Category: News Briefs

News brief: Calo Lab

How do cells respond to disruptions in splicing?

Lillian Eden | Department of Biology
March 4, 2024

New research from the Calo Lab in the Department of Biology has identified the protein Mdm2 generating a form that activates a cascade of cellular stress responses when splicing is disrupted.

To create proteins, DNA is transcribed into RNA, and that RNA is then “translated” into protein. Between the creation of the RNA and the translation to protein is often a step called splicing. During splicing, segments called introns are removed, and the remaining pieces, called exons, are joined together to form the blueprint for translation. By splicing together different exons, the cell can create different proteins from the same section of genetic code. When splicing goes awry, it can lead to diseases and cancers. 

New research recently published in Disease Models & Mechanisms from the Calo Lab in the Department of Biology at MIT has identified the mechanism for how cells respond to disruptions in splicing, which involves activating a cellular stress response. The stress response, once activated, causes widespread effects, including changes to cell metabolism. 

Researchers have discovered cellular stress responses for other core cellular processes, such as ribosome biogenesis. However, this is the first time researchers have identified how cells respond to perturbing the splicing process.

A particular protein acts as a kind of canary in a coal mine: Mdm2, which responds to a broad range of splicing disruptions. Mdm2 does not cause a stress response by itself. Rather, Mdm2 is itself spliced differently in response to splicing disruptions. Downstream, the alternative splicing of Mdm2 leads to the activation of a protein called p53, which is known to orchestrate a cascade of responses to stress.

Researchers have long wondered why some cell types seem more sensitive to splicing disruptions than others. For example, some disorders caused by mutations in proteins that perform RNA splicing, despite affecting the whole organism, induce more noticeable changes in tissues derived from the neural crest—a collection of stem cells that contributes to the formation of the face, jaw, retinas, limbs, and heart during development. Certain splicing inhibitors have also increased the effectiveness of some cancer treatments, but the mechanism is unknown. 

One of the p53-induced stress responses includes changing the metabolism of cells and how they use sugars, which may explain why some cells are more sensitive to splicing disruptions than others. Inhibiting glycolysis, the reactions that extract energy from glucose, can affect how cells divide and migrate. 

The way cells divide and migrate is critical during development; in experiments, zebrafish treated with glycolysis inhibitors exhibited similar changes to craniofacial features as those where splicing was disrupted. Cancerous cells, too, are known to require high levels of sugar metabolism and, therefore, may be especially sensitive to treatments that induce changes in the splicing pathway. 

The researchers knocked down genes to mimic milder splicing disruptions instead of knocking them out entirely. Splicing is so essential that knocking out the splicing machinery can lead to extreme responses like cell death. In organismal models like zebrafish, those severe phenotypes don’t accurately reflect how splicing disruptions present in human diseases.

First author Jade Varineau, a graduate student in the Calo lab, was drawn to the project because it allowed her to explore what was happening at the RNA and cellular level while also observing how splicing perturbations were affecting the whole organism. 

“I think this data can help us reframe the way we think about diseases and cancers that are impacted by splicing—that a treatment that works for one may work for another because all the symptoms may stem from the same cellular response,” Varineau says. 

Although the results indicate how cells broadly respond to splicing perturbations, the mechanism for how disruptions in splicing induce the alternate splicing of Mdm2 remains unclear. Senior author Eliezer Calo says the lab is also exploring how splicing mechanisms may be altered for things like cancer. Their work, he says, opens the door for further exploration of cell-type specificity of genetic disorders and improvements in cancer treatments using splicing inhibitors. 

 “We know that the sensor is encoded in the gene Mdm2—what are the molecules that allow Mdm2 to act as a sensor, and how does the sensor malfunction for things like cancer?” Calo says. “The next step is to find out how the sensor works.”  

How early-stage cancer cells hide from the immune system

A new study finds precancerous colon cells turn on a gene called SOX17, which helps them evade detection and develop into more advanced tumors.

Anne Trafton | MIT News
February 28, 2024

One of the immune system’s primary roles is to detect and kill cells that have acquired cancerous mutations. However, some early-stage cancer cells manage to evade this surveillance and develop into more advanced tumors.

A new study from MIT and Dana-Farber Cancer Institute has identified one strategy that helps these precancerous cells avoid immune detection. The researchers found that early in colon cancer development, cells that turn on a gene called SOX17 can become essentially invisible to the immune system.

If scientists could find a way to block SOX17 function or the pathway that it activates, this may offer a new way to treat early-stage cancers before they grow into larger tumors, the researchers say.

“Activation of the SOX17 program in the earliest innings of colorectal cancer formation is a critical step that shields precancerous cells from the immune system. If we can inhibit the SOX17 program, we might be better able to prevent colon cancer, particularly in patients that are prone to developing colon polyps,” says Omer Yilmaz, an MIT associate professor of biology, a member of MIT’s Koch Institute for Integrative Cancer Research, and one of the senior authors of the study.

Judith Agudo, a principal investigator at Dana-Farber Cancer Institute and an assistant professor at Harvard Medical School, is also a senior author of the study, which appears today in Nature. The paper’s lead author is MIT Research Scientist Norihiro Goto. Other collaborators include Tyler Jacks, a professor of biology and a member of MIT’s Koch Institute; Peter Westcott, a former Jacks lab postdoc who is now an assistant professor at Cold Spring Harbor Laboratory; and Saori Goto, an MIT postdoc in the Yilmaz lab.

Immune evasion

Colon cancer usually arises in long-lived cells called intestinal stem cells, whose job is to continually regenerate the lining of the intestines. Over their long lifetime, these cells can accumulate cancerous mutations that lead to the formation of polyps, a type of premalignant growth that can eventually become metastatic colon cancer.

To learn more about how these precancerous growths evade the immune system, the researchers used a technique they had previously developed for growing mini colon tumors in a lab dish and then implanting them into mice. In this case, the researchers engineered the tumors to express mutated versions of cancer-linked genes Kras, p53, and APC, which are often found in human colon cancers.

Once these tumors were implanted in mice, the researchers observed a dramatic increase in the tumors’ expression of SOX17. This gene encodes a transcription factor that is normally active only during embryonic development, when it helps to control development of the intestines and the formation of blood vessels.

The researchers’ experiments revealed that when SOX17 is turned on in cancer cells, it helps the cells to create an immunosuppressive environment. Among its effects, SOX17 prevents cells from synthesizing the receptor that normally detects interferon gamma, a molecule that is one of the immune system’s primary weapons against cancer cells.

Without those interferon gamma receptors, cancerous and precancerous cells can simply ignore messages from the immune system, which would normally direct them to undergo programmed cell death.

“One of SOX17’s main roles is to turn off the interferon gamma signaling pathway in colorectal cancer cells and in precancerous adenoma cells. By turning off interferon gamma receptor signaling in the tumor cells, the tumor cells become hidden from T cells and can grow in the presence of an immune system,” Yilmaz says.

Without interferon gamma signaling, cancer cells also minimize their production of molecules called MHC proteins, which are responsible for displaying cancerous antigens to the immune system. The cells’ insensitivity to interferon gamma also prevents them from producing immune molecules called chemokines, which normally recruit T cells that would help destroy the cancerous cells.

Targeting SOX17

When the researchers generated colon tumor organoids with SOX17 knocked out, and implanted those into mice, the immune system was able to attack those tumors much more effectively. This suggests that preventing cancer cells from turning off SOX17 could offer a way to treat colon cancer in its earliest stages.

“Just by turning off SOX17 in fairly complex tumors, we were able to essentially obliterate the ability of these tumor cells to persist,” Goto says.

As part of their study, the researchers also analyzed gene expression data from patients with colon cancer and found that SOX17 tended to be highly expressed in early-stage colon cancers but dropped off as the tumors became more invasive and metastatic.

“We think this makes a lot of sense because as colorectal cancers become more invasive and metastatic, there are other mechanisms that create an immunosuppressive environment,” Yilmaz says. “As the colon cancer becomes more aggressive and activates these other mechanisms, then there’s less importance for SOX17.”

Transcription factors such as SOX17 are considered difficult to target using drugs, in part because of their disorganized structure, so the researchers now plan to identify other proteins that SOX17 interacts with, in hopes that it might be easier to block some of those interactions.

The researchers also plan to investigate what triggers SOX17 to turn on in precancerous cells.

The research was funded by the MIT Stem Cell Initiative via Fondation MIT, the National Institutes of Health/National Cancer Institute, and a Koch Institute-Dana Farber Harvard Cancer Center Bridge Project grant.

News Brief: Vos Lab

Poise or Pause: researchers expand understanding of transcription factor’s role with newly discovered conformation

Lillian Eden | Department of Biology
February 23, 2024

New research from the Vos Lab in the Department of Biology at MIT reveals the dynamic nature of elongation factor protein key for regulating early stages of gene expression.

Transcription, the process of copying RNA from DNA, is a critical first step for cells to create proteins. The enzyme responsible for transcription is a motor protein called RNA polymerase. 

When an RNA polymerase transcribes a gene, it will begin elongating the mRNA and will then, often, pause. 

From there, the RNA polymerase will either return to elongating the mRNA or it will get stuck. For the latter occurrence, the mRNA and subsequent protein will never be made: the polymerase will go somewhere else, or restart transcription on the same gene and get stuck again. 

Pausing is thought to be governed by a protein called NELF (Negative Elongation Factor) and DRB-sensitivity inducing factor (DSIF). Previous research suggested that NELF stably clamps down onto RNA polymerase to stall the elongation process and prevent the polymerase from moving. That model contradicted cell-based experiments, however, which indicated that NELF is somehow still attached to polymerase after transcription resumes. 

New research from the Vos Lab in the Department of Biology at MIT published today in Molecular Cell reveals that NELF isn’t merely an on-off switch for transcription. Instead, NELF can change into a distinct conformation that allows the polymerase to resume transcription. The researchers dubbed this distinct conformation NELF’s “poised” state.

RNA polymerase pausing, sometimes for minutes at a time, is thought to be an important gene expression checkpoint; more than half of genes exhibit pausing, although many questions remain about the role of pausing in gene expression. Understanding both how and why the process is occurring, down to the atomic level, and what components are involved, is key to understanding how cells function, both individually and as part of an organism.

“It’s a very central question to biological research, and we still don’t fully understand it because it’s such a complex process,” says first author Bonnie G. Su, a graduate student in the Vos lab. “The bigger picture is: how does the cell decide what resources to allocate to certain biological processes? This finding might help us answer questions like that.” 

To visualize the two distinct conformations of NELF and polymerase, the researchers used a combination of biochemical and structural approaches. The previous understanding of proximal pausing was based on Cryo-Electron Microscopy (cryo-EM) images of the static complex. Cryo-EM is a powerful microscopy technique that involves freezing samples and imaging them, and that approach had captured polymerase in its paused state. 

Using the core Cryo-EM facility available at MIT.nano, Su instead added the necessary components for the polymerase to transcribe, and gathered structural data on an actively transcribing complex —allowing, for the first time, a stepwise visualization of how proximal pausing occurs. 

“What we found is that NELF, which we always thought of as static, can actually move around,” Su says. “This has updated our understanding of what pausing is, and how early gene regulation happens.” 

The structural results also provide an explanation for how polymerase may be cycling between the two states—and how one form of NELF may be forcing polymerase to pause, while the newly discovered form allows polymerase to resume transcription. 

It’s still unclear what triggers NELF to transition from the paused state to the poised state, and many questions remain about how polymerase is regulated, according to senior author Seychelle Vos, the Robert A. Swanson (1969) Career Development Professor of Life Sciences and HHMI Freeman Hrabowski Scholar. RNA polymerase can be associated with and is known to be regulated by a large repertoire of proteins. 

“We’re trying to see if we can actually lock the complex in the paused state by adding additional factors,” Vos says. “We’re also pursuing whether sequence context is affecting pausing behavior—how or if the sequence of DNA may be causing polymerase to pause.”

Protein production glitches in Huntington’s disease revealed

Research from the Jain Lab finds that, in Huntington's disease, repeats of certain nucleotides too many times in a row interferes with splicing.

January 30, 2024
Capsid of HIV-1 behaves like cell’s cargo receptor to enter the nucleus

Biologists demonstrate that HIV-1 capsid acts like a Trojan horse to pass viral cargo across the nuclear pore.

Lillian Eden | Department of Biology
January 24, 2024

Retroviruses cannot replicate on their own — they must insert their genetic code into the DNA of a host and exploit the host cell’s resources to make more copies of themselves, furthering infection. Some retroviruses only infect cells as they divide, when the nuclear envelope that protects the host’s genetic material breaks down, making it easily accessible. HIV-1 is a type of retrovirus, called a lentivirus, that can infect non-dividing cells.

HIV-1 delivers its genome into the nucleus by packaging it into a large, cone-shaped structure called a capsid — but the exact mechanism has remained elusive for decades. Travel through the nuclear envelope occurs through, and is regulated by, nuclear pores, doughnut-shaped protein assemblies. Human cells have about 2,000 nuclear pores perforating the nuclear envelope. Some earlier evidence suggested that the capsid remains intact during its delivery into the nucleus — but this created a dimensional conundrum. The cone-shaped HIV-1 capsid is about 120 nanometers long and 60 nm wide — too large, researchers thought, to fit through the opening of the nuclear pore, measured at only 43 nm wide.

Members of the Schwartz Lab at MIT, in the Department of Biology, became interested in this question when a postdoc in the lab used cryo-electron tomography, slicing up sections of frozen cells to examine structures, to show that nuclear pores in the nuclear envelope are larger than 43nm. They deflate and shrink, it turns out, when removed from their native conditions. In native conditions, the nuclear pore complex is about 60nm wide — wide enough to accommodate the HIV-1 capsid.

Knowing that it could fit, a question remained: How can the capsid navigate the dense mesh of spaghetti-like proteins that act like a sieve in the nuclear pore channel? That spaghetti-like mesh allows small cargo to diffuse through, but prevents large cargo from entering unless it is escorted by proteins called nuclear transport receptors.

In an open-access paper published today in Nature, researchers present evidence that the HIV-1 capsid mimics the cell’s transport receptors to traverse the nuclear pore.

To support that conclusion, the researchers showed three things in vitro: that an HIV-1 capsid can deliver cargo through a nuclear pore analog; that the capsid can interact with the sieve of proteins in the nuclear pore channel; and that the capsid targets the nuclear pore in the absence of native transport proteins.

Nuclear transport receptors escort large cargo through the nuclear pore by “batting away” the spaghetti-like mesh of proteins inside the channel — like someone holding your hand and guiding you across a crowded dance floor. The HIV-1 capsid interacts with the spaghetti-like proteins, but its purpose is more like a Trojan horse — the capsid encapsulates the viral cargo, protecting it from detection in the cytoplasm and as it enters the nuclear pore complex.

“What’s really amazing about cells is that they are incredibly complex. What’s really difficult about studying cells is that they are incredibly complex,” jokes co-first author Erika Weiskopf, a graduate student in the Schwartz lab. “Biochemists are constantly trying to find ways to study their system in a simplified context, but still give it a flavor of cell biology.”

To do that, the Schwartz lab collaborated with Dirk Görlich, the director of cellular logistics at the Max Planck Institute for Multidisciplinary Sciences. Görlich is a co-senior author on the paper with MIT’s Boris Magasanik Professor of Biology Thomas Schwartz. Görlich’s lab has produced concentrated droplets of the spaghetti-like proteins found inside the nuclear pore, and those droplets allow and exclude cargo the same way a nuclear pore will. In experiments, fluorescently-labeled cargo did not enter the droplets, but fluorescently-labeled cargo packaged in an HIV-1 capsid was delivered. This indicated that the capsid could deliver cargo through a nuclear pore.

Using a biophysical binding assay, the researchers also showed that the HIV-1 capsid interacts with the proteins inside the channel. Different spaghetti-like proteins are found in different channel sections, such as at the cytoplasmic side’s entrance or only inside the channel; there are 10 such proteins in human cells. The capsid is a promiscuous binder — it can interact with all the spaghetti-like proteins found in the channel.

The capsid can target the nuclear pore complex even without the cell’s transport receptors, indicating that it is not commandeering native transport receptors to find and enter the nuclear pore. The team used a classic assay in the nucleocytoplasmic transport field to collect this evidence: When cells are treated with digitonin, their membranes become porous. Everything in the cytoplasm will leak out of the cells, but the nuclear envelope will remain intact. Despite the absence of native proteins, the capsid was attracted to the nuclear pore complex, a behavior indicative of a nuclear transport receptor.

Although the capsid behaves like a nuclear transport receptor to penetrate the nuclear pore, it is fundamentally different. A transport receptor doesn’t need to conceal material for delivery the way the capsid does to avoid detection.

These findings open new lines of inquiry for what the nuclear pore complex is capable of accommodating.

“The HIV-1 capsid is one of the largest things that we now know can go through the nuclear pore complex intact,” Weiskopf says. “It raises all kinds of questions — what other things could be going through the pore that we thought was impossible?”

Schwartz said another question is whether all of the 2,000 nuclear pores in human cells are identical or whether there is something that makes certain pores more amenable to allowing the capsid through.

The capsid is also known to be unusually elastic, a property that may be key for passage through the pore. Another interesting question for the field is whether the cone-shaped capsid gains entry into the pore by squeezing through.

Although the team has shown that the capsid can enter the pore, what happens at the other end of the channel is still unknown — whether the capsid fully or partially enters the nucleus or breaks down inside the channel. Weiskopf is working on perturbing parts of the capsid or the spaghetti-like proteins to learn more about which interactions are most important for successful capsid entry.

Although these results have expanded our understanding of the nuclear pore, much remains unknown, both for HIV-1 infection and for the transport process through the nuclear pore complex.

“The nuclear pore is such an important element of cell biology, we thought it would be interesting to understand it better — and that’s how we figured out that the pore is much bigger than we anticipated,” Schwartz says. “We will certainly try to see whether we can understand the mechanism of HIV-1 infection, how the capsid is released on the other side of the channel, and what factors are important there — and to what extent you can manipulate it or influence it for therapeutic applications.”

Blood cell family trees trace how production changes with aging

A new method from the Weissman Lab provides a detailed look at the family trees of human blood cells and the characteristics of individual cells, providing insights into hematopoietic stem cells (HSCs).

January 19, 2024
How cells accurately assemble complex machinery

New research from the Cheeseman Lab reveals how cells control the location of the machinery that splits apart chromosomes during cell division.

January 2, 2024
New research explores intertwined structures of protein Met18

Research from the Drennan Lab in the Department of Biology at MIT explores how a protein called Met18, which is part of a ubiquitous pathway to transfer clusters of iron and sulfur to client proteins in the cytosol and nucleus of cells, can interact with other Met18 units to form intertwined structures

Lillian Eden | Department of Biology
December 18, 2023
Sex chromosomes responsible for much more than determining sex

Genes expressed from the X and Y chromosomes impact cells throughout the body—not just in the reproductive system—by dialing up or down the expression of thousands of genes found on other chromosomes.

December 13, 2023