Research Area: Biochemistry, Biophysics, and Structural Biology

Jarrett Smith receives Hanna Gray Fellowship from HHMI
Greta Friar | Whitehead Institute
September 12, 2018

Cambridge, Mass — Jarrett Smith, postdoctoral researcher in David Bartel’s lab at the Whitehead Institute, has been announced as a recipient of the Howard Hughes Medical Institute (HHMI)’s 2018 Hanna Gray fellowship. The fellowship supports outstanding early career scientists from groups underrepresented in the life sciences. Each of this year’s fifteen awardees will be given up to $1.4 million dollars in funding over the course of their postdoctoral program and beginning of a tenure-track faculty position.

“This program will help us retain the most diverse talent in science,” said HHMI President Erin O’Shea. “We feel it’s critically important in academia to have exceptional people from all walks of life, all cultures, and all backgrounds – people who can inspire the next generation of scientists.”

For Smith, who began his postdoctoral training in the Bartel lab in January, finding out he got the fellowship was a defining moment.

“I’m grateful for the support that the fellowship will provide during the formative years of my career,” Smith says. “This kind of opportunity gives you the confidence to set ambitious research goals and find out what you can accomplish.”

In the Bartel lab, Smith studies how cells respond to stress. When a cell is exposed to environmental stressors such as heat, UV radiation, or viral infection, proteins and RNAs in the cell may clump together into dense aggregates called stress granules. Several diseases are associated with altered stress granule formation, but the exact function of stress granules and their potential role in disease are unknown. Smith is investigating changes in the cell linked to their formation. His findings could shed light on a potential role for stress granules in cancer, viral infection, and neurodegenerative disease.

Growing up, Smith was always interested in science but no one in his family had ever received a PhD, making biology research feel like an unlikely career path for him. Nevertheless, he followed his passion, which led him to a PhD program at the Johns Hopkins University School of Medicine. Despite his strong academic performance, Smith began graduate school with doubts about his ability to become a scientist. His mentors were incredible teachers but their self-assuredness could be intimidating.

“They were absolutely my role models, but I didn’t think of them as having gone through what I was going through. In the first few years, I felt like I had a lot of catching up to do,” Smith said.

Smith says he was frequently inspired and guided by his graduate school mentor, Geraldine Seydoux. Under her tutorship he became more confident in his abilities.

“I try to pick mentors who are the kind of scientist I aspire to be,” Smith said.

With that tenet in mind, he set his sights on David Bartel’s lab for his postdoctoral research. He had heard that Bartel was a great mentor and knew the Bartel lab had expertise in all of the research techniques he wanted to learn. Since arriving at Whitehead Institute, Smith says he has experienced support not only from Bartel, but from the entire lab as well.

“Jarrett’s graduate experience with P granules in nematodes brings much appreciated expertise to our lab, and we are all excited about what he will discover here on stress-granule function,” Bartel says. “Receiving this fellowship is a well-deserved honor, and I am very happy for him.”

Smith noted that he is deeply grateful for the community he’s found at Whitehead Institute. However, he also noted that throughout his scientific career he has typically been the only black person in the room. One of the joys of applying for the fellowship was meeting the rest of the candidates, a diverse and impressive group of scientists, he says. He looks forward to seeing the other fellows again at meetings hosted by the HHMI.

“I’ve never really had a scientific role model that shared those experiences or that I could identify with in that way,” Smith says, but he hopes that future aspiring scientists won’t have to go through the same experience. His brother-in-law recently began an undergraduate major in biology. Smith enjoys being there to answer his questions about school work or life as a researcher.

“I’d never ask him if he thinks of me as a role model,” Smith says, laughing. “But I’m glad that I have the chance to help people who—like I did—might question whether they could be successful in the sciences.” With the support of the fellowship and his lab, and an exciting research question he is eager to tackle, Smith has never been more certain that he belongs right where he is.

A Summer of Science

Victor Rivera-Santana, a chemistry major at the University of Puerto Rico at Mayagüez, visited MIT Biology for 10 weeks to investigate protein form and function.

August 30, 2018

Victor Rivera-Santana grew up on the western edge of Puerto Rico, in what he refers to as an “atmosphere of science.” His mother is a professor of animal science at the University of Puerto Rico at Mayagüez, and in elementary school he would attend her lectures about the effects of environment and hormones on animal behavior. Three years ago, Rivera-Santana enrolled there as an undergraduate, and has been studying chemistry ever since — with the exception of this past summer, when he became a full-fledged member of the MIT Biology Department for 10 weeks during MIT’s Summer Research Program in Biology (MSRP­-Bio).

Rivera-Santana remembers being drawn to basic research because of its simple, pure, and noble nature, stemming from the creativity of the researcher. “Science almost always has an application, so the fact that researchers in basic science are not looking for an application per se doesn’t mean their work won’t have one in the future,” he says. “The researcher fulfills his or her own curiosity, and afterwards someone else can find a way to put that into practice in society.”

A rising senior, Rivera-Santana chose chemistry because he enjoyed analyzing the minute building blocks of life, but wasn’t sure which field he would ultimately pursue. With chemistry, he could engineer the major to encompass biology and physics as well, which would give him “a taste of everything.”

At first he didn’t know what post-graduation life might hold. However, two weeks into the MSRP-Bio program he’d made up his mind: a PhD. “I like the people, I like the passion, and most importantly I like the research — everything is so interesting it’s hard to pick,” he says.

Rivera-Santana applied to MSRP-Bio early last January because he had it on good authority from three independent sources that this was the program for him. First a good friend and former MSRP-Bio student suggested it, then his professor, and finally his father.

He had two main expectations coming in. First, that everyone would be intimidating and aloof. “Boy was I wrong,” he says. “The MIT faculty are really accessible and engage you as a potential researcher. You can stop them as they’re walking down the hall, or ask them questions during the scheduled Q&A sessions.”

Second, he expected everyone would be hardworking, irrespective of their area of focus. “I was very pleased to find that’s the case,” he says. “I have not met one person at MIT who would not go the extra mile to do their job correctly.”

Rivera-Santana worked in Thomas Schwartz’s lab, investigating an aggregate of proteins known as the nuclear pore complex (NPC), which is embedded in the nuclear envelope and controls the passage of proteins, RNAs, and even ribosomal subunits between the cytoplasm and the nucleus. Although the NPC is vital to cellular survival, its structure is not yet fully understood.

The Schwartz lab goes bit by bit, studying each of its components and their interactions with one another. Rivera-Santana concentrated on one NPC protein in particular, Nup93, parsing its role and design. He hopes this work will eventually help scientists understand the complex as a whole, “because as the name says, it’s complex.”

Rivera-Santana studied four different variants of Nup93, working to express each variant by itself in bacteria. Most of his days in lab went by “either very slowly or very quickly.” He would spend the beginning of the week growing the bacteria to express his proteins — a relatively low-key process since the bacteria “essentially take care of themselves.” The latter half of the week, though, when he began the purification process, was more fast-paced. It involved extracting and purifying the proteins from the bacterial cytosol, while at the same time taking steps to prevent the proteins from becoming damaged, such as keeping them at low temperatures by performing the purification steps in the cold room.

“Purifying is the really challenging part, but it’s also the most fun,” Rivera-Santana says. “I had to work at four degrees Celsius, and I’m from Puerto Rico so you can just imagine how I bear the temperature,” he adds.

Looking back, his most exciting summer experience was purifying his first protein. “I just felt this bundle of joy well up inside me,” he says. “When I ran it through the gel to check its identity and I saw that beautiful blob of ‘ink’ that told me I had my protein, I just felt so happy.”

While Rivera-Santana thoroughly enjoyed his experiences in lab, he was also thrilled to meet other budding researchers and explore Boston’s museums and brick buildings. His proudest moment was cooking his first meal for his new MSRP-Bio friends (a classic Puerto Rican dish: rice, beans, chicken, and plantains). He enjoys putting smiles on people’s faces, “especially when those grins are full of food.” After all, MSRP-Bio isn’t just about being at MIT; it’s also about meeting people and being part of the community.

He also learned he could live by himself, thousands of miles from his family. And he’s prepared to do it again next summer, perhaps in the same lab. “I’m definitely considering doing MSRP-Bio again,” he says. “And I’m certainly also thinking about MIT for graduate school.”

Until then, the three things he’ll miss the most — in no particularly order — are his MSRP-Bio cohort, his lab mentor, and the tasty East Coast cherries.

Photo credit: Raleigh McElvery
Exploring cancer metabolism

Matthew Vander Heiden seeks new cancer treatments that exploit tumor cells’ abnormal metabolism.

Anne Trafton | MIT News Office
August 28, 2018

Nearly 100 years ago, the German chemist Otto Warburg discovered that cancer cells metabolize nutrients differently than most normal cells. His discovery launched the field of cancer metabolism research, but interest in this area waned; by the 1970s most cancer scientists had shifted their focus to the genetic mutations that drive cancer development.

In the past decade or so, interest in cancer metabolism has resurged, and the first drugs that target cancer cells’ abnormal metabolism were approved to treat leukemia in 2017.

“Cancer metabolism is a very sophisticated field at this point,” says Matthew Vander Heiden, an associate professor of biology at MIT. “We have a lot better understanding of what nutrients cancer cells use and what determines how those nutrients are used. This has led to different ways to think about drugs.”

Vander Heiden, who is also a member of MIT’s Koch Institute for Integrative Cancer Research, is one of the people responsible for the recent surge in cancer metabolism research. As a graduate student and postdoc, he published some of the first studies of how cancer cells alter their metabolism, and now his lab at MIT is devoted to the topic.

“All of the time that I was in grad school and working as a postdoc, I was never working in a lab that was dedicated to studying metabolism. So my vision, if someone gave me a job, was to set up a lab that could really be built in a way that would allow us to ask questions about metabolism,” he says.

Metabolism and cancer

Vander Heiden grew up in a small town in Wisconsin, and unlike most of his high school classmates, he headed out of state for college, to the University of Chicago. He was interested in science, so decided on a pre-med track. A work-study job in a plant biology lab led him to discover that he also enjoyed doing research.

“At that point I already had this idea I was going to go to medical school, but then the idea of MD/PhD came up, and I ended up going down that path,” Vander Heiden says.

While in the MD/PhD program at the University of Chicago Medical School, he worked in the lab of Craig Thompson, now president of Memorial Sloan Kettering Cancer Center. At that time, Thompson was studying the biochemical regulation of apoptosis, the programmed cell death pathway. For his PhD thesis, Vander Heiden investigated the function of a protein called Bcl-x, which is a regulator of apoptosis found in the membranes of mitochondria — cell organelles responsible for generating energy.

“That project really got me thinking about how the mitochondria work and how metabolism works,” Vander Heiden recalls. “At the time, I came to the realization that we don’t understand cell metabolism anywhere near as well as we thought we did, and someone should really study this.”

After finishing his degrees, he spent five years doing clinical training, then decided to pursue research in cancer metabolism.

“Altered metabolism has been known about in cancer for 100 years, but few people were studying it,” Vander Heiden says. “The challenge was finding a lab that would allow me to study metabolism and cancer, which in 2004-2005 was not such an obvious thing to do.”

He ended up going to Harvard Medical School to work with Lewis Cantley, who studies signaling pathways in cells and was receptive to the idea of exploring cancer metabolism. There, Vander Heiden began studying an enzyme called pyruvate kinase M2 (PKM2), which is involved in regulation of glycolysis, a biochemical process that cells use to break down sugar for energy.

In 2008, Vander Heiden, Cantley, and others at Harvard Medical School reported that when cells shift between normal and Warburg (cancer-associated) metabolism, they start using PKM2 instead of PKM1, the enzyme that adult cells normally use for glycolysis. Cantley and Craig Thompson have since founded a company, Agios Pharmaceuticals, that is developing potential drugs that target PKM2, as well as other molecules involved in cancer metabolism.

While at Harvard, Vander Heiden also worked on a paper that contributed to the eventual development of drugs that target cancer cells with a mutation in the IDH gene. These drugs, the first modern FDA-approved cancer drugs that target metabolism, shut off an alternative pathway used by cancer cells with the IDH mutation.

New drug targets

In 2010, Vander Heiden became one of the first new faculty members hired after the creation of MIT’s Koch Institute, where he set up a lab focused on metabolism, particularly cancer metabolism.

His research has yielded many insights into the abnormal metabolism of cancer cells. In one study, together with other MIT researchers, he found that tumor cells turn on an alternative pathway that allows them to build lipids from the amino acid glutamine instead of the glucose that healthy cells normally use. He also found that altering the behavior of PKM2 to make it act more like PKM1 could stop tumor cell growth.

Studies such as these can offer insights that may help researchers to develop drugs that starve tumor cells of the nutrients they need, offering a new way to fight cancer, Vander Heiden says.

“If one wants to develop drugs that target metabolism, one really needs to focus on the context in which it’s happening, which is the environment of the cell plus the genetics of the cell,” he says. “That is what defines the sensitivity to drugs.”

Ankur Jain

Education

  • PhD, 2013, University of Illinois, Urbana-Champaign
  • BTech, 2007,  Biotechnology and Biochemical Engineering, Indian Institute of Technology Kharagpur

Research Summary

We study how biomolecules in a cell self-organize. In particular, we are interested in understanding how membrane-free cellular compartments such as RNA granules form and function. Our lab develops new biochemical and biophysical techniques to investigate these compartments and to understand their dysfunction in human disease.

Awards

  • Young Alumni Achiever’s Award, Indian Institute of Technology Kharagpur, 2019
  • NIH K99/R00 Pathway to Independence Award, 2017
  • Pew Scholar in the Biomedical Sciences, 2022
A tale of two projects

Graduate student Julie Monda has spent five years investigating two divergent aspects of cell division, revealing some unexpected results and new research questions.

Raleigh McElvery
July 23, 2018

To sixth year graduate student Julie Monda, dividing cells are among the most beautiful things she’s ever seen. Watching the tiny, delicate spheres split into identical versions of themselves also provides her with a visual readout for her experiments — will the process continue if she removes a certain piece of a certain protein? Will the genetic material still distribute equally between the two cells? Which molecules are crucial for cell division, and how are they regulated?

Our cells are constantly dividing in order to grow and repair themselves, although some (like skin cells) do so more often than others, say, in the brain. This process, known as mitosis, is the primary focus of Iain Cheeseman’s lab, situated in the Whitehead Institute for Biomedical Research. Most of the research in the Cheeseman lab involves the kinetochore, a group of proteins located on the chromosome where the arms join. During mitosis, long, fibrous structures, known as microtubules, attach to the kinetochore to pull apart the duplicated chromosomes as the parent cell splits in half, ensuring each daughter cell receives an exact copy of the parent’s genetic blueprint.

Before she arrived at MIT Biology in the fall of 2012, Monda worked as a research technician at St. Jude Children’s Research Hospital in Memphis, Tennessee in the lab of Brenda Schulman PhD ’96 . As she recalls, she always “preferred performing hands-on research techniques at the lab bench over being in a classroom.” So she surprised even herself when she chose MIT’s graduate program in biology precisely because it requires all first-year students to take a full course load their fall semester before beginning lab rotations.

“That structure seemed useful given that I studied biochemistry as an undergraduate at the University of Tulsa, and the degree requirements were weighted more towards chemistry than biology,” she says. “Plus, when you’re only taking classes, you spend more time interacting with your classmates. It creates a close-knit community that extends throughout your entire graduate career and beyond.”

Monda ultimately selected the Cheeseman lab because it married her interests in biochemistry and cell biology.

“The research in this lab focuses on various elements of kinetochore function and cell division, but everyone is generally working on their own distinct questions,” she explains. “I knew I would have an area that was mine to explore. It’s both exciting and challenging because no one else is thinking about your projects to the extent that you are.”

Monda’s story is a tale of two projects: one focused on the interface between the kinetochore and the array of microtubules known as the mitotic “spindle,” and another project that ended up taking both her and the lab in a slightly new direction.

The first, concerning kinetochore-microtubule interactions, represented a collaboration with former lab technician Ian Whitney. For this endeavor, Monda investigated a protein complex called Ska1, found at the outer kinetochore.

The Ska1 complex is located where the kinetochore and microtubule meet. Ska1’s role, Monda explains, is to allow the kinetochores to remain attached to the spindle during chromosome segregation, even as the microtubules that compose the spindle begin to disassemble (as they must do).

“We wanted to know how the kinetochore hangs onto this polymer that is essentially falling apart,” Monda explains. “Long story short, we ended up defining specific surfaces within the Ska1 complex that are important for holding on to the microtubule as it shrinks, and — as we were surprised to note — also as it grows”

Although Ska1 only requires a single point of contact to bind a microtubule, Monda and Whitney pinpointed multiple surfaces on Ska1 that are required to allow it to remain associated with the microtubules as they disassemble and reassemble themselves.

While her Ska1 project was very much in line with the types of questions that the Cheeseman lab traditionally pursues, Monda also worked on another endeavor that “began as a side project and slowly evolved into a more full-time effort.” This project involves a motor protein called dynein, which helps to align the chromosomes and position the spindle during mitosis.

Dynein piqued Monda’s interest because of its role in mitosis, as well as its importance throughout the entire cell cycle. Motor proteins are molecules powered by the release of chemical energy that move along surfaces, sometimes transporting cargo, sometimes performing other essential tasks. Dynein is a motor protein that walks in one direction along microtubules, even when the microtubules latch onto the kinetochore to yank apart the chromosomes during mitosis.

But dynein doesn’t act alone. There are a number of additional proteins that also play a key role in coordinating its activity and localization. Monda is studying two of these accessory regulatory proteins, Nde1 and NdeL1, which bind to dynein and help promote some of its functions. She wanted to understand how Nde1 and NdeL1 interact with dynein to activate it. Although Nde1 and NdeL1 are nearly identical in function, Monda discovered that Nde1 (but not NdeL1) binds to another complex: the 26S proteasome.

The proteasome degrades proteins within the cell, influencing virtually all aspects of cellular function, including DNA synthesis and repair, transcription, translation, and cell signaling. Given its ubiquity, it has remained a point of interest among the scientific community for years. And yet, before Monda’s research, the interaction between Nde1 and the proteasome had apparently gone unnoticed. Researchers have long studied Nde1 in relation to dynein, but it’s possible that the interaction between Nde1 and the proteasome represents a new function for Nde1 unrelated to dynein regulation. In fact, Monda’s finding may have implications for understanding the development of the human brain.

“It’s clear that patients with mutations in Nde1 have much more severe neurodevelopmental defects than scientists would have predicted,” Monda says, “so it’s possible that this new interaction between Nde1 and the proteasome could help to explain why Nde1 is so important in the brain.”

Her most recent results have been published in Molecular Biology of the Cell.

“I’ve found some exciting results over the past few years,” Monda says, “and even though a lot of my research has gone in a direction that’s not strictly mitosis-related, Iain has been great about allowing me to follow the science wherever it leads. We want to know what these proteins are actually doing, both in terms of this new interaction and also more broadly within the cell.”

Monda intends to submit and defend her thesis this summer, and assume a postdoctoral position at the University California, San Diego in the fall. Although she’s been watching cells divide for years now, the process still retains its grandeur.

“Often times biologists investigate questions at scales where we can’t really see what we’re studying as we study it,” she says. “But having this visual readout makes it more tangible; I feel like I can better appreciate what exactly it is that I’m trying to understand, as well as the beauty and complexity of the processes that sustain life.”

Decoding RNA-protein interactions

Scientists leverage one step, unbiased method to characterize the binding preferences of more than 70 human RNA-binding proteins.

Raleigh McElvery
June 7, 2018

Thanks to continued advances in genetic sequencing, scientists have identified virtually every A, T, C, and G nucleotide in our genetic code. But to fully understand how the human genome encodes us, we need to go one step further, mapping the function of each base. That is the goal of the Encyclopedia of DNA Elements (ENCODE) project, funded by the National Human Genome Research Institute and launched on the heels of the Human Genome Project in 2003. Although much has already been accomplished — mapping protein-DNA interactions and the inheritance of different epigenetic states — understanding the function of a DNA sequence also requires deciphering the purpose of the RNAs encoded by it, as well as which proteins bind to those RNAs.

Such RNA-binding proteins (RBPs) regulate gene expression by controlling various post-transcriptional processes — directing where the RNAs go in the cell, how stable they are, and which proteins will be synthesized. Yet these vital RNA-protein relationships remain difficult to catalog, since most of the necessary experiments are arduous to complete and difficult to interpret accurately.

In a new study, a team of MIT biologists and their collaborators describes the binding specificity of 78 human RBPs, using a one-step, unbiased method that efficiently and precisely determines the spectrum of RNA sequences and structures these proteins prefer. Their findings suggest that RBPs don’t just recognize specific RNA segments, but are often influenced by contextual features as well — like the folded structures of the RNA in question, or the nucleotides flanking the RNA-binding sequence.

“RNA is never naked in the cell because there are always proteins binding, guiding, and modifying it,” says Christopher Burge, director of the Computational and Systems Biology PhD Program, professor of biology and biological engineering, extramural member of the Koch Institute for Integrative Cancer Research, associate member of the Broad Institute of MIT and Harvard, and senior author of the study. “If you really want to understand post-transcriptional gene regulation, then you need to characterize those interactions. Here, we take advantage of deep sequencing to give a more nuanced picture of exactly what RNAs the proteins bind and where.”

MIT postdoc Daniel Dominguez, former graduate student Peter Freese, and current graduate student Maria Alexis are the lead authors of the study, which is part of the ENCODE project and appears in Molecular Cell on June 7.

A method for the madness

From the moment an RNA is born, it is coated by RBPs that control nearly every aspect of its lifecycle. RBPs generally contain a binding domain, a three-dimensional folded structure that can attach to a specific nucleotide sequence on the RNA called a motif. Because there are over 1,500 different RBPs found in the human genome, the biologists needed a way to systematically determine which of those proteins bound to which RNA motifs.

After considering a number of different approaches to analyze RNA-protein interactions both directly in the cell (in vivo) and isolated in a test tube (in vitro), the biologists settled on an in vitro method known as RNA Bind-n-Seq (RBNS), developed four years ago by former Burge lab postdoc and co-author Nicole Lambert.

Although Lambert had previously tested only a small subset of proteins, RBNS surpassed other approaches because it was a quantitative method that revealed both low and high affinity RNA-protein interactions, required only a single procedural step, and screened nearly every possible RNA motif. This new study improved the assay’s throughput, systematically exploring the binding specificities of more than 70 human RBPs at a high resolution.

“Even with that initial small sample, it was clear RBNS was the way to go, and over the last three-and-a-half years we’ve been gradually building on this approach,” Dominguez says. “Since a single RBP can select from billions of unique RNA molecules, our approach gives you a lot more power to detect the all those possible targets, taking into account RNA secondary structure and contextual features. It’s an extremely deep and detailed assay.”

First, the researchers purified the human RBPs, mixing them with randomly-generated synthetic RNAs roughly 20 nucleotides long, which represented virtually all the RNAs an RBP could bind to. Next, they extracted the RBPs along with their bound RNAs and sequenced them. With the help of their collaborators from the University of California at San Diego and University of Connecticut Health, the team conducted additional assays to glean what these RNA-protein interactions might look like in an actual cell, and infer the cellular function of the RBPs.

The researchers expected most RBPs to bind to a unique RNA motif, but to their surprise they found the opposite: Many of the proteins, regardless of structural class, seemed to prefer similar short, unfolded nucleotide sequence motifs.

“Human cells express hundreds of thousands of distinct transcripts, so you might think that each RBP would bind a slightly different RNA sequence in order to distinguish between targets,” Alexis says. “In fact, one might assume that having distinct RBP motifs would ensure maximum flexibility. But, as it turns out, nature has built in substantial redundancy; multiple proteins seem to bind the same short, linear sequences.”

Redundant motifs with distinct targets and functions

This overlap in RBP binding preference suggested to the scientists that there must be some other indicator besides the sequence of the motif that signaled RBPs which RNA to target. Those signals, it turned out, stemmed from the spacing of the motifs as well as which nucleotide bases flank its binding sites. For the less common RBPs that targeted non-linear RNA sequences, the precise way the RNA folded also seemed to influence binding specificity.

The obvious question, then, is: Why might RBPs have evolved to rely on contextual features instead of just giving them distinct motifs?

Accessibility seems like one of the more plausible arguments. The researchers reasoned that linear RNA segments are physically easier to reach because they are not obstructed by other RNA strands, and they found that more accessible motifs are more likely to be bound. Another possibility is that having many proteins target the same motif creates some inter-protein competition. If one protein increases RNA stability and another decreases it, whichever binds the strongest will prevent the other from binding at all, enabling more pronounced changes in gene activity between cells or cell states. In other scenarios, proteins with similar functions that target the same motif could provide redundancy to ensure that regulation occurs in the cell.

“It’s definitely a difficult question, and one that we may never truly be able to answer,” Dominguez says. “As RBPs duplicated over evolutionary time, perhaps altering recognition of the contextual features around the RNA motif was easier than changing the entire RNA motif. And that would give new opportunities for RBPs to select different cellular targets.”

This study marks one of the first in vitro contributions to the ENCODE Project. While in vivo assays reveal information specific to the particular cell line or tissue in which they were conducted, RBNS will help define the basic rules of RNA-protein interactions — so fundamental they are likely to apply across many cell types and tissues.

The research was funded by the National Institutes of Health ENCODE Project, an NIH/NIGMS grant, the National Defense Science and Engineering Graduate Fellowship, Kirschstein National Research Service Award, Burroughs Wellcome Postdoctoral Fund, and an NIH Individual Postdoctoral Fellowship.

Network of diverse noncoding RNAs acts in the brain

Scientists identify the first known network consisting of three types of regulatory RNAs.

Nicole Giese Rura | Whitehead Institute
June 7, 2018

Scientists at MIT’s Whitehead Institute have identified a highly conserved network of noncoding RNAs acting in the mammalian brain. While gene regulatory networks are well described, this is the first documented regulatory network comprised of three types of noncoding RNA: microRNA, long noncoding RNA, and circular RNA. The finding, which is described online this week in the journal Cell, expands our understanding of how several noncoding RNAs can interact to regulate each other.

This sophisticated network, which is conserved in placental mammals, intrigued Whitehead Member David Bartel, whose lab identified it.

“It has been quite an adventure to unravel the different elements of this network,” says Bartel, who is also a professor of biology at MIT and investigator with the Howard Hughes Medical Institute. “When we removed the long noncoding RNA, we saw huge increases in the microRNA, which, with the help of a second microRNA turned out to reduce the levels of the circular RNA.”

RNA may be best known for acting as a template during protein production, but most RNA molecules in the cell do not actually code for proteins. Many play fundamental roles in the splicing and translation of protein-coding RNAs, whereas others play regulatory roles. MicroRNAs, as the name would suggest, are small, about 22 nucleotides (nucleotides are the building blocks of RNA); long noncoding RNAs (lncRNAs) are longer than 200 nucleotides; and circular RNAs (circRNAs) are looped RNAs formed by atypical splicing of either lncRNAs or protein-coding RNAs. These three types of noncoding RNAs have been shown previously to be vital for controlling protein-coding gene expression, and in some instances their dysregulation is linked to cancer or other diseases.

Previous work by Bartel and Whitehead member and MIT Professor Hazel Sive identified hundreds of lncRNAs conserved in vertebrate animals, including Cyrano, which contains an unusual binding site for the microRNA miR-7.

In the current research, Ben Kleaveland, a postdoc in Bartel’s lab and first author of the Cell paper, delves into Cyrano’s function in mice. His results are surprising: a regulatory network centered on four noncoding RNAs — a lncRNA, a circRNA, and two microRNAs — acting in mammalian neurons. The network employs multiple interactions between these noncoding RNAs to ultimately ensure that the levels of one microRNA, miR-7, are kept extremely low and the levels of one circRNA, Cdr1as, are kept high.

Several aspects of this highly tuned network are unique. The lncRNA Cyrano targets miR-7 for degradation. Cyrano is exceptionally efficient, and in some cells, reduces miR-7 by an astounding 98 percent — a stronger effect than scientists have ever documented for this phenomenon, called target RNA-directed microRNA degradation. In the described network, unchecked miR-7 indirectly leads to degradation of the circRNA Cdr1as. CircRNAs such as this one are usually highly stable because the RNA degradation machinery needs to latch onto the end of an RNA molecule before the machinery can operate. In the case of Cdr1as, the circRNA contains a prodigious number of sites that can interact with miR-7: 130 in mice and 73 in humans. As these sites are bound by miR-7, another microRNA, miR-671, springs into action and directs slicing of the Cdr1as. This renders Cdr1as vulnerable to degradation.

The network’s precise function still eludes researchers, but evidence suggests that it may be important in brain function. All four components of the network are enriched in the brain, particularly in neurons, and recently, Cdr1as has been reported to influence neuronal activity in mice.

“We’re in the early stages of understanding this network, and there’s so much left to discover,” Kleaveland says. “Our current hypothesis is that Cdr1as is not only regulated by miR-7 but also facilitates miR-7 function by delivering this microRNA to neuronal synapses.”

This work was supported by the National Institutes of Health and the Howard Hughes Medical Institute.

Structure of key growth regulator revealed

Researchers identify the molecular structure of the GATOR1 protein complex, which regulates growth signals in human cells, using cryo-electron microscopy.

Nicole Davis | Whitehead Institute
March 28, 2018

A team of researchers from Whitehead Institute and the Howard Hughes Medical Institute has revealed the structure of a key protein complex in humans that transmits signals about nutrient levels, enabling cells to align their growth with the supply of materials needed to support that growth. This complex, called GATOR1, acts as a kind of on-off switch for the “grow” (or “don’t grow”) signals that flow through a critical cellular growth pathway known as mTORC1.

Despite its importance, GATOR1 bears little similarity to known proteins, leaving major gaps in scientists’ understanding of its molecular structure and function. Now, as described online on March 28 in the journal Nature, Whitehead scientists and their colleagues have generated the first detailed molecular picture of GATOR1, revealing a highly ordered group of proteins and an extremely unusual interaction with its partner, the Rag GTPase.

“If you know something about a protein’s three-dimensional structure, then you can make some informed guesses about how it might work. But GATOR1 has basically been a black box,” says senior author David Sabatini, a member of Whitehead Institute, a professor of biology at MIT, and investigator with the Howard Hughes Medical Institute (HHMI). “Now, for the first time, we have generated high-resolution images of GATOR1 and can begin to dissect how this critical protein complex works.”

GATOR1 was first identified about five years ago. It consists of three protein subunits (Depdc5, Nprl2, and Nprl3), and mutations in these subunits have been associated with human diseases, including cancers and neurological conditions such as epilepsy. However, because of the lack of similarity to other proteins, the majority of the GATOR1 complex is a molecular mystery. “GATOR1 has no well-defined protein domains,” explains Whitehead researcher Kuang Shen, one of the study’s first authors. “So, this complex is really quite special and also very challenging to study.”

Because of the complex’s large size and relative flexibility, GATOR1 cannot be readily crystallized — a necessary step for resolving protein structure through standard, X-ray crystallographic methods. As a result, Shen and Sabatini turned to HHMI’s Zhiheng Yu. Yu and his team specialize in cryo-electron microscopy (cryo-EM), an emerging technique that holds promise for visualizing the molecular structures of large proteins and protein complexes. Importantly, it does not utilize protein crystals. Instead, proteins are rapidly frozen in a thin layer of vitrified ice and then imaged by a beam of fast electrons inside an electron microscope column.

“There have been some major advances in cryo-EM technology over the last decade, and now, it is possible to achieve atomic or near atomic resolution for a variety of proteins,” explains Yu, a senior author of the paper and director of HHMI’s shared, state-of-the-art cryo-EM facility at Janelia Research Campus. Last year’s Nobel Prize in chemistry was awarded to three scientists for their pioneering efforts to develop cryo-EM.

GATOR1 proved to be a tricky subject, even for cryo-EM, and required some trial-and-error on the part of Yu, Shen, and their colleagues to prepare samples that could yield robust structural information. Moreover, the team’s work was made even more difficult by the complex’s unique form. With no inklings of GATOR1’s potential structure, Shen and his colleagues, including co-author Edward Brignole of MIT, had to derive it completely from scratch.

Nevertheless, the Whitehead-HHMI team was able to resolve near-complete structures for GATOR1 as well as for GATOR1 bound to its partner proteins, the Rag GTPases. (Two regions of the subunit Depdc5 are highly flexible and therefore could not be resolved.) From this wealth of new information as well as from the team’s subsequent biochemical analyses, some surprising findings emerged.

First is the remarkable level of organization of GATOR1. The protein is extremely well organized, which is quite unusual for proteins that have no predicted structures. (Such proteins are usually quite disorganized.) In addition, the researchers identified four protein domains that have never before been visualized. These novel motifs — named NTD, SABA, SHEN, and CTD — could provide crucial insights into the inner workings of the GATOR1 complex.

Shen, Sabatini, and their colleagues uncovered another surprise. Unlike other proteins that bind to Rag GTPases, GATOR1 contacts these proteins at at least two distinct sites. Moreover, one of the binding sites serves to inhibit — rather than stimulate — the activity of the Rag GTPase. “This kind of dual binding has never been observed — it is highly unusual,” Shen says. The researchers hypothesize that this feature is one reason why GATOR1 is so large — because it must hold its Rag GTPase at multiple sites, rather than one, as most other proteins of this type do.

Despite these surprises, the researchers acknowledge that their analyses have only begun to scratch the surface of GATOR1 and the mechanisms through which it regulates the mTOR signaling pathway.

“There is much left to discover in this protein,” Sabatini says.

This work was supported by the National Institutes of Health, Department of Defense, National Science Foundation, the Life Sciences Research Foundation, and the Howard Hughes Medical Institute.