Author: Lillian Eden
Whitehead Institute Member Jonathan Weissman and colleagues used large-scale systematic genetic screens to identify the molecules and pathways that populate the mitochondrial surface with important and diverse signaling proteins. They deciphered the logic by which the cell ensures the proper delivery of these proteins. These findings may have important implications for understanding the impact on health and disease when these processes go awry.
Greta Friar | Whitehead Institute
February 26, 2024
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.”
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 Negative Elongation Factor, NELF, 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 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.”
Research from the Jain Lab finds that, in Huntington's disease, repeats of certain nucleotides too many times in a row interferes with splicing.
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Lillian Eden | Department of Biology
January 4, 2024
