Author: Lillian Eden

Alumni News: Mission: Protecting the Planet

MIT Alum Catharine Conley, SB ’88, who earned two bachelor's degrees in biology and the humanities, spent more than a decade as NASA's planetary protection officer, working on protocols to prevent biological contamination on Earth and beyond.

Kathryn M. O'Neill | MIT Technology Review
August 20, 2024

When the space shuttle Columbia disintegrated during reentry in 2003, the disaster killed the human crew of seven—but not every creature onboard.

A collection of roundworms (a.k.a. nematodes) survived and was found in the debris, surprising everyone and prompting Catharine Conley ’88—principal investigator on the experiment—to publish a paper on the implications for astrobiology. It also led Conley to a new NASA role: planetary protection officer.

“Planetary protection is about trying to prevent Earth organisms from getting to other planets and, more importantly, making sure there’s nothing nasty when you bring material back to Earth,” says Conley, who held the job from 2006 to 2017 and helped ensure US compliance with the Outer Space Treaty, the international agreement that governs space exploration.

Conley got an early start on science thanks to a geneticist mother and mathematician father, and then completed two MIT majors—in biology and the humanities, focusing on Russian and French translation—and two bachelor’s degrees. That language study would prove useful: “Translation is essential when communicating with people from very different backgrounds—politicians, managers, bureaucrats, engineers, scientists—so for being planetary protection officer that was probably my most valuable training.”

After earning a PhD in plant sciences from Cornell, Conley studied a protein involved in muscle contraction as a postdoc at the Scripps Research Institute. That work led to NASA, where the Columbia experiment was designed to test the effects of low gravity on nematodes’ muscle tissue (muscle atrophy is a known problem for astronauts).

As the nematodes showed, Earth organisms are hard to kill. So a planetary protection officer must develop protocols not only to prevent biological contamination here but also to ensure that any “alien” life forms discovered elsewhere aren’t actually from Earth. “We have found signs of intelligent life on Mars,” Conley notes wryly. “But it’s us.”

Some scientists theorize that life on Earth actually came from Mars, Conley points out, which would increase the risk of importing something infectious: novel yet related organisms can quickly wreak havoc, as the recent pandemic illustrated.

Conley is currently visiting at the Carnegie Institution for Science, working to develop an analytical framework for assessing whether a space sample is indigenous life, Earth contamination, or just chemistry.

New approach enables a closer look at brain cell organelle

Microglia are involved in brain development, as well as neurodegeneration and brain cancer. A new approach from the Jaenisch Lab allows researchers to isolate and analyze microglia phagosomes.

Greta Friar | Whitehead Institute
August 14, 2024

Microglia are the immune system’s front-line enforcers in the brain. They are cells that patrol the brain and destroy anything harmful that they encounter, from invading bacteria to cellular debris. They also remove plaques and prune dysfunctional synapses between neurons. Microglia eliminate their targets by eating them: they envelope material and seal it in bubble-like organelles called phagosomes. A phagosome can then fuse with other organelles that break down its contents.

Microglial phagosomes play important roles in brain development, brain function and a plethora of brain diseases, including neurodegeneration and brain cancer. Therefore, understanding microglial phagosome biology could help to develop new therapies for currently untreatable brain diseases. However, microglia and their organelles have been difficult to study because existing stem cell and animal models insufficiently resemble microglia in the human brain, and because microglia, as vigilant immune patrollers, react to even subtle stimuli and so experimental conditions can trigger changes in the cells that confound analyses.

To overcome those issues, Whitehead Institute Founding Member Rudolf Jaenisch, also a professor of biology at the Massachusetts Institute of Technology; University of Freiburg Professor of Neuropathology Marco Prinz; and University of Freiburg neuropathologist Emile Wogram, who began this project as a postdoctoral researcher in Jaenisch’s lab, have developed a method to isolate and analyze microglia phagosomes in a rapid, gentle, and unbiased fashion.

In research shared in the journal Immunity on August 15, the researchers describe how they can isolate and profile phagosomes from stem cell-derived microglia and fresh human brain tissue. They also share new insights into phagosome biology in the human brain, regarding synaptic pruning and generation of NAD+, a broadly used molecule in the brain, by microglia.

The method that the researchers developed to isolate phagosomes from cells uses immunoprecipitation, in which antibodies latch on to a specific target protein on an organelle’s surface. When the antibodies are collected, they pull the organelles with them. This technique avoids many chemical perturbations that might alter the microglial profile. Sometimes researchers genetically engineer a target for the antibodies, but in order to isolate phagosomes from human brain tissue, Wogram had to find a naturally expressed target. Eventually, he and colleagues found one: the protein CD68.

The researchers first isolated phagosomes from stem cell-derived microglia. They co-cultured the microglia with other brain cell types to create a more brain-like environment, which led to a better match between brain and stem cell-derived microglia gene expression. They triggered some of the microglia to enter an inflammatory or disease-like state to see how that affected the phagosomes. Additionally, Wogram collaborated with the neurosurgery department at the University of Freiburg to get access to brain tissues immediately after their removal during surgery. He isolated phagosomes from brain tissue within a half hour of its removal, allowing him to profile the organelles before their contents could change much.

The profiles that the researchers built included what proteins and metabolites the phagosomes contained, and the whole-cell gene expression profile. The profiles differed significantly between sets of phagosomes, but the researchers identified a core of consistent proteins, including many known and also some unknown phagosome proteins. The results showed that phagosomes contain sensitive signaling molecules that allow them to react quickly to even subtle environmental stimuli.

Additionally, the protein contents of the co-cultured microglia provided strong evidence that when microglia prune synapses, they predominantly prune the side that sends a signal and not the side that receives one. This insight could be useful for understanding how microglia interact with synapses in health and disease.

The researchers also gained insights into a key metabolic pathway that occurs inside of microglia. In excess, the molecule quinolinic acid can be toxic to neurons; it is implicated as involved in many neurodegenerative diseases. However, cells can use quinolinic acid to make NAD+, a molecule broadly used to carry out essential cellular functions. Microglia are the only brain cells that generate NAD+. Wogram and colleagues found that key steps in this process occur in phagosomes. Phagosomes are therefore necessary both for removing excess quinolinic acid to prevent toxicity and for helping to generate NAD+ in the brain.

Finally, Wogram used brain tissues to compare phagosomes from within a tumor to those in the surrounding healthy tissue. The phagosomes in the tumor contained excess quinolinic acid. Although follow-up studies would be needed to confirm the results, these findings are consistent with research that suggests cancer cells use quinolinic acid to fuel their growth.

Collectively, these findings illuminate aspects of phagosome biology and the roles that phagosomes may play in normal brain development and maintenance, as well as in cancer and neurodegeneration. The researchers also anticipate that their method could prove useful for profiling other organelles, especially when the organelles need to be rapidly isolated from human tissue.

Talented high schoolers excel while they explore the brain

Over six years of operation, pre-college outreach programs administered by Mandana Sassanfar, Senior Lecturer and Director of Diversity and Outreach, have placed seven exceptional pre-college students, often from underserved or underrepresented backgrounds, with research groups in The Picower Institute.

David Orenstein | The Picower Institute for Learning and Memory
August 14, 2024

During the pandemic, when many classes delivered online could barely hold students’ attention, Presley Simelus became captivated by the subject of biology thanks to their boundless curiosity and their uncommonly engaging teacher at Prospect Hill Academy Charter School in Cambridge. Meanwhile for Eli Hanechak, the science bug must have bit her very early. She’s wanted to be a doctor for as long as she can remember and in fifth grade built a model of a space station the size of a car out of duct tape, cardboard and broomsticks.

Not every teenager is expected to want to spend their summer breaks exploring science at a bench in an MIT lab, but each year students like Simelus and Hanechak, who have a distinct passion for research, can bring that to The Picower Institute and other research entities around MIT. Over six years of operation, pre-college outreach programs administered by Mandana Sassanfar, Director of Diversity and Outreach, have placed seven exceptional pre-college students, often from underserved or underrepresented backgrounds, with research groups in The Picower Institute. Despite their relative lack of experience compared to the technicians, graduate students, postdocs and professors around them, the students typically thrive.

“Eli has been a wonderful addition to our lab for the summer,” said Kendyll Burnell, the graduate student in the lab of Professor Elly Nedivi who has been working closely with Hanechak. “She is a hard worker, has caught on to techniques quickly, and is constantly asking excellent questions about science and doing research.”

Simelus, too, has been not only learning but also contributing, said their summer host, Yire Jeong, a postdoc in the lab of Associate Professor Gloria Choi.

“Presley has been amazing in our lab, and I was impressed by Presley’s eagerness to learn so much about neuroscience,” Jeong said. “Even when facing technical difficulties, Presley diligently worked to overcome them and achieved meaningful results.”

‘Dive into it’

Simelus, who hails from Everett, Mass., and will be enrolling in Swarthmore College this fall to study biochemistry, first came to MIT through the Leah Knox Scholars Program. Friends who’d been in the program before encouraged them to apply and they got in. During five weeks last summer Simelus and their cohort of fellow Leah Knox high-schoolers had the geeky pleasure of extracting bacteria out of the Charles River and performing a battery of tests to genetically characterize the novel organisms they found. Sassanfar noted that Simelus did the lab work exceptionally well, which is something she looks for when determining whom she might invite back the next summer to do research in an MIT Brain and Cognitive Sciences or Biology lab.

This spring when it came time for Simelus to decide where they might like to take that opportunity, they chose the Choi lab, which studies how the central nervous systems and immune systems interact, sometimes with consequences relevant to disorders including autism. Those keywords intrigued Simelus but really they made the choice because of the potential to learn something entirely new.

“It was all this stuff I just simply wasn’t familiar with and I wanted to learn more about it,” Simelus said. “With Gloria’s lab I was truly mystified and I wanted to dive into it. That’s the reason I chose it.”

This summer Simelus has been working with Jeong on a study of how brain cell activity differs when mice are sick vs. when they are well. The project has involved imaging neurons in the brain to detect telltale signs of recent activation, expression of a protein called c-fos. Learning about neuroscience and gaining skills like preparing, staining and imaging tissue have been a very fulfilling outcome of the internship, Simelus said.

“I truly have learned so much about neuroscience,” they said. “I feel like the field, anything related to the brain or neuroscience, is always under this sort of veil and nobody really knows what’s going on. But I feel like my time at the Choi lab has really allowed me to see what neuroscience is about. It’s taught be more about the brain itself and also more about different biology techniques and skills I might need.”

Now the only problem, Simelus said, is that there are even more things to be deeply curious about. Simelus feels committed to harnessing the life sciences in some way in the future to sustain human life and experience. And as someone who not only plays the viola but also composes, they’ve begun thinking more about how the brain responds to music.

There will no doubt be many chances to continue exploring these interests at Swarthmore, but during the summer at MIT, Simelus said they’ve expanded their horizons while still hanging out with friends, some of whom have been working in other nearby labs.

“I don’t think I would have changed my summer,” Simelus said.

‘The perfect opportunity’

Hanechak lives in the tiny Western Massachusetts town of Russell (population: 1,643) and commutes 45 minutes to Pope Francis Preparatory School in Springfield, where she is a rising senior.

In her freshman year at a different school, she yearned for an extra challenge so she got involved in science fair. Interested in medicine, but eager for a project in which she could make a difference without having clinical credentials, she chose to work on reducing pollution by developing a microbe-derived enzyme that could biodegrade plastics. She had read about such enzymes in the research literature and learned that they don’t work as well as engineers have hoped. In successive years she has scrounged lab space and general supervision in labs at Westfield State University and UMass Amherst to create and screen beneficial mutations in the enzyme and to synthesize structures that might help the enzyme work better. The enzyme she presented at the International Science and Engineering Fair last year can degrade plastics in 24 hours.

Sasssanfar, who also directs the Massachusetts Junior Academy of Science (MassJAS), learned of Hanechak’s award-winning science fair presentation and invited her to present at the MassJAS symposium, held at MIT last October. Hanechak did so well, Sassanfar said, she earned a spot present at the American Junior Academy of Science meeting (adjacent to the American Association for the Advancement of Science Annual Meeting) in Denver in February. She also earned Sassanfar’s invitation to join a lab this summer at MIT.

Hanechak has long had an MIT pennant on her wall at home and has admired MIT as a place where regardless of one’s background, if one has a passion for science and technology, that’s what matters.

“No one in my family has gone to college and no one has been involved in a science-related career of any kind,” she said. “One of the reasons MIT has always stood out to me is that there are especially great minds here, but they didn’t all come from established families or super prestigious backgrounds or anything like that. They kind of just were able to make their own way.”

Moreover, the chance to come to MIT to learn about the brain in the Nedivi lab seemed like a great step to take toward that longer-term goal of medicine.

“It seemed like the perfect opportunity to start transitioning into what I want my career to look like and to get some experience doing neuroscience research,” Hanechak said. “I’m very glad I’m able to have this summer experience, like learning the techniques. When I go into my college major of neuroscience, I will have a good background of what I’m doing, besides just my environmental research.”

With Burnell, Hanechak is working on finding a DNA promoter specific for a rare but interesting kind of neuron in the visual cortex, where the brain processes what the eyes see. Finding this genetic signature would allow the lab to label these cells and image them under the microscope, so that they could see how the cells contribute to visual processing.

Hanechak acknowledged she was anxious at first about joining a bigger lab with scientists who have much more experience.

“But my entire summer has been incredibly gratifying and exciting—just being able to work in Cambridge, and live in this area, and experience city life, and then also be in a lab environment where it’s so collaborative and everyone’s very friendly,” she said.

For many teens, summer provides a chance to do what they want to do. Simelus and Hanechak chose the opportunity to explore the brain at The Picower Institute and have made the most of it.

Two Whitehead Institute graduate researchers awarded the 2024 Regeneron Prize for Creative Innovation

Whitehead Institute graduate student researchers Christopher Giuliano (Lourido Lab) and Julian Roessler (Hrvatin Lab) have been awarded the 2024 Regeneron Prize for Creative Innovation.

Merrill Meadow | Whitehead Institute
July 30, 2024

Whitehead Institute graduate student researchers Christopher Giuliano and Julian Roessler have been awarded the 2024 Regeneron Prize for Creative Innovation. In addition, postdoctoral researcher Chen Weng was selected as a finalist in the postdoctoral fellows competition.

The Regeneron Prize, sponsored by global biotechnology company Regeneron Pharmaceuticals, Inc., is a competitive award designed to recognize and honor exceptional talent and originality in biomedical research. Individual graduate students and postdoctoral fellows in the biomedical sciences are nominated by the nation’s top research universities. Then, nominees outline their “Dream Projects” — potentially groundbreaking research projects that they would pursue given unrestricted access to resources and state-of-the-art technology.

The “Dream Project” proposals, presented by the nominees to a selection committee comprised of Regeneron’s leading scientists, are used to evaluate a trainee’s scientific merit, elegance, precision, and creativity. Novel research ideas and out-of-the-box thinking is encouraged — although the proposal must include a strong rationale, basic methodology and design for the project, and a discussion of how its results could advance the field. Both Giuliano and Roessler have been awarded $50,000 for their proposals, which can be used in any way the winners choose. In addition, Weng was awarded $5,000 as a finalist, and Regeneron has made a $10,000 grant to the Whitehead Institute as the home institute of the winners to support its seminar series.

This year’s awards are distinctive in that the two winners are from the same institution: Both Giuliano and Roessler are pursuing their PhDs at Massachusetts Institute of Technology (MIT) and conducting their doctoral research at Whitehead Institute.

Giuliano is a researcher in the lab of Whitehead Institute Member Sebastian Lourido, who is also an associate professor of biology at MIT and holds the Landon Clay Career Development Chair at Whitehead Institute. Giuliano’s Dream Project seeks to address the unique challenges posed by genetically based muscle disorders. “An obstacle in using current gene therapies to treat these conditions,” he explains, “is that muscle tissue comprises large syncytial cells, which contain hundreds of nuclei in a shared cytoplasm. Even when a gene therapy is able to reach an individual muscle cell, it often isn’t able to spread to every nucleus within that cell.” However, certain parasites, like Toxoplasma gondii, thrive because they have the capacity to successfully gain access to and manipulate muscle cells. T. gondii, the primary focus of the Lourido lab’s work, may infect nearly one third of all humans. “My project,” Giuliano says, “would identify the specific biological mechanisms used by the parasites to spread their virulence factor proteins throughout the cell. Using genetic screens for protein spread, we would work toward applying these protein features to improve the efficiency of muscle-directed gene therapies, and ultimately test our system in a mouse model of Duchenne muscular dystrophy.”

Roessler is a researcher in the lab of Whitehead Institute Member Siniša Hrvatin, who is also an assistant professor of biology at MIT. While Roessler’s doctoral research focuses on the neuronal circuitry underlying torpor and hibernation in small mammals, his Dream Project seeks to identify the sensory circuitry regulating the “diving reflex” displayed in land- and sea-dwelling mammals, including humans. The diving reflex occurs when an animal’s face is immersed in cold water, prompting an array of organs to reduce their function in ways that, scientists believe, privileges the flow of oxygen to the brain and muscles. “That this reflex has been conserved across millions of years of mammalian evolution suggests an extraordinary genetic advantage,” Roessler says. “Yet, researchers have given comparatively little attention to the neuronal circuits underlying this reflex, and we don’t understand even the fundamental mechanisms by which the nervous system coincidently detects both cold temperature and the presence of water.” Beyond elucidating a foundational aspect of mammalian biology, Roessler’s projects could, if pursued, underpin new interventions for conditions ranging from migraine headaches to cardiac arrhythmia that might be ameliorated by artificial stimulation or inhibition of the diving response.

Weng is a postdoctoral researcher in the lab of Whitehead Institute Member Jonathan Weissman, who is also a professor of biology at MIT, the Landon T. Clay Professor of Biology at Whitehead Institute, and an Investigator of the Howard Hughes Medical Institute. His Dream Project — which proposes a new approach to using single-cell genealogy to understand factors driving cell line evolution — is an extension of his current work. Indeed, this past year he co-developed a technology that details the family trees of human blood cells and provides new insights into the differences between lineages of hematopoietic stem cells. The technology gives researchers unprecedented access to any human cells’ histories — and a path to resolving previously unanswerable questions.

In immune cells, X marks the spot(s)

By researching the effects of sex chromosomes on two types of immune cells, researchers in the Page Lab explore the biological underpinnings of sex biases in immunity and autoimmune disease

Greta Friar | Whitehead Institute
August 6, 2024

There are many known sex differences in health and disease: cases in which either men or women are more likely to get a disease, experience a symptom, or have a certain drug side effect. Some of these sex differences are caused by social and environmental factors: for example, when men smoked more than women, men were more likely to develop lung cancer. However, some have biological underpinnings. For example, men are more likely to be red-green colorblind because the relevant gene is on the X chromosome, of which men with XY chromosomes have no backup copy for a dysfunctional version.

Often, the specific factors contributing to a sex difference are hard to tease apart; there may not be a simple way to tell what is caused by sex chromosomes versus sex hormones versus environment. To address this question, researchers in Whitehead Institute Member David Page’s lab previously developed an approach to identify the contributions of the sex chromosomes to sex differences. Now, Page and former postdoc in his lab Laura Blanton have built on that work by measuring the effects of the sex chromosomes on two types of immune cells. The work, published in the journal Cell Genomics on August 6, shows that sex chromosome gene expression is consistent across cell types, but that its effects are cell type specific.

Sex differences are common in the function and dysfunction of our immune system. Examples include the typically weaker male immune response to pathogens and vaccines, and the female-biased frequency of autoimmune diseases. Page and Blanton’s work in immune cells examines several genes that have been implicated in such sex differences.

Developing a method to measure sex chromosome influence

The approach that the researchers used is based on several facts about sex chromosomes. Firstly, although females typically have two X chromosomes and males typically have one X and one Y, there are people with rare combinations of sex chromosomes, who have anywhere from 1-5 X chromosomes and 0-4 Y chromosomes. Secondly, there are two types of X chromosome: The active X chromosome (Xa) and the inactive X chromosome (Xi). They are genetically identical, but many of the genes on Xi are either switched off or have their expression level dialed way down.

Xa does not really function as a sex chromosome since everyone in the world has exactly one Xa regardless of their sex. In people with more than one X chromosome, any additional X chromosomes are always Xi. Furthermore, Page and Blanton’s research demonstrates that Xa responds to gene expression by Xi and Y—the sex chromosomes—in the same manner as do the other 22 pairs of non-sex chromosomes—the autosomes.

With these facts in mind, the researchers collected cells from donors with different combinations of sex chromosomes. Then they measured the expression of every gene in these cells, across the donor population, and observed how the expression of each gene changed with the addition of each Xi or Y chromosome.

This approach was first shared in a Cell Genomics paper by Page and former postdoc Adrianna San Roman in 2023. They had cultured two types of cells, fibroblasts and lymphoblastoid cell lines, from donor tissue samples. They found that the effects of Xi and Y were modular—each additional chromosome changed gene expression by about the same amount. This approach allowed the researchers to identify which genes are sensitive to regulation by the sex chromosomes, and to measure the strength of the effect for each responsive gene.

In that and a following paper, Page and San Roman looked at how Xi and Y affect gene expression from Xa and the autosomes. Blanton expanded the study of Xi and Y by using the same approach in two types of immune cells, monocytes and CD4+ T cells, taken directly from donors’ blood. Studying cells taken directly from the body, rather than cells cultured in the lab, enabled the researchers to confirm that their observations applied in both conditions.

In all three papers, the researchers found that the sex chromosomes have significant effects on the expression levels of many genes that are active throughout the body. They also identified a particular pair of genes as driving much of this effect in all four cell types. The genes, ZFX and ZFY, found on the X and Y chromosomes respectively, are transcription factors that can dial up the expression of other genes. The pair originates from the same ancestral gene, and although they have grown slightly apart since the X and Y chromosomes diverged, they still perform the same gene regulatory function. The researchers found that they tended to affect expression of the same gene targets by similar though not identical amounts.

In other words, the presence of either sex chromosome causes roughly the same effect on expression of autosomal and Xa genes. This similarity makes sense: carefully calibrated gene regulation is necessary in every body, and so each sex chromosome must maintain that function. It does, however, make it harder to spot the cases in which sex chromosomes contribute to sex differences in health and disease.

“Sex differences in health and disease could stem from the rare instances in which one gene responds very differently to Xi versus Y—we found cases where that occurs,” Blanton says. “They could also stem from subtle differences in the gene expression changes caused by Xi and Y that build up into larger effects downstream.”

Blanton then combined her and San Roman’s data in order to look at how the effects of sex chromosome dosage—how many Xs or Ys are in a cell—compared across all four cell types.

The effects of sex chromosomes on immune cells

 Blanton found that gene expression from the sex chromosomes was consistent across all four cell types. The exceptions to this rule were always X chromosome genes that are only expressed on Xa, and so could be regulated by Xi and Y in the way that autosomal genes are. This contrasts with speculation that different genes on Xi might be silenced in different cells.

However, each cell type had a distinct response to this identical sex chromosome gene expression. Different biological pathways were affected, or the same biological pathway could be affected in the opposite direction. Key immune cell processes affected by sex chromosome dosage in either monocytes or T cells included production of immune system proteins, signaling, and inflammatory response.

The cell type specific responses were due to different genes responding to the sex chromosomes in each cell type. The researchers do not yet know the mechanism causing the same gene to respond to sex chromosome dosage in one cell type but not another. One possibility is that access to the genes is blocked in some of the cell types. Regions of DNA can become tightly packed so that a gene, or a DNA region that regulates the gene, becomes inaccessible to transcription factors such as ZFX and ZFY, and so they cannot affect the gene’s expression. Another possibility is that the genes might require specific partner molecules in order for their expression level to increase, and that these partners may be present in one cell type but not the other.

Blanton also measured how X chromosome dosage affected T cells in their inactive state, when there is no perceived immune threat, versus their activated state, when they begin to produce an immune response and replicate themselves. Increases in X chromosome dosage led to heightened activation, with increased expression of genes related to proliferation. This finding highlights the importance of looking at how sex chromosomes affect not just different cell types, but cells in different states or scenarios.

“As we learn what pathways the sex chromosomes influence in each cell type, we can begin to make sense of the contributions of the sex chromosomes to each cell type’s functions and its roles in disease,” Blanton says.

Although Page and Blanton found that the presence of an Xi or Y chromosome had very similar effects on most genes, the researchers did identify one interesting case in which response to X and Y differed. FCG2RB is a gene involved in immunity that has been implicated in and thought to contribute to the female bias in developing systemic lupus erythematosus (SLE). Blanton found that unlike most genes, FCGR2B is sensitive to X and not Y chromosome dosage. This strengthens the case that higher expression of FCGR2B could be driving the SLE female bias.

“FCGR2B provides a promising opportunity to study the contributions of the sex chromosomes to a sex bias in disease, and to learn more about the biology of a chronic disease that affects many people around the world,” Page says.

In other cases, the researchers found that genes which have been suspected to contribute to female bias in disease did not have a strong response to X chromosome dosage. For example, TLR7 is thought to contribute to female bias in developing autoimmunity, and CD40LG is thought to contribute to female bias in developing lupus. Neither of the genes showed increased expression as X chromosome dosage increased. This suggests that other mechanisms may be driving the sex bias in these cases.

Because of the limited pool of donors, the researchers were not able to identify every gene that responds to sex chromosome dosage, and future research may uncover more sex-chromosome-sensitive genes of interest. Meanwhile, the Page lab continues to investigate the sex chromosomes’ shared role as regulators of gene expression throughout the body.

“We’ve got to recalibrate our thinking from the view that X and Y are mainly involved in differentiating males and females, to understanding that they also have largely shared functions that are important throughout the body,” Page says. “At the same time, I think that uncovering the biology of Xi is going to be incredibly important for understanding women’s health and sex differences in health and disease.”

News Brief: Lamason Lab uncovers seven novel effectors in Rickettsia parkeri infection

The enemy within: new research reveals insights into the arsenal Rickettsia parkeri uses against its host

Lillian Eden | Department of Biology
July 29, 2024

Identifying secreted proteins is critical to understanding how obligately intracellular pathogens hijack host machinery during infection, but identifying them is akin to finding a needle in a haystack.

For then-graduate student Allen Sanderlin, PhD ’24, the first indication that a risky, unlikely project might work was cyan, tic tac-shaped structures seen through a microscope — proof that his bacterial pathogen of interest was labeling its own proteins.  

Sanderlin, a member of the Lamason Lab in the Department of Biology at MIT, studies Rickettsia parkeri, a less virulent relative of the bacterial pathogen that causes Rocky Mountain Spotted Fever, a sometimes severe tickborne illness. No vaccine exists and definitive tests to diagnose an infection by Rickettsia are limited.

Rickettsia species are tricky to work with because they are obligately intracellular pathogens whose entire life cycles occur exclusively inside cells. Many approaches that have advanced our understanding of other bacterial infections and how those pathogens interact with their host aren’t applicable to Rickettsia because they can’t be grown on a plate in a lab setting. 

In a paper recently published in Nature Communications, the Lamason Lab outlines an approach for labeling and isolating R. parkeri proteins released during infection. This research reveals seven previously unknown secreted factors, known as effectors, more than doubling the number of known effectors in R. parkeri. 

Better-studied bacteria are known to hijack the host’s machinery via dozens or hundreds of secreted effectors, whose roles include manipulating the host cell to make it more susceptible to infection. However, finding those effectors in the soup of all other materials within the host cell is akin to looking for a needle in a haystack, with an added twist that researchers aren’t even sure what those needles look like for Rickettsia.  

Approaches that worked to identify the six previously known secreted effectors are limited in their scope. For example, some were found by comparing pathogenic Rickettsia to nonpathogenic strains of the bacteria, or by searching for proteins with domains that overlap with effectors from better-studied bacteria. Predictive modeling, however, relies on proteins being evolutionarily conserved. 

“Time and time again, we keep finding that Rickettsia are just weird — or, at least, weird compared to our understanding of other bacteria,” says Sanderlin, the paper’s first author. “This labeling tool allows us to answer some really exciting questions about rickettsial biology that weren’t possible before.”

The cyan tic tacs

To selectively label R. parkeri proteins, Sanderlin used a method called cell-selective bioorthogonal non-canonical amino acid tagging. BONCAT was first described in research from the Tirrell Lab at Caltech. The Lamason Lab, however, is the first group to use the tool successfully in an obligate intracellular bacterial pathogen; the thrilling moment when Sanderlin saw cyan tic-tac shapes indicated successfully labeling only the pathogen, not the host. 

Sanderlin next used an approach called selective lysis, carefully breaking open the host cell while leaving the pathogen, filled with labeled proteins, intact. This allowed him to extract proteins that R. parkeri had released into its host because the only labeled proteins amid other host cell material were effectors the pathogen had secreted. 

Sanderlin had successfully isolated and identified seven needles in the haystack, effectors never before identified in Rickettsia biology. The novel secreted rickettsial factors are dubbed SrfA, SrfB, SrfC, SrfD, SrfE, SrfF, and SrfG. 

“Every grad student wants to be able to name something,” Sanderlin says. “The most exciting — but frustrating — thing was that these proteins don’t look like anything we’ve seen before.”

Special delivery

Theoretically, Sanderlin says, once the effectors are secreted, they work independently from the bacteria — a driver delivering a pizza does not need to check back in with the store at every merge or turn.

Since SrfA-G didn’t resemble other known effectors or host proteins the pathogen could be mimicking during infection, Sanderlin then tried to answer some basic questions about their behavior. Where the effectors localize, meaning where in the cell they go, could hint at their purpose and what further experiments could be used to investigate it. 

To determine where the effectors were going, Sanderlin added the effectors he’d found to uninfected cells by introducing DNA that caused human cell lines to express those proteins. The experiment succeeded: he discovered that different Srfs went to different places throughout the host cells.  

SrfF and SrfG are found throughout the cytoplasm, whereas SrfB localizes to the mitochondria. That was especially intriguing because its structure is not predicted to interact with or find its way to the mitochondria, and the organelle appears unchanged despite the presence of the effector. 

Further, SrfC and SrfD found their way to the endoplasmic reticulum. The ER would be especially useful for a pathogen to appropriate, given that it is a dynamic organelle present throughout the cell and has many essential roles, including synthesizing proteins and metabolizing lipids. 

Aside from where effectors localize, knowing what they may interact with is critical. Sanderlin showed that SrfD interacts with Sec61, a protein complex that delivers proteins across the ER membrane. In keeping with the theme of the novelty of Sanderlin’s findings, SrfD does not resemble any proteins known to interact with the ER or Sec61. 

With this tool, Sanderlin identified novel proteins whose binding partners and role during infection can now be studied further. 

“These results are exciting but tantalizing,” Sanderlin says. “What Rickettsia secrete — the effectors, what they are, and what they do is, by and large, still a black box.” 

There are very likely other effectors in the proverbial cellular haystack. Sanderlin found that SrfA-G are not found in every species of Rickettsia, and his experiments were solely conducted with Rickettsia at late stages of infection — earlier windows of time may make use of different effectors. This research was also carried out in human cell lines, so there may be an entirely separate repertoire of effectors in ticks, which are responsible for spreading the pathogen.

Expanding Tool Development

Becky Lamason, the senior author of the Nature Communications paper, noted that this tool is one of a few avenues the lab is exploring to investigate R. parkeri, including a paper in the Journal of Bacteriology on conditional genetic manipulation. Characterizing how the pathogen behaves with or without a particular effector is leaps and bounds ahead of where the field was just a few years ago when Sanderlin was Lamason’s first graduate student to join the lab.

“What I always hoped for in the lab is to push the technology, but also get to the biology. These are two of what will hopefully be a suite of ways to attack this problem of understanding how these bacteria rewire and manipulate the host cell,” Lamason says. “We’re excited, but we’ve only scratched the surface.”

Unusual Labmates: Meet tardigrades, the crafters of nature’s ultimate survival kit

Whitehead Institute Member Siniša Hrvatin is studying tardigrades to decode the mechanisms enabling their survival in extreme environmental conditions. Learn about the biology of these microscopic “water bears” and what makes them a particularly fascinating model organism.

Shafaq Zia | Whitehead Institute
July 23, 2024

Tardigrades, also affectionately known as “water bears” or “moss piglets”, are remarkable microscopic organisms that have captured the imagination of scientists and nature enthusiasts alike.

With adults measuring anywhere from 0.2 to 1.2 millimeters in length — as big as a grain of salt — tardigrades possess the astounding ability to survive harsh environmental conditions. These resilient creatures have been found in habitats ranging from the depths of oceans and hot radioactive springs to the frigid expanses of Antarctica. It is their unparalleled adaptability that makes them invaluable as a model organism for researchers like Whitehead Institute Member Siniša Hrvatin, who’s studying physiological adaptation in animals with a focus on states that can slow down tissue damage, disease progression, and even aging.

Follow along to learn what’s behind tardigrades’ nearly indestructible nature, how researchers at Whitehead Institute — and beyond — are studying them, and what insights this work can offer into long-term organ preservation, space exploration, and more.

Big discovery of a tiny creature

In 1773, German naturalist Johann August Ephraim Goeze was analyzing moss samples under a microscope when he stumbled upon an unusual creature. Captivated by its peculiar appearance, he continued his observations and documented the discovery of Kleiner Wasserbär, translating to “little water bear”, in his publication. This work also featured the first-ever drawing of a tardigrade.

Since then, researchers’ understanding of this remarkable organism has evolved alongside advancements in imaging technology. Today, tardigrades are recognized as bilaterally symmetrical invertebrates with two eyes and eight chubby legs adorned with hook-like claws. Often described as a mix between nematodes and insects, these extremophiles are able to withstand freezing, intense radiation, vacuum of outer space, desiccation, chemical treatments, and possibly more.

And the best part? Despite their otherworldly appearance and surprising capabilities, tardigrades share plenty of similarities with larger, more complex organisms, including possessing a primordial brain, muscles, and even a digestive system.

The biology of an extremophile

Researchers trace the evolutionary origins of tardigrades back to panarthropods, a group that includes now-extinct worm-like organisms called lobopodians. To date, over a thousand species of tardigrades have been identified, with terrestrial species inhabiting environments like moss, leaf litter, and lichen, grassland, and deserts while aquatic ones are found in both fresh and saltwater.

Little is known about tardigrades’ diet but researchers are particularly drawn to herbivorous ones that like to munch on single-celled algae and thrive in water. There’s good reason for it: algae are inexpensive to grow in the lab with just light and basic nutrients. But it’s not just their diet that makes tardigrades an attractive model organism — they also have a short generation time (11 to 14 days), with eggs hatching within a four-day span. In fact, some species are able to reproduce without sexual reproduction through a process called parthenogenesis, during which the female egg undergoes cell division without fertilization by a male gamete.

Although genomic resources for studying tardigrades are limited to only a few species, researchers from Keio University and University of Edinburgh have successfully sequenced the genome of a moss-residing tardigrade commonly used in research called Hypsibius exemplaris. Its genome is less than half the size of a Drosophila melanogaster genome, consisting of 105 million base pairs that serve as the building blocks of DNA.

In spite of their small genome — and only a few thousand cells in the body — tardigrades have a well-defined miniaturized body plan, consisting of a head and four segments, that holds valuable insights for researchers looking to decode their adaptation prowess.

Inside tardigrade research at Whitehead Institute

In 2022, as Hrvatin was setting up his lab at Whitehead Institute, a question lingered in his mind. “I was trying to find animals that can survive being frozen for long periods of time and then continue living,” he says. “But there are not that many that fit the bill.”

Then, an undergraduate student at Massachusetts Institute of Technology (MIT) expressed her enthusiasm for astrobiology — the study of life across the universe — and highlighted tardigrades as a favorite among space researchers. Hrvatin was intrigued.

Up until this point, his research had centered upon two states of dormancy, or reduced metabolic activity, in animals: hibernation and a shorter, less intense torpor. But tardigrades possessed a survival mechanism unlike any other. When faced with harsh conditions like dehydration, they would expel water, retract their head and legs, and curl up in a small, dry ball, entering a state of suspended animation called crytobiosis or tun formation.

For decades, researchers hypothesized that the tun state might be responsible for tardigrades’ unparalleled ability to withstand a myriad of environmental assaults, including extremely low temperature. However, recent work has revealed that these animals utilize a separate and unique adaptation, distinct from the tun state, to survive being frozen for extended periods. In fact, preliminary evidence from a preprint by a team of scientists at UC Berkeley and UC San Francisco illustrates unique patterns of how tardigrades survive freezing while hydrated in water.

This phenomenon is markedly different from hibernation and its cousin torpor. “Unlike animals lowering their body temperature, we’re talking about putting tardigrades at minus 180 degrees Celsius, and then thawing them,” says Hrvatin. In fact, cryobiosis is so intense that tardigrades’ metabolic activity drops to undetectable levels, rendering them virtually, but not quite, dead. The organisms can then remain in this state from months to years, only to revive as healthy when conditions become favorable once again.

Frozen in time

In 2014, a group of Japanese researchers at Tokyo’s National Institute for Polar Research undertook an intriguing experiment. They began by thawing moss samples collected from East Antarctica in November 1983. Then, they carefully teased apart each sample using tweezers to retrieve tardigrades that might be nestled within. Among the tardigrades the researchers found, two stood out: Sleeping Beauty 1 and Sleeping Beauty 2 who were believed to be undergoing cold induced-dormancy. Turns out, the researchers were right — within the first day of being placed in the Petri dish with water, the tardigrades began exhibiting slow movements despite having been frozen for over 30 years.

The Swiss army knife in tardigrades’ toolbox

Yet, the remarkable resilience of tardigrades continues to baffle scientists. Recently, they’ve uncovered what could be another potential weapon in the creatures’ arsenal: intrinsically disordered proteins or IDPs. Picture them as putty — a group of proteins that do not have a well-defined three-dimensional structure and can interact with other molecules to produce a range of different outcomes. Some researchers have linked these tardigrade-specific IDPs to the animals extraordinary resilience: under extreme heat, these proteins remain stable. And when desiccated, they form protective glasses that shield cells and vital enzymes from dehydration.

If confirmed, the implications of this work would extend beyond tardigrades’ survival, potentially revolutionizing dry vaccine storage and the development of drought-resistant crops.

Mary Gehring named 2024 HHMI investigator

The Gehring Lab studies plant epigenetics — the heritable information that influences cellular function but is not encoded in the DNA sequence itself.

Merrill Meadow | Whitehead Institute
July 23, 2024

Whitehead Institute Member Mary Gehring has been selected as an Investigator of the Howard Hughes Medical Institute (HHMI), one of just 26 scientists appointed in 2024. Considered one of the most prestigious positions in biomedical research, HHMI Investigators receive substantial direct support over a renewable seven-year term.

Gehring, who is also a professor of biology at Massachusetts Institute of Technology (MIT) and the David Baltimore Chair in Biomedical Research at Whitehead Institute, is a widely respected plant biologist who studies how plant epigenetics modulate plant growth and development. Her long-term goal is to uncover the essential genetic and epigenetic elements of plant seed biology, providing the scientific foundations for engineering alternative modes of seed development and improving plant resiliency.

“I’m pleased that HHMI has been expanding its support for plant biology, and gratified that our lab will benefit from its generous support,” Gehring says. “The appointment gives us the freedom to step back, take a fresh look at the scientific opportunities before us, and pursue the ones that most interest us. And that’s a very exciting prospect.”

Whitehead Institute Director Ruth Lehmann —  a previous HHMI Investigator herself — says, “Mary is an extraordinary scientist. This appointment will help fuel her continuing discoveries, which advance the field of plant biology and hold great promise to impact discovery broadly.”

In practical terms, the appointment will provide the Gehring lab with new, unrestricted funds. “Because we can count on those funds for an extended period, we will be able to pursue a range of opportunities,” Gehring explains. “The new resources will enable us to add researchers and technological capacities, to expand on existing projects, and to explore areas that are new for the lab such as synthetic biology.”

At the same time, Gehring notes, “ I’m very much looking forward to becoming a member of the HHMI community and building connections with this amazing group of scientists.”

With Gehring’s appointment, six Whitehead Institute Members are current HHMI Investigators: David Bartel, David Page, Peter Reddien, Jonathan Weissman, and Yukiko Yamashita.

Gene-Wei Li named 2024 HHMI Investigator

HHMI award will help Department of Biology faculty unravel the mysteries of precision gene expression across the proteome

Noah Daly | Department of Biology
July 23, 2024

To better understand how cells precisely control the levels of their proteins, Associate Professor Gene-Wei Li utilizes rigorous quantitative analysis to improve our molecular understandings of life. With the support he’ll receive as an HHMI Investigator, Li will explore how genomes are sculpted to allow lifeforms to survive in a competitive environment.

As versatile and durable as cells are, their every function depends on producing precise quantities of proteins. These proteins enable the cells to perform their functions, their organelles to work, and tell the cells when to grow or decompose. Without precise instructions for how much protein they need to generate, organisms would struggle to self-regulate efficiently, rendering them incapable of becoming competitive life forms. These “recipes” for protein production are written into the genetic code of all life. Recent advances in DNA sequencing have identified every protein an organism can produce–every “ingredient” in the genetic cookbook. Despite these significant advances, researchers still don’t know how to read the instructions. 

Since opening his lab at MIT in 2015, Associate Professor of Biology Gene-Wei Li has been working, among other things, on quantifying the amount of proteins cells produce and how that process is orchestrated within the cell. 

“The goal that we hope to achieve,” Li says, “is to read the genomic sequence and accurately tell you not just what types of proteins are made, but also how many of them will be made.” 

Li was recently named a 2024 Howard Hughes Medical Institute Investigator, one of 26 newly appointed Investigators hailing from 19 institutions. Each HHMI Investigator will receive roughly $11 million in support over a seven-year term, potentially renewable indefinitely. This support includes their full salary and benefits, a generous research budget, scientific equipment, and additional resources. 

“I feel grateful for the extremely supportive environment in my department,” Li says. “This award is also a recognition for the hard work and risk-taking by my lab’s current and past trainees.” 

Other MIT School of Science faculty joining the 2024 cohort include Mary Gehring, Professor of Biology and Core Member and David Baltimore Chair in Biomedical Research at the Whitehead Institute; Steven Flavell, Associate Professor of Brain and Cognitive Sciences and Investigator in the Picower Institute for Learning and Memory; and Mehrdad Jazayeri, Professor of Brain and Cognitive Sciences at the McGovern Institute.  

Of the nearly 1,000 researchers who applied to be HHMI investigators this year, successful applicants were selected for their singular accomplishments in scientific research. They receive extensive resources to continue their work at their home institution. HHMI enables scientists to pursue their work with extraordinary freedom, allowing them to expand their current efforts, pivot focus as needed, and execute original ideas. 

One of the hallmarks of Li’s lab is the devoted attention he gives to his students. Each member of the lab receives extensive guidance and mentorship, enabling them to pursue careers in science while sharing their ideas and concerns with fellow lab members and Li. For this inclusive culture, Li was honored by MIT as “Committed to Caring” for 2020-2021. 

“When scientists create environments in which others can thrive, we all benefit,” says HHMI President Erin O’Shea. “These newest HHMI Investigators are extraordinary, not only because of their outstanding research endeavors but also because they mentor and empower the next generation of scientists to work alongside them at the cutting edge.”  

In his lab, Li has emphasized the interweaving of individual achievement and the success of the group, creating a space for lab members to learn from one another, freely question their principal investigator, and ultimately make breakthroughs together. 

Discovery through Collaboration 

While Li’s lab was built around the question of quantifying a cell’s protein synthesis–that is, the amounts of all the proteins produced in a cell—his background is in physics. He approaches his work by making quantitative and systematic measurements (mainly with high-throughput DNA sequencing tools) and using that information to uncover fundamental molecular mechanisms in gene expression. 

The Li lab’s early work utilizing this methodology demonstrated that proteins that go on to form complexes are made in the correct ratios to immediately form complexes with few extra copies. 

Li’s team went on to discover that metabolic proteins are synthesized at precise ratios that are conserved across evolutionarily distant species, such as the two bacterial model organisms E. coli and B. subtilis. However, despite their shared output of protein production, the billions of years of evolution gave rise to two completely different ways to control protein quantity. 

In 2020, this line of research produced a study that contradicted the longstanding dogma of molecular biology that the machinery of protein synthesis and RNA polymerase work side by side in bacteria, which it does in E coli. 

According to Li, two of his graduate student researchers found that, in B. subtilis, the ribosome lags far behind RNA polymerase, a process the lab termed “runaway transcription.” They found that the coordination between transcription and translation is fundamentally different between E. coli and B. subtilis. They then identified bioinformatic signatures, revealing that this kind of uncoupling between transcription and translation is widespread across many species of bacteria. The students, Grace Johnson, a former graduate student in the Department of Biology, and Jean-Benoît Lalanne, a former graduate student in the Department of Physics, were the lead authors of the paper, which appeared in Nature. 

 “This is very exciting stuff, but all the credit goes to my grad students,” Li chuckles. 

Finding the Room to Be Bold 

The support from Howard Hughes Medical Institute enables Li and his team the flexibility to pursue the basic research that leads to discoveries. 

“Having this award really allows us to be bold and to do things at a scale that wasn’t possible before. The discovery of runaway transcription is a great example of this,” Li says.  

Li plans on using the funds made available from HHMI to help determine how functionally related genes differ in their expression and how signals are encoded in the genome at the DNA and RNA levels. According to Li, the collection of high-quality and system-wide data is essential to making discoveries in his field. 

“I’m incredibly grateful to HHMI for encouraging us to pursue this work and follow the science wherever it leads us,” he says. 

Li and his team are as eager as ever to understand life’s coded cookbook. 

“The work of science begins with great people,” Li says. “This award will help ensure our lab continues to be a place where incredible young scientists can work together to achieve miraculous things.” 

Gene silencing tool has a need for speed

Small changes in the molecular machines that carry out RNA interference can lead to big differences in the efficacy of gene silencing. These new findings from the Bartel Lab have implications for the design of gene-silencing therapeutics.

Greta Friar | Whitehead Institute
July 17, 2024

RNA interference (RNAi) is a process that many organisms, including humans, use to decrease the activity of target RNAs in cells by triggering their degradation or slicing them in half. If the target is a messenger RNA, the intermediary between gene and protein, then RNAi can decrease or completely silence expression of the gene. Researchers figured out how to tailor RNAi to target different RNAs, and since then it has been used as a research tool to silence genes of interest. RNAi is also used in a growing number of therapeutics to silence genes that contribute to disease.

However, researchers still do not understand some of the biochemistry underlying RNAi. Slight differences in the design of the RNAi machinery can lead to big differences in how effective it is at decreasing gene expression. Through trial and error, researchers have worked out guidelines for making the most effective RNAi tools without understanding exactly why they work. However, Whitehead Institute Member David Bartel and graduate student in his lab Peter Wang have now dug deeper to figure out the mechanics of the main cellular machine involved in RNAi. The researchers’ findings, shared in Molecular Cell on July 17, not only provide explanations for some of the known rules for RNAi tool design, but also provide new insights that could improve future designs.

Slicing speed is highly variable

The cellular machine that carries out RNAi has two main parts. One is a guide RNA, a tiny RNA typically only 22 bases or nucleotides long. RNA, like DNA, is made of four possible bases, although RNA has the base uracil (U) instead of the DNA base thymine (T). RNA bases bind to each other in certain pairings—guanines (G) pair to cytosines (C) and adenines (A) pair to U’s—and the sequence of bases in the guide RNA corresponds to a complementary sequence within the target RNA. When the guide RNA comes across a target, the corresponding bases pair up, binding the RNAs. Then the other part of the RNAi machine, an Argonaute protein bound to the guide RNA, can slice the target RNA in half or trigger the cell to break it down more gradually.

In humans, AGO2 is the Argonaute protein that is best at slicing. Only a couple dozen RNA targets actually get sliced, but these few targets play essential roles in processes such as neuron signal control and accurate body shape formation. Slicing is also important for RNAi tools and therapeutics.

In order for AGO2 to slice its target, the target must be in the exact right position. As the guide and target RNAs bind together, they go through a series of motions to ultimately form a double helix. Only in that configuration can AGO2 slice the target.

Researchers had assumed that AGO2 slices through different target RNAs at roughly the same rate, because most research into this process used the same few guide RNAs. These guide RNAs happen to have similar features, and so similar slicing kinetics—but they turn out not to be representative of most guide RNAs.

Wang paired AGO2 with a larger variety of guide RNAs and measured the rate at which each AGO2-guide RNA complex sliced its targets. He found big differences. Whereas the commonly used guide RNAs might differ in their slicing rate by 2-fold, the larger pool of guide RNAs differed by as much as 250-fold. The slicing rates were often much slower than the researchers expected. Previously, researchers thought that all targets could be sliced relatively quickly, so the rate wasn’t considered as a limiting factor – other parts of the process were thought to determine the overall pace – but Wang found that slicing can sometimes be the slowest step.

“The important consideration is whether the slicing rate is faster or slower than other processes in the cell,” Wang says. “We found that for many guide RNAs, the slicing rate was the limiting factor. As such, it impacted the efficacy.”

The slower AGO2 is to slice targets, the more messenger RNAs will remain intact to be made into protein, meaning that the corresponding gene will continue being expressed. The researchers observed this in action: the guide RNAs with slower slicing rates decreased target gene expression by less than the faster ones.

Small changes lead to big differences in slicing rate

Next, the researchers explored what could be causing such big differences in slicing rate between guide RNAs. They mutated guide RNAs to swap out single bases along the guide RNA’s sequence—say, switching the 10th base in the sequence from a C to an A—and measured how this changed the slicing rate.

“The important consideration is whether the slicing rate is faster or slower than other processes in the cell,” Wang says. “We found that for many guide RNAs, the slicing rate was the limiting factor. As such, it impacted the efficacy.”

The researchers found that slicing rate increased when the base at position 7 was an A or a U. The bases A and U pair more weakly than C and G. The researchers found that having a weak A-U pair at that position, or a fully mismatched pair at position 6 or 7, may allow a kink to form in the double helix shape that actually makes the target easier to slice. Wang also found that slicing rate increases with certain substitutions at the 10th and the 17th base positions, although the researchers could not yet determine possible underlying mechanisms.

These observations correspond to existing recommendations for RNAi design, such as not using a G at position 7. The new work demonstrates that the reason these recommendations work is because they affect the slicing rate, and, in the case of position 7, the new work further identifies the specific mechanism at play.

Interplay between regions matters

People designing synthetic guide RNAs thought that the bases at the tail end, past the 16th position, were not very important. This is because in the case of the most commonly used guide RNAs, the target will be rapidly cleaved even if all of the tail end positions are mismatches that cannot pair.

However, Wang and Bartel found that the identity of the tail end bases are only irrelevant in a specific scenario that happens to be true of the most commonly used guide RNAs: when the bases in the center of the guide RNA (positions 9-12) are strong-pairing Cs and Gs. When the center pairings are weak, then the tail end bases need to be perfect matches to the target RNA. The researchers found that guide RNAs could have up to a 600-fold difference in tolerance for tail end mismatches based on the strength of their central pairings.

The reason for this difference has to do with the final set of motions that the two RNAs must perform in order to assume their final double helix shape. A perfectly paired tail end makes it easier for the RNAs to complete these motions. However, a strong enough center can pull the RNAs into the double helix even if the tail ends are not ideally suited for doing so.

The observation that weak central pairing requires perfect or near perfect tail end matches could provide a useful new guideline for designing synthetic RNAs. Any guide RNA runs the risk of sometimes binding other messenger RNAs that are similar enough to the intended target RNA. In the case of a therapy, this off-target binding can lead to negative side effects. Bartel and Wang suggest that researchers could design guide RNAs with weak centers, which would require more perfect pairing in the tail end, so that the guide RNA will be less likely to bind non-target RNAs; only the perfect pairing of the target’s RNA sequence would suffice.

Altogether, Wang and Bartel’s findings explain how small differences between guide RNAs can make such large differences in the efficacy of RNAi, providing a rationale for the long-standing RNAi design guidelines. Some of the findings even suggest new guidelines that could help with future synthetic guide RNA designs.

“Discovering the interplay between the center and tail end of the guide RNA was unexpected and satisfying,” says Bartel, who is also a professor at the Massachusetts Institute of Technology and a Howard Hughes Medical Investigator. “It explains why, even though the guidelines suggested that tail-end sequence doesn’t matter, the target RNAs that are sliced in our cells do have pairing to the tail end. This observation could prove useful to reduce off-target effects in RNAi therapeutics.”