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Error-prone or adaptive? The role of topoisomerases during DNA replication and transcription conflicts

By Alexandria Oviatt

Replication—copying a cell’s DNA—and transcription—making RNA from DNA—are not separated spatially or temporally in a cell and can happen simultaneously. That means that the protein complexes (also called “machineries”) responsible for replication and those responsible for transcription sometimes have conflicts with one another. Such conflicts can be harmful to the cell, leading to mutations in the DNA or even cell death.

3D rendering of an RNA polymerase transcribing DNA. The polymerase is orange and looks vaguely like a hand holding a purple, double-stranded DNA within it. Tiny red molecules are floating about, and they represent RNA nucleotides that are coming together and forming a single-stranded RNA that's coming out of the polymerase.
RNA polymerase II transcribing DNA into RNA. Juan Gartner, Adobe Stock.

One type of conflict is the head-on conflict, which happens when the two sets of machineries approach each other from opposite directions. Kevin Lang, a postdoctoral fellow in the lab of Houra Merrikh, professor of biochemistry, recently described a critical cause of the deleterious effects of head-on conflicts—and a surprising benefit—through the use of a bacterial model.

Separation of the DNA strands during replication and transcription results in overwinding, or tight coiling, of the DNA double helix ahead of the two sets of machineries. When the two approach each other, there is a buildup of overwound DNA; the tight coils in the DNA must be removed or the cell cannot complete its replication and transcription tasks.

The researchers found that the enzymes gyrase and topoisomerase IV are needed to unwind the tight DNA coils that accumulate at regions of head-on conflicts. When both of those enzymes were inhibited, the DNA at the head-on conflict remained overwound and replication stalled. Inhibiting one or both enzymes resulted in much lower bacterial cell survival, suggesting that both enzymes are necessary to resolve head-on conflicts.

Headshot of Kevin Lang. He's wearing a Ski-themed collared shirt (that old Microsoft game) and a nose ring.
First author: Kevin Lang, postdoctoral fellow. Photo by Stephen Doster.

Previous work by the team had shown that R-loops, or regions where RNA and DNA hybridize to each other during transcription, contribute to the negative effects of head-on conflicts if they are not resolved at conflict regions. Thus, they tested whether the presence of gyrase and topoisomerase IV could influence formation of R-loops. They found that the enzymes, particularly gyrase, can promote R-loop formation and, consequently, stalling at head-on conflicts.

Lang and Merrikh’s work adds to our under-standing of the dynamics of DNA replication and transcription, which are central to the functioning of cells. At first glance, this mechanism seems counterintuitive: Why would a process exist that results in potentially harmful conflicts? A large body of work from the Merrikh lab has shown that head-on conflicts increase mutation rates, and, although increased mutagenesis is often detrimental to cells, it can help bacteria rapidly adapt to changes in their environment.

Lang, K.S., Merrikh, H. (2021). Topological stress is responsible for the detrimental outcomes of head-on replication-transcription conflicts. Cell Reports 34, 108797.

The case against rapid change

By Marissa Shapiro

Headshot of Alexander Thiemicke. He's wearing a plaid, collared shirt and is posing in front of some trees/greenery.
First author: Alexander Thiemicke, recent Ph.D. graduate. Photo by Gregor Neuert.

Pioneering research led by Alexander Thiemicke, former graduate student in chemical and physical biology, and Gregor Neuert, assistant professor of molecular physiology and biophysics, reveals that cells respond differently to environments in which conditions change gradually as opposed to rapidly. The investigators developed a cell culture environment that more closely mimics incremental physiological changes—such as in hormone or nutrient levels—as opposed to conventional approaches in which sudden alterations are made. Their goal is to provide researchers with a more relevant context to inform both basic research and drug discovery efforts.

In their most recent study, the researchers changed—first gradually and then suddenly—the amount of salt surrounding immune cells that typically exist in the kidney and intestine. This alteration causes osmosis, the movement of water across the cell membrane. Neuert and Thiemicke then monitored deviations in 25 markers of cell stress and death. They determined that 60 percent of cells died when exposed to gradual stress, whereas 90 percent of cells acutely exposed to the same change died.

3D rendering of human cell or embryonic stem cells. The cells look like they're floating and are transparent with a red nucleus. The nucleus looks like a bumpy sphere.
3D rendering of cells. Anusorn, Adobe Stock.

Cells exposed to a gradual salt increase took up protective amino acids to defend themselves against the extracellular stress. Upon acute stress, however, a cell death program was triggered before the cells had time to protect themselves. The researchers also found that, if acutely stressed cells were given those protective amino acids, they survived at rates similar to those of the gradually stressed cells.

“Looking at how cells respond to perturbations in their environments in this way is an entirely new approach in biomedical research,” said Neuert, also a Dean’s Faculty Fellow and an NIH New Innovator Awardee. “This has serious implications as we unravel fundamental biological questions and for how drug development is pursued.”

Thiemicke, A., Neuert, G. (2021). Kinetics of osmotic stress regulate a cell fate switch of cell survival. Science Advances 7, eabe1122.

How a key antidepressant works

By Marissa Shapiro

A black and white photograph of a person with long hair sitting on the floor, hugging their knees, and holding their head on their knees. The image is a visualization of someone who is depressed.
In 2019, an estimated 7.8% of all U.S. adults (19.4 million people) had at least one major depressive episode.

The recent discovery of ketamine’s remarkable antidepressant activity has led to considerable excitement among neuroscientists. Used as an anesthetic for more than 50 years, ketamine can provide, in as little as two hours, up to weeks of antidepressant relief for individuals resistant to typical antidepressants. In contrast, typical anti-depressants take four to five weeks to work, if they work at all. However, along with the excitement comes a mystery—how does ketamine work as a rapid-acting antidepressant in the brain?

Lisa Monteggia, professor of pharmacology and Barlow Family Director of the Vanderbilt Brain Institute, along with her collaborators, is addressing that question.

Synapses are structures within the nervous system by which two neurons communicate with one another. Communication occurs when one neuron releases a chemical neurotransmitter that travels across the synapse and binds to a protein receptor on the other, thereby triggering a physiological response in that neuron. Monteggia’s work supports the current understanding that ketamine blocks glutamate N-methyl-D-aspartate—or NMDA—receptors, triggering a neural biochemical pathway that elicits the rapid antidepressant effect.

Headshot of Ji-Woon Kim, who is wearing a white, collared dress shirt, a tie, and a black jacket.
First author: Ji-Woon Kim, postdoctoral fellow. Photo courtesy of Ji-Woon Kim.

Monteggia’s team, which included Joachim Herz, a professor of molecular genetics, neurology, and neuroscience at UT Southwestern, also looked into how Reelin—a protein that controls cells’ interactions in the brain—is involved in ketamine’s action. They found that mutations or impairments to Reelin could result in a patient not experiencing ketamine’s antidepressant activity.

Understanding why ketamine works in some patients but not others is important for everyone. “Depression is a heterogenous disorder; it is rather remarkable that antidepressants and ketamine work in as many patients as they do,” Monteggia said. “By understanding the target and how mutations or impairments affect the drug’s mechanism, we can develop fast-acting antidepressants that target other populations.”

Kim, J.-W., Herz, J., Kavalali, E.T., Monteggia, L.M. (2021). A key requirement for synaptic Reelin signaling in ketamine-mediated behavioral and synaptic action. Proceedings of the National Academy of Sciences 118, e2103079118.

Single-cell data curation with machine learning

By Cody Heiser

Single-cell RNA sequencing, or scRNA-seq, allows researchers to explore cell-to-cell variability in healthy and diseased tissues—and even entire organisms—lending insight into normal development and complex pathologies such as cancer.

The lab of Ken Lau, associate professor of cell and developmental biology, in collaboration with Jake Hughey, assistant professor of biomedical informatics, recently developed a fully automated machine learning tool to help researchers maintain high quality in scRNA-seq experiments. The work, published in Genome Research, will help biologists better extract information from the large amount of data generated by this method.

Recent advances in single-cell technologies have made such data accessible to researchers working on diverse biological problems. The most common platforms make use of small, liquid-flow channels that capture individual cells in a water-oil emulsion from which RNA molecules can be isolated and sequenced. Because of the large number of cell-free droplets generated to ensure the isolation and capture of single cells, a major challenge is distinguishing cells from “empty droplets” based on the detected RNA alone.

The automated tool, called “dropkick,” performs quality control and cell identification on scRNA-seq data. After determining batch-specific metrics that describe droplet quality, dropkick learns a gene-based representation of real cells and background noise, calculating a cellular probability score for each droplet. Thanks to dropkick, researchers can interpret the biological significance of background RNA expression and fine-tune the filtering cutoff and downstream analysis accordingly.

Headshot of Cody Heiser. He's wearing a blue and white striped polo shirt with a green collar. There is greenery in the background.
First author: Cody Heiser, Ph.D. student. Photo by Tracy Heiser.

The dropkick tool was benchmarked against existing filtering approaches on simulated and real-world data and was shown to reproducibly recover rare cell types and exclude empty droplets, especially in high-background datasets. These characteristics make dropkick extremely valuable for biologists by protecting downstream analyses from noise that may skew interpretations and rescuing rare cell populations, contributing to robust biological discovery.

Heiser, C. N., Wang, V. M, Chen, B., Hughey, J. J., & Lau, K.S. (2021). Automated quality control and cell identification of droplet-based single-cell data using dropkick. Genome Research, Article gr.271908.120.

RNA-protein interactions mediate an innate immune response

By Nicole Kendrick

The innate immune system is made up of receptors that, upon detection of a potential pathogen, trigger a robust transcriptional response in cells. This means that hundreds of mRNAs are made that encode for pro-inflammatory and anti-viral proteins that protect cells from infection. However, dysregulation of the mRNAs can lead to improper levels of those pro-inflammatory proteins, which can be detrimental to cells and tissues. To prevent damage, cells have evolved mechanisms to carefully balance the levels of immune-relevant mRNAs to promote a strong immune response but avoid rampant overstimulation.

Headshot of Katie Rothamel. She's wearing a navy blue shirt and her hair down to her shoulders.
First author: Katherine Rothamel, recent Ph.D. graduate. Photo by Manuel Ascano.

Overactivation of the immune system among patients is a primary concern of health care workers who treat infections such as COVID-19. A better understanding of the mechanisms that prevent immune overstimulation could lead to better treatments for patients with autoimmune disorders or for those who develop hyperactive reactions to bacterial or viral infections, and even vaccinations.

One way the cell regulates gene expression is through the control of mRNAs by RNA-binding proteins, or RBPs. RBPs can target specific mRNAs to alter their stability, location inside the cell, and ability to be translated into proteins. RBPs that bind to mRNAs that get made into innate immune proteins influence the duration and intensity of the immune response.

Researchers from groups of Manuel Ascano, assistant professor of biochemistry, and Neelanjan Mukherjee, assistant professor of biochemistry and molecular genetics at the University of Colorado School of Medicine, showed that the RBP ELAVL1 binds to a specific region within mRNAs that stabilizes them during an innate immune response.

Before a cell receives a signal from the innate immune system, ELAVL1 is located in the nucleus, where it binds to introns, or non-coding sections of mRNA. Upon receiving a signal, ELAVL1 moves to the cytoplasm, where it binds to a set of innate immunity-related mRNAs at a very specific location called the 3′ untranslated region. This transition is required to stabilize the mRNAs that encode for the innate immune proteins.

Overall, this study provides a novel example of how the activation status of an immune cell affects not only the identity of the mRNAs that RBPs bind, but also the function of those RBPs on their targets. The redistribution of ELAVL1 stabilizes certain mRNAs, switching immune cells from a resting to an activated state, and demonstrates how an RBP targeting specific mRNAs can positively regulate the immune response and can significantly influence a cell’s resilience to infection.

Rothamel, K., Arcos, S., Kim, B., Reasoner, C., Lisy, S., Mukherjee, N., Ascano, M. (2021). ELAVL1 primarily couples mRNA stability with the 3′ UTRs of interferon-stimulated genes. Cell Reports 35, 109178.

Finding better structures

By Diego del Alamo

Proteins carry out vital functions throughout the cell, and many of them need to undergo structural changes to accomplish their tasks. Bacterial drug exporters, for example, adopt several distinct structural poses to grab and eject toxic drugs from within the cell. Knowing the detailed structures of these poses can help researchers design drugs that can, for example, circumvent bacterial drug resistance mechanisms. Unfortunately, proteins are rarely captured in the full breadth of structural states they adopt.

Close-up of Diego del Alamo, who appears to be on a ski lift. He is wearing a gray jacket and a brown/black patterned scarf.
First author: Diego del Alamo, Ph.D. student. Photo by Selena Chacón Simon.

The labs of Hassane Mchaourab, professor of molecular physiology and biophysics, and Jens Meiler, professor of chemistry, recently teamed up and tackled this challenge by integrating experimental spectroscopy and computational modeling.

Our research, published in PLOS Computational Biology, adapted a technique called multilateration. Multilateration is used in everyday life for things such as positioning cellphones using GPS distance data.

The central idea of the research is that specific structural states that may be poorly understood can be detected and monitored using an experimental technique called electron paramagnetic resonance—or EPR—spectroscopy, which measures distances between pairs of probes attached to a protein. Although these probes are quite flexible, we showed that a multilateration algorithm can calculate their precise positions.

We combined this algorithm with Rosetta, a modeling platform, to model structural rearrangements in a well-studied bacterial multidrug exporter. We found substantial improvements in the accuracy and precision of the model, allowing us to recapitulate the change in the structure of the transporter that leads to bacteria ejecting a drug.

This algorithm promises to address fundamental challenges to modeling protein structures using data obtained through EPR, which has previously been restricted to monitoring low-resolution structural movements. The work also opens the door to improved knowledge of the relationship between protein structure and function.

del Alamo, D., Jagessar, K.L., Meiler, J., Mchaourab, H.S. (2021). Methodology for rigorous modeling of protein conformational changes by Rosetta using DEER distance restraints. PLOS Computational Biology 17, e1009107.

RNA editing enzymes as internal pH sensors

By Wendy Bindeman

Single-stranded RNA. nobeastsofierce, Adobe Stock.

Cells have to regulate gene expression to function properly, and they have many tools to do this. Many of these tools are dependent on the DNA or RNA sequences or have to do with controlling whether the RNA is made in the first place. However, one cellular tool called “RNA editing” is employed after the RNA is produced but before it is used to make a protein. The regulation of RNA editing is not yet well understood, but recent findings from the lab of Professor of Pharmacology Ron Emeson show that it is dependent on the pH of the cell.

RNA is made up of four bases: adenosine (A), guanosine (G), thymidine (T), and uracil (U). One common RNA editing event occurs when a cell replaces an A with inosine (I), a rare base that looks and acts like a G; this change is called “A-to-I editing.” It can be present in any cell but is particularly important and occurs frequently in the nervous system. Alterations in A-to-I editing are associated with a variety of neurological disorders, such as epilepsy, amyotrophic lateral sclerosis, and schizophrenia.

A recent paper from the Emeson lab identified intracellular acidification as an important regulator of RNA editing. They found that the enzymes responsible for A-to-I editing, called ADARs (adenosine deaminases acting on RNA), are pH sensitive and become more active at a lower, or more acidic, pH than is normally found in the cell. This is due to a structural change that occurs at acidic pH that makes ADARs better at stabilizing the RNA.

Headshot of Turnee Malik. She is wearing a navy blue jacket and is standing in front of a brick wall.
First author: Turnee Malik, recent Ph.D. graduate. Photo by Kayla Shumate.

Based on these results, Emeson and colleagues suggest that ADARs might act like pH sensors, allowing cells to modulate their RNA editing to respond to environmental changes. For instance, stressors such as hypoxia or stroke disturb the pH balance in neurons, leading to acidification of their internal environments. Acidification can cause hyperexcitability in neurons, which can damage or kill cells if unchecked. Higher levels of ADARs have been shown to decrease neuron excitability, and the RNA of many proteins that help control membrane excitability in neurons is a target of A-to-I editing. Therefore, the authors speculate that RNA editing may provide a pH-dependent protective mechanism for neurons by facilitating the rapid editing of RNAs that encode membrane proteins to correct hyperexcited states and thereby limit damage.

Currently, RNA editing is an attractive way to try to repair mutations associated with some genetic diseases. Careful study of the regulation of RNA editing, such as that carried out by the Emeson lab, is necessary to develop safe and effective strategies for precisely targeting RNA editing enzymes to the specific sequences that need repair.

Malik, T.N., Doherty, E.E., Gaded, V.M., Hill, T.M., Beal, P.A., Emeson, R.B. (2021). Regulation of RNA editing by intracellular acidification. Nucleic Acids Research 49, 4020–4036.

Unveiling the life cycle of a microvillus

By Colbie Chinowsky

Headshot of Isabella Gaeta. She is wearing a white t-shirt and a green cardigan.
First author: Isabella Gaeta, Ph.D. student. Photo by Matt Tyska.

The surface of the intestinal tract is the sole site of nutrient absorption—a life-sustaining process—and disturbances to this tissue have the potential for deadly consequences. The small intestine has evolved a variety of structures that maximize the surface area available for nutrient uptake, including microvilli, fingerlike projections that protrude from the cell. The collection of microvilli on the surface of intestinal cells forms a larger structure known as the brush border, which resembles the bristles on a toothbrush.

In a recent Current Biology paper, graduate student Isabella Gaeta, from the lab of Professor of Cell and Developmental Biology Matt Tyska, reveals how microvilli are born and begin to grow. The assembly of new microvilli is critical for the function of the intestine, but until now little was known about how cells build these structures.

The intestine is different from most other tissues because of its high rate of cell turnover—old cells constantly die off and are replaced by new, robust cells. Each new cell, however, also must build brand-new microvilli. This process is driven by the end-to-end assembly of a cellular protein known as actin, and the resulting actin “filaments” are in turn bundled together to form a strong support for the cell membrane. Using live-cell microscopy, Gaeta observed the various proteins that come to the cell surface to “birth” a microvillus and revealed that EPS8, a protein known to help form actin structures, is required for microvilli to continue to grow.

Gaeta also showed that a “mother microvillus” is capable of birthing a “daughter” by creating a new branch off an existing projection that in turn splits into an independent structure. This exciting new finding illuminates a novel pathway for microvilli formation, and additional studies along these lines will lead to a better understanding of the birth and growth of microvilli. This work will also allow researchers to understand how the complex structures of the intestine may be disrupted in diseases characterized by loss of microvilli, such as enteric (intestinal) infections with pathogenic Escherichia coli.

Gaeta, I.M., Meenderink, L.M., Postema, M.M., Cencer, C.S., Tyska, M.J. (2021). Direct visualization of epithelial microvilli biogenesis. Current Biology 31, 2561–2575.e6.