Part II: Making Sense of Nonsense
The central dogma of biology is that genetic instructions in DNA are transcribed into messenger RNAs (mRNAs) that deliver the instructions to ribosomes, which then translate that information into the proteins that carry out myriad functions throughout the body. The production of mRNA is a crucial component of gene expression.
In simplest terms, disease is gene expression gone awry, and Lynne Maquat has devoted her career to elucidating one of the most important and incredibly complex aspects of gene expression: quality control.
As in most areas of life, mistakes in cells happen all the time. Humans have 20,000 protein-coding genes but are able to produce many more than 20,000 proteins through alternative pre-mRNA splicing. A single gene can generate multiple proteins by encoding a pre-mRNA from which various exons (including protein-coding regions) can be mixed and matched (alternatively spliced).
This ability to diversify greatly increases the margin of error, but fortunately, the human body has built-in systems to eliminate potentially harmful slip-ups.
Maquat is credited with discovering one of the most prominent of these quality control systems: nonsense-mediated mRNA decay, or NMD.
One common flaw in gene expression is the introduction of an early “stop” signal.
Normally, this stop signal (termination codon) appears at the end of the genetic instructions in mRNA for protein synthesis to indicate that the instructions have been read start-to-finish and that all of the information has been translated into a full-length, functional protein.
Early stop signals, called “premature termination codons” or “nonsense codons,” prevent the genetic instructions in mRNA from being read completely. Consequently, protein synthesis is cut short, resulting in an incomplete or truncated protein that doesn’t function normally, and worse, could be toxic.
Similar to installing a sink with only half of the instructions, or baking a cake with half of a recipe, premature termination codons lead to undesired, and oftentimes dire, results in a cell.
Before Maquat officially arrived on the scientific scene in 1981 with her breakthrough manuscript, “Unstable beta-globin mRNA in mRNA-deficient beta0 thalassemia,” published in Cell, scientists didn’t understand the molecular cause of
this disease.
Heading her own research lab, Maquat then set out to define the mechanism that helps cells detect the difference between a premature stop signal and a normal stop signal, and extended her studies to other diseases. Her studies de-mystified NMD’s crucial role in gene expression, revealing how it works to identify and eliminate mRNAs containing premature termination codons and thereby prevents production of truncated proteins. Her paper also opened the flood- gates for scientists to pursue an entirely new and auspicious field of research into how mRNAs are monitored and regulated, with Maquat leading the way.
“Before Lynne, nobody could come up with a logical explanation for how NMD worked,” says colleague Steitz, who follows Maquat’s work at Yale. “When everyone else abandoned ship, she stuck with it, and slowly made significant advances. It was hard, and at times the answers weren’t coming, but she really wanted a scientific explanation, so she kept at it. I admire her persistence, and her uncanny ability to keep thinking of the next experiment that would lead to answers.”
A Lucky Bout of Food Poisoning
Maquat’s novel understanding of NMD’s role in human cells had its genesis in her 1980 study of bone marrow aspirates from four children in Jerusalem suffering from thalassemia major, the most severe form of thalassemia. Still a young researcher in Jeffrey Ross’ University of Wisconsin lab, Maquat set out to learn why the children’s marrow contained no beta-globin protein.
However, the study almost ended before it even began.
After retrieving and spending days working up the bone marrow samples provided by Israeli hematologist Eliezer Rachmilewitz, MD, Maquat stopped for lunch with a colleague at Israel’s Weizmann Institute of Science in Rehovot on her way to the Ben Gurion airport.
“We ate at an outdoor café in the blazing heat, and my colleague suggested I try the pickles, which were delicious,” she recalls. “However, I realized later that they had probably been marinating in the hot sun for who knows how long.”
On the cab ride to the airport, Maquat’s stomach began to revolt and she fell ill with what was most likely food poisoning.
“I got to the airport, feeling horrible, just wanting desperately to get home, with my precious RNA and DNA samples sealed tightly in a Styrofoam container,” she says. “But after getting my boarding pass, security officers wanted to search the container, which would have been very bad. Everything in a tube looks dangerously foreign to non-scientists, and they would have contaminated my samples.”
Fortunately, just as she was about to be violently sick again, security was momentarily distracted by a scuffle at another checkpoint, and Maquat bolted to the bathroom, container in hand.
“I honestly wasn’t trying to escape them, but I had a real emergency,” she says.
It worked nonetheless. She quietly left the bathroom and proceeded to the gate unnoticed.
“Having food poisoning probably saved me and my samples from being detained,” she says. “The airline was very kind, and let me sit by the bathroom on the flight back to the states. I must have looked a complete wreck. When my Columbia University colleague met me at JFK airport, his first words were, ‘What the hell happened to you?’”
From Little World to Big Impressions
Right before her trip to Jerusalem, Maquat’s 1980 publication in Proceedings of the National Academy of Sciences began turning heads in the scientific community, as it was the first to show that a human disease (beta+ thalassemia) could be due to a pre-mRNA splicing defect.
But her landmark study arose from the series of carefully executed experiments she conducted on the bone marrow samples she retrieved from Jerusalem. Published in Cell in 1981, it was the first demonstration of a human disease (beta0 thalassemia) caused by unstable mRNA.
Maquat’s 1981 discovery marked the beginning of years of investigation to understand the NMD mechanism, and more than 35 years later, insights are still emerging. mRNA surveillance was, and still is, such a notoriously difficult field, beset with such time-consuming, mind-bending questions, that many scientists simply gave up in frustration. But not Maquat.
“She doesn’t like run-of-the-mill projects that aren’t going to add much,” says Reyad Elbarbary, PharmD, PhD, who recently finished his post-doctoral fellowship in Maquat’s Rochester lab, and is now an assistant professor in the Center for Orthopaedic Research and Translational Science at Penn State University College of Medicine. “She gravitates toward the most difficult, ambitious projects that she thinks will contribute new and significant insights to existing knowledge.”
Maquat has not only identified the act of molecular gymnastics that is called NMD, but—much like analyzing a successful football play—she has identified the molecular “players,” and the routes and patterns they need to follow, for NMD to work properly.
Among her most noteworthy contributions is her discovery of the exon-junction complex (EJC), a splicing-dependent “mark” that tags an mRNA so the cell can define which termination codons are premature and should trigger NMD. She defined the 50-55-nucleotide rule, which determines which mRNAs containing premature termination codons are subject to degradation by NMD. She also discovered the “pioneer round of translation,” during which NMD occurs.
Despite what most of her peers now describe as “flawless” experiments, there were doubters along the way, including Maquat herself.
Einstein professor Robert H. Singer, PhD, first met a “ruminating” Maquat more than two decades ago in a café following one of her early presentations on NMD.
“She was nervous about how the presentation went and concerned about whether she’d be taken seriously,” Singer recounts. “She asked me, ‘Do you think people will believe it?’ I told her to keep on going, and fortunately, she did.”
When contradictory data emerged, Maquat pushed forward, evaluating and addressing opposing analyses with thoughtful answers in published reviews. Her science was so precise and her understanding so thorough, it was difficult to argue against her.
“Lynne’s grasp of detail is unparalleled,” says Singer. “The level of specificity that she reaches in her research is beyond what most scientists are capable of doing, or want to do. She drives ideas into the ground to their ultimate conclusion.”
Just as the creativity of many artists and musicians is fueled by past relationships, Maquat says her relentless research focus began in the aftermath of a failed marriage in her early 30s.
“At that time I lived in my own little world,” says Maquat, who conducted much of her early research as the only RNA-centric biologist at Roswell Park Cancer Institute in Buffalo from 1982 to 2000. “I could seek guidance and encouragement from faculty members when I needed it, but other than that I worked in relative isolation with my grad students and post-docs. I didn’t have people questioning me, or telling me ‘no,’ or that an idea I had was crazy. I just put my head down and focused on what I thought was right.”
Only at scientific meetings did she emerge from her bubble to share her findings with the world. It was at those meetings and through peer-reviewed publications that she slowly began to make her mark.
Anita Hopper, PhD, professor of Molecular Genetics at The Ohio State University, recalls a presentation Maquat gave at an RNA Society meeting in Banff, Canada, more than 15 years ago. Maquat presented on the pioneer round of translation, the stage of gene expression where NMD occurs.
“The talk was so beautiful and the experiments so smart, that the room fell silent,” says Hopper. “You could hear a pin drop. She nailed it, and everyone knew she had done something special.”