Research Area: Genetics

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.”

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.

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.”

“Vaults” within germ cells offer more than safekeeping

Ribonucleoprotein (RNP) granules are believed to preserve maternal mRNA within eggs and developing embryos. The Lehman Lab reveals that a specific type of RNP granule also plays an active role in translating the mRNA that is crucial for specifying germ cells.

Shafaq Zia | Whitehead Institute
July 2, 2024

Maternal messenger RNAs (mRNAs), located within the cytoplasm of an immature egg, are crucial for jump starting development. Following fertilization, these mRNAs are passed onto the zygote, the first newly formed cell. Having been read from the maternal DNA genetic code, they serve as the sole templates for protein production essential for early development until the zygote’s own genes become active and take over.

Many maternal mRNAs are stored in ribonucleoprotein (RNP) granules, which are a type of membrane-less compartments, or condensates, within eggs and developing embryos. These granules are believed to preserve the mRNA in a “paused” state until the encoded proteins are needed for specific developmental processes upon fertilization of the egg cell. Then, certain developmental signals kick in to instruct the RNP granules to release the stored mRNA so the instructions can be translated into a functional protein.

One type of RNP granules called germ granules is found in embryo germplasm, a cytoplasmic region that gives rise to germ cells, which become the eggs or sperms of adult flies. Whitehead Institute Director Ruth Lehmann studies how germ cells form and transmit their genetic information across generations. Her lab is particularly interested in understanding how germ granules in embryos localize and regulate maternal mRNAs.

Now, Lehmann, along with graduate student Ruoyu Chen and colleagues, has uncovered that the role of germ granules in fruit flies (Drosophila melanogaster) extends beyond safeguarding maternal mRNAs. Their findings, published in the journal Nature Cell Biology on July 4, demonstrate that germ granules also play an active role in translating, or making into protein, a specific maternal mRNA, called nanos, crucial for specifying germ cells and the abdomen of the organism.

“Traditionally, scientists have thought of RNP granules as a dead zone for translation,” says Chen. “But through high-resolution imaging, we’ve challenged this notion and shown that the surface of these granules is actually a platform for translation of nanos mRNA.”

RNP granules act as vaults

Within a developing embryo, various fate-determining proteins dictate whether a cell will become a muscle, nerve, or skin cell in a fully-formed body. Nanos, a gene with conserved function in Drosophila and humans, guides the production of Nanos protein which instructs cells to develop into germline. Mutations in the nanos gene cause sterility in animals.

During early embryonic development, Nanos protein also helps establish the body plan of the fruit fly embryo — it specifies the posterior end or abdominal region, and guides the ordered development of tissues along the length of the body, from head to tail. In embryos with impaired Nanos function, the consequences are fatal.

“When Nanos protein isn’t functioning properly, the fruit fly embryos are really short,” says Chen. “This is because the embryo has no abdomen, which is basically half of its body. Nanos also has a second function that is conserved from flies to humans. This function is very local and instructs the cells with lots of Nanos to become germ cells. ”

Given Nanos’ vital role, embryos must safeguard instructions for its production until the embryo reaches a specific stage of development, when it is time to define the posterior region. Previous work has indicated that germ granules in the germplasm and germ cells can act like vaults, shielding the nanos mRNA from degradation or premature translation.

However, while the mRNA instructions for building the protein are distributed throughout the embryo, Nanos protein is found only in regions where germ granules reside. The mRNA does not get translated elsewhere in the embryo because of a regulatory protein called Smaug, named after the golden dragon depicted in J. R. R. Tolkien’s 1937 novel The Hobbit. Smaug binds to a non-protein coding segment of the mRNA known as the 3’ untranslated region (3’ UTR), extending beyond the protein-coding sequence, effectively suppressing the translation process.

For Lehmann, Chen, and their colleagues, this hinted at an intriguing relationship between nanos mRNA and germ granules. Are the granules essential for translating nanos mRNA into a functional protein? And if they are, is their role primarily to serve as a safekeeping place to evade repression by Smaug or do they actively facilitate the translation of nanos mRNA too?

To answer these questions, the researchers combined high-resolution imaging with a technique called the SunTag system to directly visualize the translation of nanos mRNA within Drosophila germ granules at the single-molecule level.

Unlike green fluorescent protein tagging, where a single fluorescent molecule is used, the SunTag system allows scientists to recruit multiple GFP copies for an amplified signal. First, a small protein tag, known as the SunTag, is fused with the protein-producing region of the nanos mRNA. As the mRNA instructions undergo translation, GFP molecules stick to the newly synthesized SunTag-Nanos protein, resulting in a bright fluorescent signal. Overlying this translation signal with fluorescent probes specifically labeling the mRNA then allows researchers to precisely visualize and track when and where the translation process is taking place.

“Using this system, we’ve discovered that when nanos mRNA is translated, it protrudes slightly from the surface of the granules like snakes peeking out of a box,” says Chen. “But they can’t fully emerge; a part of their sequence, specifically their “back” end, the 3’ UTR, remains tucked inside the granules. When the RNA is not translated, like during oogenesis, the tip coils back and is hidden inside the granule.”

With their high-resolution SunTag imaging technique, Lehmann, Chen and their colleagues have directly added to the work of other researchers with similar observations: mRNAs in the process of translation are in an extended configuration, while the 5’UTR curls back to the 3’UTR when the mRNAs are repressed.

Flipping on nanos translation

Then, the researchers went on to take a closer look at how these granules help initiate translation, while Smaug is able to inhibit the same nanos mRNA molecules from being translated in other areas of the embryo. They hypothesized that the untranslated region (UTR) of nanos mRNA, which remains concealed within the granules, might be playing a pivotal role in the translation process by localizing the mRNA instructions within germ cell granules. This localization, they speculated, protects the mRNA from Smaug’s inhibitory actions and facilitates Nanos protein production, so the posterior region can develop properly.

However, counter-intuitive to a simple protection model, they found that rather than being depleted, Smaug is enriched within germ granules, indicating that additional mechanisms within the RNP granule must counteract Smaug’s inhibitory effects. To explore this, the researchers turned to another regulatory protein called Oskar, which is known to interact with Smaug.

Discovered by Lehmann in a 1986 study, and named after a character in the German novel The Tin Drum, the oskar gene in Drosophila is known to help with the development of the posterior region. Later research has revealed that, during the development of oocytes, Oskar acts as a scaffold protein by initiating the formation of germ granules in germ cells and directing mRNA molecules, including nanos, towards the granules.

To gain a deeper understanding of Oskar’s full role in translational regulation in germ granules and its interaction with Smaug, the researchers engineered a modified version of Oskar protein. This altered Oskar protein retained its ability to initiate the formation of germ granules and localize nanos mRNA within them. However, Smaug no longer localized to the germ granules assembled by this altered Oskar.

The researchers then studied whether the mutant protein had any effect on nanos mRNA translation. In the germ cells with this mutant version of Oskar, the researchers saw a significant reduction in the translation of nanos mRNA. These findings, combined, suggested that Oskar regulates nanos translation in fruit fly embryos by recruiting Smaug to the granules and then counteracting its repression of translation.

“Condensates composed of RNAs and proteins are found in the cytoplasm of pretty much every cell and are thought to mediate mRNA storage or transport,” says Lehmann, who is also a professor of biology at the Massachusetts Institute of Technology. “But our results provide new insights into condensate biology by suggesting that condensates can be also used to specifically translate stored mRNAs.”

Indeed, in the oocyte, the germ granules are silent and only become activated when the egg is fertilized.

“This suggests that there might also be other ‘on and off switches’ governing translation within condensates during early development,” adds Lehmann. “How this is achieved and whether we could engineer this to happen at will in these and other granules is a question for the future.”

Taking RNAi from interesting science to impactful new treatments

Alnylam Pharmaceuticals is translating the promise of RNA interference (RNAi) research into a new class of powerful, gene-based therapies. These days Alnylam is not the only company developing RNAi-based medicines, but it is still a pioneer in the field. The company’s founders — MIT Institute Professor Phil Sharp, Professor David Bartel, Professor Emeritus Paul Schimmel, and former MIT postdocs Thomas Tuschl and Phillip Zamore — see Alnylam as a champion for the field more broadly.

Zach Winn | MIT News
May 13, 2024

There are many hurdles to clear before a research discovery becomes a life-changing treatment for patients. That’s especially true when the treatments being developed represent an entirely new class of medicines. But overcoming those obstacles can revolutionize our ability to treat diseases.

Few companies exemplify that process better than Alnylam Pharmaceuticals. Alnylam was founded by a group of MIT-affiliated researchers who believed in the promise of a technology — RNA interference, or RNAi.

The researchers had done foundational work to understand how RNAi, which is a naturally occurring process, works to silence genes through the degradation of messenger RNA. But it was their decision to found Alnylam in 2002 that attracted the funding and expertise necessary to turn their discoveries into a new class of medicines. Since that decision, Alnylam has made remarkable progress taking RNAi from an interesting scientific discovery to an impactful new treatment pathway.

Today Alnylam has five medicines approved by the U.S. Food and Drug Administration (one Alnylam-discovered RNAi therapeutic is licensed to Novartis) and a rapidly expanding clinical pipeline. The company’s approved medicines are for debilitating, sometimes fatal conditions that many patients have grappled with for decades with few other options.

The company estimates its treatments helped more than 5,000 patients in 2023 alone. Behind that number are patient stories that illustrate how Alnylam has changed lives. A mother of three says Alnylam’s treatments helped her take back control of her life after being bed-ridden with attacks associated with the rare genetic disease acute intermittent porphyria (AIP). Another patient reported that one of the company’s treatments helped her attend her daughter’s wedding. A third patient, who had left college due to frequent AIP attacks, was able to return to school.

These days Alnylam is not the only company developing RNAi-based medicines. But it is still a pioneer in the field, and the company’s founders — MIT Institute Professor Phil Sharp, Professor David Bartel, Professor Emeritus Paul Schimmel, and former MIT postdocs Thomas Tuschl and Phillip Zamore — see Alnylam as a champion for the field more broadly.

“Alnylam has published more than 250 scientific papers over 20 years,” says Sharp, who currently serves as chair of Alnylam’s scientific advisory board. “Not only did we do the science, not only did we translate it to benefit patients, but we also described every step. We established this as a modality to treat patients, and I’m very proud of that record.”

Pioneering RNAi development

MIT’s involvement in RNAi dates back to its discovery. Before Andrew Fire PhD ’83 shared a Nobel Prize for the discovery of RNAi in 1998, he worked on understanding how DNA was transcribed into RNA, as a graduate student in Sharp’s lab.

After leaving MIT, Fire and collaborators showed that double-stranded RNA could be used to silence specific genes in worms. But the biochemical mechanisms that allowed double-stranded RNA to work were unknown until MIT professors Sharp, Bartel, and Ruth Lehmann, along with Zamore and Tuschl, published foundational papers explaining the process. The researchers developed a system for studying RNAi and showed how RNAi can be controlled using different genetic sequences. Soon after Tuschl left MIT, he showed that a similar process could also be used to silence specific genes in human cells, opening up a new frontier in studying genes and ultimately treating diseases.

“Tom showed you could synthesize these small RNAs, transfect them into cells, and get a very specific knockdown of the gene that corresponded to that the small RNAs,” Bartel explains. “That discovery transformed biological research. The ability to specifically knockdown a mammalian gene was huge. You could suddenly study the function of any gene you were interested in by knocking it down and seeing what happens. … The research community immediately started using that approach to study the function of their favorite genes in mammalian cells.”

Beyond illuminating gene function, another application came to mind.

“Because almost all diseases are related to genes, could we take these small RNAs and silence genes to treat patients?” Sharp remembers wondering.

To answer the question, the researchers founded Alnylam in 2002. (They recruited Schimmel, a biotech veteran, around the same time.) But there was a lot of work to be done before the technology could be tried in patients. The main challenge was getting RNAi into the cytoplasm of the patients’ cells.

“Through work in Dave Bartel and Phil Sharp’s lab, among others, it became evident that to make RNAi into therapies, there were three problems to solve: delivery, delivery, and delivery,” says Alnylam Chief Scientific Officer Kevin Fitzgerald, who has been with the company since 2005.

Early on, Alnylam collaborated with MIT drug delivery expert and Institute Professor Bob Langer. Eventually, Alnylam developed the first lipid nanoparticles (LNPs) that could be used to encase RNA and deliver it into patient cells. LNPs were later used in the mRNA vaccines for Covid-19.

“Alnylam has invested over 20 years and more than $4 billion in RNAi to develop these new therapeutics,” Sharp says. “That is the means by which innovations can be translated to the benefit of society.”

From scientific breakthrough to patient bedside

Alnylam received its first FDA approval in 2018 for treatment of the polyneuropathy of hereditary transthyretin-mediated amyloidosis, a rare and fatal disease. It doubled as the first RNAi therapeutic to reach the market and the first drug approved to treat that condition in the United States.

“What I keep in mind is, at the end of the day for certain patients, two months is everything,” Fitzgerald says. “The diseases that we’re trying to treat progress month by month, day by day, and patients can get to a point where nothing is helping them. If you can move their disease by a stage, that’s huge.”

Since that first treatment, Alnylam has updated its RNAi delivery system — including by conjugating small interfering RNAs to molecules that help them gain entry to cells — and earned approvals to treat other rare genetic diseases along with high cholesterol (the treatment licensed to Novartis). All of those treatments primarily work by silencing genes that encode for the production of proteins in the liver, which has proven to be the easiest place to deliver RNAi molecules. But Alnylam’s team is confident they can deliver RNAi to other areas of the body, which would unlock a new world of treatment possibilities. The company has reported promising early results in the central nervous system and says a phase one study last year was the first RNAi therapeutic to demonstrate gene silencing in the human brain.

“There’s a lot of work being done at Alnylam and other companies to deliver these RNAis to other tissues: muscles, immune cells, lung cells, etc.,” Sharp says. “But to me the most interesting application is delivery to the brain. We think we have a therapeutic modality that can very specifically control the activity of certain genes in the nervous system. I think that’s extraordinarily important, for diseases from Alzheimer’s to schizophrenia and depression.”

The central nervous system work is particularly significant for Fitzgerald, who watched his father struggle with Parkinson’s.

“Our goal is to be in every organ in the human body, and then combinations of organs, and then combinations of targets within individual organs, and then combinations of targets within multi-organs,” Fitzgerald says. “We’re really at the very beginning of what this technology is going do for human health.”

It’s an exciting time for the RNAi scientific community, including many who continue to study it at MIT. Still, Alnylam will need to continue executing in its drug development efforts to deliver on that promise and help an expanding pool of patients.

“I think this is a real frontier,” Sharp says. “There’s major therapeutic need, and I think this technology could have a huge impact. But we have to prove it. That’s why Alnylam exists: to pursue new science that unlocks new possibilities and discover if they can be made to work. That, of course, also why MIT is here: to improve lives.”

Q&A: Pulin Li on recreating development in the lab

In the whirlwind of activity that occurs simultaneously in a developing embryo, it can be difficult for scientists to pinpoint critical moments of a particular trait. In this Q&A, Pulin Li discusses how her lab ventures beyond mere observation to actually engineer developmental events in a petri dish, and why this approach is vital for understanding health and disease more broadly.

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Scientists develop a rapid gene-editing screen to find effects of cancer mutations

With the new technique, MIT researchers hope to identify mutations that could be targeted with new cancer therapies.

Anne Trafton | MIT News
March 12, 2024

Tumors can carry mutations in hundreds of different genes, and each of those genes may be mutated in different ways — some mutations simply replace one DNA nucleotide with another, while others insert or delete larger sections of DNA.

Until now, there has been no way to quickly and easily screen each of those mutations in their natural setting to see what role they may play in the development, progression, and treatment response of a tumor. Using a variant of CRISPR genome-editing known as prime editing, MIT researchers have now come up with a way to screen those mutations much more easily.

The researchers demonstrated their technique by screening cells with more than 1,000 different mutations of the tumor suppressor gene p53, all of which have been seen in cancer patients. This method, which is easier and faster than any existing approach, and edits the genome rather than introducing an artificial version of the mutant gene, revealed that some p53 mutations are more harmful than previously thought.

This technique could also be applied to many other cancer genes, the researchers say, and could eventually be used for precision medicine, to determine how an individual patient’s tumor will respond to a particular treatment.

“In one experiment, you can generate thousands of genotypes that are seen in cancer patients, and immediately test whether one or more of those genotypes are sensitive or resistant to any type of therapy that you’re interested in using,” says Francisco Sanchez-Rivera, an MIT assistant professor of biology, a member of the Koch Institute for Integrative Cancer Research, and the senior author of the study.

MIT graduate student Samuel Gould is the lead author of the paper, which appears today in Nature Biotechnology.

Editing cells

The new technique builds on research that Sanchez-Rivera began 10 years ago as an MIT graduate student. At that time, working with Tyler Jacks, the David H. Koch Professor of Biology, and then-postdoc Thales Papagiannakopoulos, Sanchez-Rivera developed a way to use CRISPR genome-editing to introduce into mice genetic mutations linked to lung cancer.

In that study, the researchers showed that they could delete genes that are often lost in lung tumor cells, and the resulting tumors were similar to naturally arising tumors with those mutations. However, this technique did not allow for the creation of point mutations (substitutions of one nucleotide for another) or insertions.

“While some cancer patients have deletions in certain genes, the vast majority of mutations that cancer patients have in their tumors also include point mutations or small insertions,” Sanchez-Rivera says.

Since then, David Liu, a professor in the Harvard University Department of Chemistry and Chemical Biology and a core institute member of the Broad Institute, has developed new CRISPR-based genome editing technologies that can generate additional types of mutations more easily. With base editing, developed in 2016, researchers can engineer point mutations, but not all possible point mutations. In 2019, Liu, who is also an author of the Nature Biotechnology study, developed a technique called prime editing, which enables any kind of point mutation to be introduced, as well as insertions and deletions.

“Prime editing in theory solves one of the major challenges with earlier forms of CRISPR-based editing, which is that it allows you to engineer virtually any type of mutation,” Sanchez-Rivera says.

When they began working on this project, Sanchez-Rivera and Gould calculated that if performed successfully, prime editing could be used to generate more than 99 percent of all small mutations seen in cancer patients.

However, to achieve that, they needed to find a way to optimize the editing efficiency of the CRISPR-based system. The prime editing guide RNAs (pegRNAs) used to direct CRISPR enzymes to cut the genome in certain spots have varying levels of efficiency, which leads to “noise” in the data from pegRNAs that simply aren’t generating the correct target mutation. The MIT team devised a way to reduce that noise by using synthetic target sites to help them calculate how efficiently each guide RNA that they tested was working.

“We can design multiple prime-editing guide RNAs with different design properties, and then we get an empirical measurement of how efficient each of those pegRNAs is. It tells us what percentage of the time each pegRNA is actually introducing the correct edit,” Gould says.

Analyzing mutations

The researchers demonstrated their technique using p53, a gene that is mutated in more than half of all cancer patients. From a dataset that includes sequencing information from more than 40,000 patients, the researchers identified more than 1,000 different mutations that can occur in p53.

“We wanted to focus on p53 because it’s the most commonly mutated gene in human cancers, but only the most frequent variants in p53 have really been deeply studied. There are many variants in p53 that remain understudied,” Gould says.

Using their new method, the researchers introduced p53 mutations in human lung adenocarcinoma cells, then measured the survival rates of these cells, allowing them to determine each mutation’s effect on cell fitness.

Among their findings, they showed that some p53 mutations promoted cell growth more than had been previously thought. These mutations, which prevent the p53 protein from forming a tetramer — an assembly of four p53 proteins — had been studied before, using a technique that involves inserting artificial copies of a mutated p53 gene into a cell.

Those studies found that these mutations did not confer any survival advantage to cancer cells. However, when the MIT team introduced those same mutations using the new prime editing technique, they found that the mutation prevented the tetramer from forming, allowing the cells to survive. Based on the studies done using overexpression of artificial p53 DNA, those mutations would have been classified as benign, while the new work shows that under more natural circumstances, they are not.

“This is a case where you could only observe these variant-induced phenotypes if you’re engineering the variants in their natural context and not with these more artificial systems,” Gould says. “This is just one example, but it speaks to a broader principle that we’re going to be able to access novel biology using these new genome-editing technologies.”

Because it is difficult to reactivate tumor suppressor genes, there are few drugs that target p53, but the researchers now plan to investigate mutations found in other cancer-linked genes, in hopes of discovering potential cancer therapies that could target those mutations. They also hope that the technique could one day enable personalized approaches to treating tumors.

“With the advent of sequencing technologies in the clinic, we’ll be able to use this genetic information to tailor therapies for patients suffering from tumors that have a defined genetic makeup,” Sanchez-Rivera says. “This approach based on prime editing has the potential to change everything.”

The research was funded, in part, by the National Institute of General Medical Sciences, an MIT School of Science Fellowship in Cancer Research, a Howard Hughes Medical Institute Hanna Gray Fellowship, the V Foundation for Cancer Research, a National Cancer Institute Cancer Center Support Grant, the Ludwig Center at MIT, a Koch Institute Frontier Award, the MIT Research Support Committee, and the Koch Institute Support (core) Grant from the National Cancer Institute.

De-tail-ing RNA regulation in eggs and early embryos

For a brief period during embryonic development, cells must rely on messenger RNAs provided by the maternal genome. In Developmental Cell, Bartel Lab members detail how cells regulate this limited supply of genetic material.

Greta Friar | Whitehead Institute
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