GN/ New technique to unravel gene regulation
Genetics biweekly vol.25, 23d March — 7th April
TL;DR
- How is the activity of genes regulated by the packaging of DNA? To answer this question, researchers developed a technique to measure both gene expression and DNA packaging at the same time.
- With the help of the CRISPR/Cas molecular scissors, genetic information in a plant can be modified to make the latter more robust to pests, diseases, or extreme climatic conditions. Researchers have now developed this method further to eliminate the complete DNA of specific cell types and, thus, prevent their formation during plant development. This will also help researchers better understand development mechanisms in plants.
- To test if a single gene could affect an entire ecosystem, a research team conducted a lab experiment with a plant and its associated ecosystem of insects. They found that plants with a mutation at a specific gene foster ecosystems with more insect species. The discovery of such a ‘keystone gene’ could change current biodiversity conservation strategies.
- From hot volcanic springs where the water is nearly boiling acid, scientists have discovered how lemon-shaped viruses got their form. And that discovery could lead to new and better ways to deliver drugs and vaccines.
- Tetrahymena, a tiny single celled-organism, turns out to be hiding a surprising secret: it’s doing respiration — using oxygen to generate cellular energy — differently from other organisms such as plants, animals or yeasts. The discovery highlights the power of new techniques in structural biology and reveals gaps in our knowledge of a major branch of the tree of life.
- Medusavirus, a giant virus, is more closely related to eukaryotic cells than other giant viruses are. In an exciting new study, scientists have used electron microscopy and time-course analysis to discover four different types of medusavirus particles within and outside infected amoeba cells, representing four different stages of virus maturation. Their results indicate that the medusavirus has a unique maturation process, providing new insights into the structural and behavioral diversity of giant viruses.
- Researchers have revealed how poxviruses build their scaffold — a temporary protein coat that forms and disappears as the virus matures.
- Until now, proteins have been the target of most medications for the prevention and treatment of human disease. Drug developers have perceived RNA to be too unstable to target with drug therapy. However, a screen of 50,000 compounds has revealed drug-like activity against an RNA prototype called Xist, a result that opens the door for development of new medications.
- Researchers have identified a four-protein complex that appears to play a key role in generating ribosomes — organelles that serve as protein factories for cells — as well as a surprising part in neurodevelopmental disorders. The findings could lead to new ways to manipulate ribosome production, which could impact a variety of conditions that affect human health.
- Researchers have combined macro photography with DNA metabarcoding to create a new botanical “CSI” tool that may hold the key to safeguarding the future of Australia’s critically endangered carnivorous plants.
- And more!
Overview
Genetic technology is defined as the term which includes a range of activities concerned with the understanding of gene expression, advantages of natural genetic variation, modifying genes and transferring genes to new hosts. Genes are found in all living organisms and are transferred from one generation to the next. Gene technology encompasses several techniques including marker-assisted breeding, RNAi and genetic modification. Only some gene technologies produce genetically modified organisms.
Modern genetic technologies like genome editing would not be possible without all the previous generations of genetic technologies that have enabled scientists to discover what genes are, what they do and how DNA can be modified to add, remove or replace genes. You can find major genetic technologies development milestones via the link.
Gene Technology Market
- The valuation of the genetic engineering market is projected to escalate to USD 6.90 MN by the end of 2027.
- Global Genetic Engineering Market is projected to grow at 12.48% CAGR during the assessment period (2017–2027).
- North America holds the largest share in the global genetic engineering market, followed by Europe and the Asia Pacific, respectively.
- Another research provider, MarketsandMarkets, forecasts the genome editing, genome engineering market to grow from USD 3.19 billion in 2017 to USD 6.28 billion by 2022, at a compounded annual growth rate (CAGR) of 14.5% during the forecast period. The key factors propelling market growth are rising government funding and growth in the number of genomics projects, high prevalence of infectious diseases (like COVID-19) and cancer, technological advancements, increasing production of genetically modified (GM) crops, and growing application areas of genomics.
Latest News & Research
Single-cell profiling of transcriptome and histone modifications with EpiDamID
by Franka J. Rang, Kim L. de Luca, Sandra S. de Vries, Christian Valdes-Quezada, Ellen Boele, Phong D. Nguyen, Isabel Guerreiro, Yuko Sato, Hiroshi Kimura, Jeroen Bakkers, Jop Kind in Molecular Cell
How is the activity of genes regulated by the packaging of DNA? To answer this question, a technique to measure both gene expression and DNA packaging at the same time was developed by Franka Rang and Kim de Luca, researchers from the group of Jop Kind (group leader at the Hubrecht Institute and Oncode Investigator). This method, EpiDamID, determines the location of modified proteins around which the DNA is wrapped. It is important to gather information about these modifications, because they influence the accessibility of DNA, thereby affecting the gene activity. EpiDamID is therefore valuable for research into the early development of organisms.
In order to fit DNA into the nucleus of a cell, it is tightly packed around nuclear proteins: histones. Depending on the tightness of this winding, the DNA can be (in)accessible to other proteins. This therefore determines whether the process of gene expression, translation of DNA into RNA and eventually into proteins, can take place.
The tightness of DNA winding around histones is regulated by the addition of molecular groups, so-called post-translational modifications (PTMs), to the histones. For example, if certain molecules are added to the histones, the DNA winding is loosened. This makes the DNA more accessible for certain proteins and causes the genes in this part of the DNA to become active, or expressed. Furthermore, proteins that are crucial for gene expression can directly recognize and bind the PTMs. This enables transcription: the process of DNA copying.
The regulation of gene expression, for instance through PTMs, is also known as epigenetic regulation. Since all cells in a body have the same DNA, regulation of gene expression is needed to (de)activate specific functions in individual cells. For instance, heart muscle cells have different functions than skin cells, thus require different genes to be expressed.
To understand how PTMs affect gene expression, first authors Franka Rang and Kim de Luca designed a new method to determine the location of the modifications. Using this approach, called EpiDamID, researchers can analyze single cells, whereas previous methods were only able to measure a large group of cells. Analysis on such a small scale results in knowledge on how DNA winding differs per cell, rather than information on the average DNA winding of many cells.
EpiDamID is based on DamID, a technique which is used to determine the binding location of certain DNA-binding proteins. Using EpiDamID, the binding location of specific PTMs on histone proteins can be detected in single cells. Compared to others, a great advantage of this technique is that researchers need very limited material. Furthermore, EpiDamID can be used in combination with other methods, such as microscopy, to study regulation of gene expression on different levels.
Following the development of this technique, the Kind group will focus on the role of PTMs from the point of view of developmental biology. Because single cells are analyzed using EpiDamID, only a limited amount of material is needed to generate enough data. This allows researchers to study the early development of organisms from its first cell divisions, when the embryo consists of only a few cells.
Using CRISPR-Kill for organ specific cell elimination by cleavage of tandem repeats
by Angelina Schindele, Fabienne Gehrke, Carla Schmidt, Sarah Röhrig, Annika Dorn, Holger Puchta in Nature Communications
With the help of the CRISPR/Cas molecular scissors, genetic information in a plant can be modified to make the latter more robust to pests, diseases, or extreme climatic conditions. Researchers of Karlsruhe Institute of Technology (KIT) have now developed this method further to eliminate the complete DNA of specific cell types and, thus, prevent their formation during plant development. This will also help scientists better understand development mechanisms in plants.
By means of molecular scissors, the DNA — the carrier of genetic information — can be modified in plants. So far, the CRISPR/Cas method co-developed in plants by Professor Holger Puchta, molecular biologist at KIT’s Botanical Institute has already been used to specifically insert, exchange or combine genes. The goal is to increase the plant’s resistance to diseases and environmental impacts. CRISPR (stands for Clustered Regularly Interspaced Short Palindromic Repeats)/Cas are molecular scissors that can specifically recognize and cut DNA sequences. “We have studied molecular scissors for plant use for 30 years now. In the beginning, we applied them to modify individual genes. Two years ago, we were the first worldwide to restructure complete chromosomes,” Puchta says. For his research, the pioneer of genome editing twice received the Advanced Grant of the European Research Council (ERC). “We were able to optimize this method. With CRISPR-Kill, we have reached now an entirely new level of development: We can eliminate certain plant cell types and prevent the formation of specific plant organs.”
The experiments carried out by the scientists concentrated on secondary roots and petals of the model plant thale cress (Arabidopsis thaliana). “These are classical examples in biology. Here, we know the genetic program and the cell types that are important for the formation of these plant organs,” the molecular biologist explains. After the elimination of these cells, CRISPR-Kill plants no longer formed any petals or secondary roots, whereas the control plants exhibited normal growth.
Contrary to other methods that eliminate cells with cytotoxins or laser radiation, CRISPR-Kill induces multiple cuts in the genome. A genome consists of a certain number of chromosomes, on which the individual genes are arranged in fixed order.
“So far, CRISPR/Cas has aimed for exactly one location and has cut once or twice to modify a gene or chromosome,” Puchta says. “Now, we have reprogrammed our molecular scissors. They no longer address the genomic DNA only once, but aim in the respective cell type for a sequence that is encountered often in the genome and that is essential for the survival of the cell. In this way, many cuts are induced at the same time — too many for the cell to repair them. The cell will die.”
The work of the KIT researchers can be classified as fundamental research. “By studying what happens when a certain cell type is eliminated, we learn more about the development processes in plants. How does the plant react? How flexible is the plant during development? Can we remove parts of plants that are not necessary in agriculture, for instance?,” Puchta adds. In the long term, food production and pharmaceutical applications might profit from this technology when the plant is prevented from forming cells that produce toxins, for instance. Moreover, the technology might be applied in multi-cellular organisms for the specific modification of tissues.
Spindle-shaped archaeal viruses evolved from rod-shaped ancestors to package a larger genome
by Fengbin Wang, Virginija Cvirkaite-Krupovic, Matthijn Vos, Leticia C. Beltran, Mark A.B. Kreutzberger, Jean-Marie Winter, Zhangli Su, Jun Liu, Stefan Schouten, Mart Krupovic, Edward H. Egelman in Cell
From hot volcanic springs where the water is nearly boiling acid, scientists have discovered how lemon-shaped viruses got their form. And that discovery could lead to new and better ways to deliver drugs and vaccines.
While the vast majority of viruses are either rod-like or spherical (such as the coronavirus responsible for COVID-19), scientists have been puzzled by the unusual forms of viruses found in some of the harshest environments on Earth. The researchers were studying one such virus when they discovered it has strange properties that let it alter its shape. While it normally resembles a lemon or spindle, the virus can grow tails. The structure that lets it do that, the scientists realized, likely explains how ancient rod-like viruses gave rise to all the spindle-shaped viruses seen today.
“We can now understand a new principle in how proteins can form the shell that packages the DNA in a virus,” said lead researcher Edward H. Egelman, PhD, of the University of Virginia School of Medicine. “This has implications for not only understanding how certain viruses evolved but potentially can be used for new ways to deliver everything from drugs to vaccines.”
The virus Egelman and his colleagues were studying, Sulfolobus monocaudavirus 1 (SMV1), has a protein shell surrounding the DNA that is spindle- or lemon-shaped. But it has been a puzzle for almost 20 years exactly how that many copies of the same protein can come together to form such a shape. Egelman and his team were able to reveal the strange properties of SMV1 using high-tech cryo-electron microscopy and advanced image processing. (Egelman was elected to the National Academy of Sciences for his pioneering work using cryo-electron microscopy and 3D modeling to map out the world that is far too small for even the most powerful light microscopes to see.)
SMV1, the researchers found, contains strands of proteins that that slip and slide past each other, due to the fact that they are “greasy.” These seven strands of proteins were found in both the body and tail of the virus, and they give the virus a remarkable ability to shapeshift. Rather than having a fixed shape, it can balloon up like a pufferfish to accommodate genetic material. At the same time, these strands form an impenetrable barrier to prevent the acid which surrounds them from destroying the DNA inside the virus. The virus is a formidable threat to the single-celled organisms it infects. Once infected, the host organisms turn into giant factories churning out more virus. These host cells grow up to 20 times larger before a new army of viruses bursts forth.
Based on their findings, Egelman and his collaborators conclude that today’s viruses shaped like spindles or lemons likely evolved from ancient rod-shaped ancestors. The rod-shaped viruses could only contain a limited amount of DNA, and the “greasy” properties that let SMV1 shapeshift would have let the ancestral viruses package more genetic material — a useful trait for viruses, from an evolutionary perspective.
“Viruses can pose great threats to human health, as we see from the COVID-19 pandemic,” said Egelman, of UVA’s Department of Biochemistry and Molecular Genetics. “It is thus crucial that we understand more about how viruses have evolved. But we can also learn from viruses, and create new technologies based upon the principles found in these very simple structures.”
Structures of Tetrahymena ’s respiratory chain reveal the diversity of eukaryotic core metabolism
by Long Zhou, María Maldonado, Abhilash Padavannil, Fei Guo, James A. Letts in Science
Tetrahymena, a tiny single celled-organism, turns out to be hiding a surprising secret: it’s doing respiration — using oxygen to generate cellular energy — differently from other organisms such as plants, animals or yeasts. The discovery highlights the power of new techniques in structural biology and reveals gaps in our knowledge of a major branch of the tree of life.
“We thought we knew about respiration from studying other organisms, but this shows us how much we still don’t know,” said Maria Maldonado, a postdoctoral researcher in the Department of Molecular and Cellular Biology at the University of California, Davis and co-first author on the paper.
Tetrahymena is a genus of free-living, single-celled organisms usually found quietly swimming around ponds by beating their coat of tiny hairs, or cilia. Like us, they are eukaryotes, with their genetic material in a nucleus. They belong to a large and diverse group of organisms called the SAR supergroup. With a few exceptions, such as the malaria parasite Plasmodium, the SAR supergroup is little studied.
“It’s a huge proportion of the biosphere, but we don’t think about them much,” Maldonado said.
Like all other eukaryotes — and some bacteria — Tetrahymena consume oxygen to generate energy through respiration, said James Letts, assistant professor of molecular and cellular biology in the UC Davis College of Biological Sciences.
Oxygen comes in at the end of the series of chemical reactions involved in respiration. Electrons are passed through a chain of proteins located in structures called cristae in the inner membrane of the mitochondrion. This drives formation of water from oxygen and hydrogen atoms, pumping protons across the membrane, which in turn drives formation of the ATP, a store of chemical energy for the cell. This electron transport chain is fundamental to oxygen-based respiration in humans and other eukaryotes.
There were clues that there is something different about the electron transport chain in Tetrahymena, Letts said. In the 1970s and 80s, scientists discovered that its electron-carrying protein — cytochrome c — and oxygen consuming enzyme at the end of the chain — terminal oxidase — function differently than those in plants and animals. Until now, it wasn’t clear exactly how or why these enzymes differed in Tetrahymena when they were conserved across other studied eukaryotes.
Maldonado, Letts and co-first author Long Zhou used new approaches in structural biology to uncover the Tetrahymena electron transport chain. These included a cryo-electron microscopy structural proteomics approach — working out the structures of large number of proteins in a mixed sample at the same time.
Cryo-electron microscopy freezes samples to extremely low temperatures, creating images at almost atomic resolution. Instead of imaging a single, purified protein, the team worked with mixed samples isolated from mitochondrial membranes and then taught an algorithm to recognize related structures.
In this way, they were able to scan through hundreds of thousands of protein images and identify the structures of 277 proteins in three large assemblies, representing the Tetrahymena electron transport chain at near atomic resolution. Some of these proteins have no matching gene in the known Tetrahymena genome database — showing that there must be gaps in the available reference genome.
By revealing the gaps in our knowledge of a fairly common organism, the work shows our blind spots with respect to biodiversity, Letts said. It also shows the potential of these new methods in structural biology as a discovery tool, he said.
A keystone gene underlies the persistence of an experimental food web
by Matthew A. Barbour, Daniel J. Kliebenstein, Jordi Bascompte in Science
More than 50 years ago on the shoreline of a rocky tide pool, the US ecologist Robert Paine found out that the removal of a single species from an ecosystem could dramatically alter its structure and function. He had discovered that starfish act as a keystone species in that their presence and role as a top predator maintained the coexistence of diverse species in the rocky intertidal zone.
A team of ecologists and geneticists at the University of Zurich (UZH) and the University of California, Davis have now found that a mutation at a single gene can also dramatically alter the structure and function of an ecosystem. The study suggests that a gene not only encodes information that determines an organism’s fitness, but can also influence the persistence of interacting species in an ecological community. The discovery of Jordi Bascompte, professor at the UZH Department of Evolutionary Biology and Environmental Studies, and his team was made using an experimental ecosystem in the lab with a predator (a parasitic wasp), two herbivores (aphids), and the plant Arabidopsis thaliana — a genetic model organism.
The researchers tested the effect of three plant genes that control the plant’s natural arsenal of chemical defenses against herbivores. They found that the herbivores and predator in their experimental community were more likely to survive on plants with a mutation at a single gene called AOP2. “This natural mutation at AOP2 not only affected the plant’s chemistry, but also made the plant grow faster, which in turn helped the herbivores and predator coexist, thereby preventing the ecosystem from collapsing,” UZH scientist and first author Matt Barbour explains. Similar to a keystone species such as the starfish, AOP2 acts as a “keystone gene” that is critical to the survival of the experimental ecosystem.
The discovery of a keystone gene is likely to have implications on how to conserve biodiversity in a changing world. In particular, knowledge from genetics and ecological networks should be included when it comes to predicting the consequences of genetic change for the persistence of biodiversity across scales. Individuals with different variants of a gene or even genetically modified organisms could be added to existing populations to foster more diverse and resilient ecosystems. However, a seemingly small genetic change could unleash a cascade of unintended consequences for ecosystems if not studied in detail first.
“We’re only just beginning to understand the implications of genetic change on how species interact and coexist. Our findings show that the current loss of genetic diversity may have cascading effects that lead to abrupt and catastrophic shifts in the persistence and functioning of terrestrial ecosystems,” says Barbour.
Particle Morphology of Medusavirus Inside and Outside the Cells Reveals a New Maturation Process of Giant Viruses
by Ryoto Watanabe, Chihong Song, Yoko Kayama, Masaharu Takemura, Kazuyoshi Murata in Journal of Virology
Giant viruses represent a unique group of viruses that are similar in size to small bacteria. Medusavirus — a special type of giant virus — was first isolated from a hot spring in Japan. Interestingly, genetic studies showed that medusavirus was more closely related to mature organisms called eukaryotes than to other giant viruses, suggesting that it may hold the key to understanding eukaryotic evolution. Although the details of medusavirus morphology and maturation in infected cells have so far remained elusive, the researchers behind its initial discovery now have some answers.
In a recent study, a team of Japanese scientists led by Prof. Kazuyoshi Murata from the National Institutes of Natural Sciences and Prof. Masaharu Takemura from Tokyo University of Science has revealed, for the first time, a unique four-stage maturation process that the medusavirus undergoes within host cells.
Prof. Takemura comments, “From an evolutionary perspective, the medusavirus is extremely interesting, as its replication process and genome are different from those of other viruses. Interestingly, medusavirus also has a unique particle structure. In this study, we wanted to make additional inroads towards elucidating the biology of this virus by characterizing its morphology and maturation process.”
To do this, the researchers used two techniques that allow the high-resolution visualization of viral infection — conventional transmission electron microscopy (C-TEM) and cryo-electron microscopy (cryo-EM). Using these techniques, they observed the detailed particle morphology of medusavirus in infected amoeba cells.
Their first and rather surprising discovery was the presence of four types of medusavirus particles both within and outside the infected host cells. Based on their features, these particles were named pseudo-DNA-empty (p-Empty, i.e., filled with spongy material but no DNA), DNA-empty (Empty, i.e., no spongy material or DNA), semi-DNA-full (s-Full, i.e., half-filled with DNA), and DNA-full (Full, i.e., completely filled with DNA) particles.
Subsequently, they performed time-course analysis, in which the gene expression was measured at several time points during maturation, and discovered that the four types of particles represented four consecutive stages of viral maturation. They found that unlike in other viruses, the viral capsid or shell of medusavirus was produced independently in the host cell’s cytoplasm, while the viral DNA was produced in the nucleus. Further, only empty capsids present near the host nucleus could incorporate viral DNA and become s-Full or DNA-full particles. These findings suggested that the medusavirus had a unique maturation process.
To observe the detailed structure of the four types of medusavirus particles, the team used the cryo-EM technique. They found that all the different particle types had a comparable outer structure, with the presence of three different spikes. The configuration of the capsid shell was also consistent with the structure of the membrane layer within the capsid. However, while s-Full and Full particles showed a complete internal membrane, p-Empty and Empty particles had “open membrane structures,” meaning the membrane had a gap at one end.
“Viruses are smart and can replicate and mature in various ways. Our findings reveal the unique way in which the medusavirus matures. The open membranes we observed in p-Empty and Empty particles were particularly interesting. We believe that the membrane gaps indicate an incompleteness and represent a state in which viral particles have not yet matured. The gaps are likely used to exchange DNA and proteins required for medusavirus maturation and disappear as the virus reaches its final stage,” explains Prof. Takemura.
These new insights not only demonstrate a novel mechanism of particle formation and maturation in medusavirus but also shed light on the great structural and behavioral diversity of giant viruses. They represent a “giant” leap in our knowledge of virus biology and call for further research into giant viruses, which could help answer numerous questions about evolution and infection.
Targeting Xist with compounds that disrupt RNA structure and X inactivation
by Rodrigo Aguilar, Kerrie B. Spencer, Barry Kesner, Noreen F. Rizvi, Maulik D. Badmalia, Tyler Mrozowich, Jonathan D. Mortison, Carlos Rivera, Graham F. Smith, Julja Burchard, Peter J. Dandliker, Trushar R. Patel, Elliott B. Nickbarg, Jeannie T. Lee in Nature
RNA (ribonucleic acid) plays many roles in human health, and now a study offers powerful evidence that RNA could also be a viable target for drug development. This work, led by researchers at Massachusetts General Hospital (MGH), suggests that a new class of biological factors numbering in the thousands can be targeted and thereby heralds a new era in drug development.
Nearly all drugs currently available target one of approximately 700 disease-related proteins among the roughly 20,000 human proteins identified by the Human Genome Project. However, in recent years there has been growing interest in expanding the list of “druggable” targets to include RNA. In cells, DNA (deoxyribonucleic acid) carries the genetic code for forming proteins. A segment of DNA is copied, or transcribed, into a “coding” RNA, which is in turn translated into protein. However, the vast majority of RNA in the human genome — 98 percent — is “noncoding.”
“These noncoding RNAs play very important roles in the genome, and we now understand that mutations in this noncoding space can result in disease,” says the senior author of the paper, Jeannie Lee, MD, PhD, of the Department of Molecular Biology at MGH. “And there may be far more of these RNA genes than there are protein-coding genes. If we could target these RNAs, we would hugely increase the universe in which we can find drugs to treat patients.”
However, the pharmaceutical industry has historically been hesitant to pursue RNA as a drug target. Proteins tend to have stable shapes, or conformations, which make them optimal targets: Drugs bind to proteins like a key in a lock. By contrast, explains Lee, RNA tends to be highly flexible, or “floppy,” and capable of assuming multiple conformations. “If a lock is constantly changing shape, your key is not going to work,” says Lee. Noncoding RNA’s unstable nature has made companies reluctant to invest in trying to develop medications that target it. However, it’s known that some regions on RNA retain stable conformations, despite all of that shape-shifting, but finding such regions has been a challenge.
Lee directs a molecular biology lab at MGH, where she and her team study RNA and its role in a biological process called X-chromosome inactivation (XCI), which deactivates one copy of the X chromosome in female mammals and is necessary for normal development. In a study led by postdoctoral fellow Rodrigo Aguilar, PhD, Lee’s group collaborated with colleagues at Merck Research Laboratories to find out if RNA could be a viable drug target. The focus of the study was a form of noncoding RNA called Xist, which silences genes on the X chromosome. Finding a way to interfere with this process and reactivate a dormant X chromosome could help guide development of treatments for genetic disorders caused by mutations on the X chromosome (known as X-linked disorders), such as Rett syndrome and Fragile X syndrome.
Together with Merck scientists Kerrie Spencer and Elliott Nickbarg, the MGH team screened Xist against a library of 50,000 small molecule compounds and found several that bind to a region called Repeat A (RepA) on Xist. One compound, which Lee’s team named X1, had particularly interesting qualities: It prevented several key proteins, PRC2 and SPEN, from binding to RepA, which is necessary for Xist to silence the X chromosome. “As a result, X inactivation cannot take place,” says Lee. To understand why, the team collaborated with structural biologists led by Trushar Patel of the University of Lethbridge in Canada. Normally, Xist’s RepA can assume 16 different conformations, but X1 caused it to adopt a more uniform shape. This structural change prevented RepA from binding with PRC2 and SPEN. The approach employed in this study could be used to identify other RNA-targeting drugs.
“This really opens up a large universe for new drug development,” says Lee. “Now we don’t just have 700 proteins to target using small molecules. In the future, we may have tens and possibly hundreds of thousands of RNAs to target to cure disease.”
Labeling of heterochronic ribosomes reveals C1ORF109 and SPATA5 control a late step in human ribosome assembly
by Chunyang Ni, Daniel A. Schmitz, Jeon Lee, Krzysztof Pawłowski, Jun Wu, Michael Buszczak in Cell Reports
UT Southwestern researchers have identified a four-protein complex that appears to play a key role in generating ribosomes — organelles that serve as protein factories for cells — as well as a surprising part in neurodevelopmental disorders. These findings could lead to new ways to manipulate ribosome production, which could impact a variety of conditions that affect human health.
“Ribosomes are fundamental for life, but we’ve had an incomplete understanding of how they’re put together and how the process of ribosome production is regulated,” said lead author Michael Buszczak, Ph.D., Professor of Molecular Biology and member of the Harold C. Simmons Comprehensive Cancer Center at UT Southwestern. “Our findings shed significant light on these questions.”
Dr. Buszczak explained that ribosomes are present in varying amounts in every cell of every organism on Earth. Because of their key role as protein producers, he added, variations from these natural set points can have deleterious consequences. For example, cancer cells tend to increase ribosome production to boost protein production necessary for unchecked cell division. In addition, a group of rare diseases known as ribosomopathies — characterized by abnormal ribosome production — manifests with a variety of symptoms including anemia, craniofacial defects, and intellectual disability.
Although every species has ribosomes, most of what’s known about ribosome biogenesis has come from the popular lab model, yeast. The basics of this process are the same for human ribosome biogenesis, Dr. Buszczak said, but the specifics are not. Consequently, the details that make human ribosome generation unique have been unknown.
To learn more about this process, Dr. Buszczak, Chunyang Ni, a graduate student in the Buszczak lab, and their colleagues, including Jun Wu, Ph.D., Assistant Professor of Molecular Biology at UTSW, started by developing a technique that prompted old ribosomes to glow red and newly generated ribosomes to glow green. The researchers used this tool on several different human cell types, confirming different rates of ribosome production in each. Using the gene editing tool called CRISPR, the researchers inactivated individual genes to identify those that might be key players in ribosome biogenesis. Their search turned up four genes known as CINP, SPATA5L1, C1orf109, and SPATA5. Further research showed that these genes come together into a complex that strips a placeholder protein from ribosomes when assembly is almost complete, allowing a different protein to take its place for ribosome maturation.
Previously, SPATA5’s function in cells had been unknown; however, mutations in this gene have been associated with neurodevelopmental disorders including microcephaly, hearing loss, epilepsy, and intellectual disability. When the researchers inserted two of these mutations into cells, causing them to create a mutant SPATA5 protein, the cells couldn’t generate the normal level of functional ribosomes — suggesting that these neurodevelopmental disorders could stem from ribosome problems.
Dr. Buszczak said that he and his colleagues plan to study why the central nervous system appears to be more sensitive than other cell types to ribosomal disruptions. He added that these findings could eventually lead to new treatments for cancer, ribosomopathies, and other conditions affected by over- or under-production of proteins.
Assembly mechanism of the pleomorphic immature poxvirus scaffold
by Jaekyung Hyun, Hideyuki Matsunami, Tae Gyun Kim, Matthias Wolf in Nature Communications
Researchers at the Okinawa Institute of Science and Technology Graduate University (OIST) have revealed how poxviruses build their scaffold — a temporary protein coat that forms and disappears as the virus matures.
The scientists revealed the structure of a protein called D13, in near-atomic resolution, and showed how it assembles with other copies of D13 to form scaffold-like structures.
“D13 is a key target for research, because if you know how the scaffold is assembled, you can design new drugs that prevent it from forming,” said Professor Jaekyung Hyun, a former staff scientist in the OIST Molecular Cryo-Electron Microscopy Unit, and now Assistant Professor at Pusan National University in South Korea. “If the scaffold can’t form, then replication of the virus stops.”
D13 is a trimer protein, as it is formed from three identical protein chains. Once synthesized, it acts as a scaffold building block for the Vaccinia virus — a harmless strain developed in the laboratory as a vaccine against smallpox. Researchers now use the Vaccinia virus as a model for all poxviruses.
“Smallpox is the most famous and lethal disease caused by a poxvirus, with 1 in 3 infected people dying,” said Professor Wolf, who leads the Molecular Cryo-Electron Microscopy Unit. “But while smallpox has been eradicated in the wild, there are fears that it could be used as a bioweapon. Also, numerous other poxviruses still infect humans and livestock, so further research into how these viruses replicate is essential.”
The scaffold seen in immature poxviruses is of particular interest to scientists, as the structure differs from the protein coats typically seen in viruses. While most viruses have regular and symmetrical structures, poxvirus scaffolds have roughly spherical honeycomb lattices, which vary in shape between each viral particle.
To determine how these blocks assemble into these spherical honeycomb lattices, the research team used cryo-electron microscopy (cryo-EM) — a technique in which samples are frozen in liquid nitrogen and probed by electrons — to generate 3D images of both single D13 protein trimers and two connected D13 protein trimers, at the highest resolution seen so far. They found that the two proteins joined together with a slight twist between their trimer axes, creating a curve that is key for forming a spherical shape. However, when the team used computer modelling to extend the interaction to multiple D13 proteins, they didn’t fit together properly.
“This told us that there must be at least one other way for the two proteins to interact, that we hadn’t yet seen,” said Prof. Hyun.
The researchers also found that when they compared the single D13 protein to the two D13 proteins joined together, a small helix structure at the end of the protein chains had shifted. Previously, the helix structure was buried deep into the pocket where the two proteins interact, suggesting that its repositioning was critical for the two proteins to connect. To explore the role of the helix structure further, the research team made modified D13 proteins and then used cryo-electron tomography to look at how they self-assembled when placed in solution. When a purification tag was added to the helix, the proteins formed spherical structures similar to the immature poxvirus scaffold. And when the helix was completely removed, the researchers were surprised to see the formation of cylindrical tubes.
Using cryo-EM, the research team were able to capture high-res images of these cylinders and zoom in on the honeycomb structure. They identified a second way for the dimers to interact and found that when they modelled alternating patterns of interaction, the D13 proteins fit together to form the hexagonal honeycomb pattern. Both modes of how the proteins interacted required the small helix structure to shift, with the proteins then held together by the attraction between positively and negatively charged amino acids. Overall, the researchers proposed that displacement of the helix was essential for forming poxvirus scaffold, and likely acted as a trigger for the assembly to begin.
“When the poxvirus replicates inside cells, the scaffold forms in association with a lipid membrane,” explained Prof. Wolf. “The helix structure is hydrophobic, which means that it would shift towards to the water-free environment of the lipid membrane, freeing up the pocket where the D13 proteins interact.”
The discovery of the helix’s role in assembly could be a promising new avenue of research for antiviral drug discovery, emphasized Prof. Hyun.
“If we can design a drug that binds really strongly into the pocket where the helix usually sits, it would interfere with formation of the scaffold,” he said. “This is one of my next goals.”
A novel approach for reliable qualitative and quantitative prey spectra identification of carnivorous plants combining DNA metabarcoding and macro photography
by Thilo Krueger, Adam T. Cross, Jeremy Hübner, Jérôme Morinière, Axel Hausmann, Andreas Fleischmann in Scientific Reports
Researchers have combined macro photography with DNA metabarcoding to create a new botanical “CSI” tool that may hold the key to safeguarding the future of Australia’s critically endangered carnivorous plants.
The new technology — developed by researchers from Curtin University, the Botanical and Zoological Natural History Collections in Munich and the University of Munich — enables experts to take a sophisticated look inside the stomachs of carnivorous plants, overcoming a hurdle that had previously stumped entomologists.
Researchers set off on a 6,000km journey to Western Australia’s remote Kimberley region to test the new method, capturing macro photographs of carnivorous plants of the genus Drosera, known as sundews. Lead author Mr Thilo Krueger, a PhD student from Curtin’s School of Molecular and Life Sciences, said understanding how many and what kinds of insects that carnivorous plants ate was critical to their survival.
“Western Australia has — by far — the highest number of carnivorous plant species in the world and many of them are critically endangered, threatened by habitat destruction, environmental pollution and climate change,” Mr Krueger said. “Quite often, several carnivorous plant species are found in one habitat, and the question arises if different species may rely upon different food sources. To develop conservation plans that protect their future, it is essential to understand their biology, which includes what they eat — their natural prey spectra.
“Studying the prey spectra of carnivorous plants has previously been hampered by the fact that digested insect prey is often hard to identify, even by trained entomologists. Soft-bodied insects such as midges often turn into unidentifiable crumbs during digestion on the leaves.”
Co-author Dr Adam Cross, a Botanist and Restoration Ecologist from Curtin’s School of Molecular and Life Sciences, said the new method combined macro photography of the captured insects with DNA metabarcoding, a cutting-edge insect identification tool.
“Any insect that is captured by a carnivorous plant will contain traces of its genetic material or DNA, even after digestion by the plant. This DNA can be detected and compared with DNA libraries of known insects, thus identifying the prey,” Dr Cross said.
“Because DNA metabarcoding is prone to contaminations and does not allow us to estimate the quantity of prey, we carefully controlled our data using macro photographs of the prey items to achieve an unprecedented completeness of prey spectra data.”
Senior author Dr Andreas Fleischmann, from the Botanical Natural History Collection and the University of Munich, said this new method of DNA metabarcoding was so sensitive that it even detected tiny amounts of insect DNA that were not obvious to researchers from field examination and macro photographs.
“Hence, our study of carnivorous prey spectra using genetic DNA fingerprints from the captured insects resembled reconstructing a crime scene — except our crime scene investigation was about finding out what a set of carnivorous plants had for lunch,” Dr Fleischmann said.
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