribner, B.H. (1960). Cannulation of blood vessels for prolonged hemodialysis. Trans. ASAIO. 6, 104–113.
Scribner, B.H., Buri, R., Caner, J.E.Z., Hegstrom, R., and Burnell, J.M. (1960) The treatment of chronic uremia by means of intermittent hemodialysis: a preliminary report. Trans. ASAIO. 6, 114–122.
Pendras, J.P., Cole, J.J., Tu, W.H., and Scribner, B.H. (1961). Improved technique of continuous flow hemodialysis. Trans. ASAIO. 7, 27–36.
Murray, J.S., Tu, W.H., Albers, J.B., Burnell, J.M., and Scribner, B.H. (1962). A community hemodialysis center for the treatment of chronic uremia. Trans. ASAIO. 8, 315–319.
Eschback, J.W., Wilson, W.E., Peoples, R.W., Wakefield, A.W., Babb, A.L., and Scribner, B.H. (1966). Unattended overnight home hemodialysis. Trans. ASAIO. 12, 346–356.