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Tag: Nucleotides

  • Targeting a non-encoding stretch of RNA may shrink pediatric brain tumors in mice

    Targeting a non-encoding stretch of RNA may shrink pediatric brain tumors in mice

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    Targeting a non-encoding stretch of RNA may help shrink tumors caused by an aggressive type of brain cancer in children, according to new research in mice reported March 8 in Cell Reports by Johns Hopkins Kimmel Cancer Center investigators. 

    Medulloblastoma are the most common type of malignant brain cancer in children. The most aggressive and difficult-to-treat form of the disease is group 3 medulloblastoma, which is often fatal. By targeting long, noncoding genetic material called lnc-RNAs that drive the expression of cancer-causing genes, the study’s senior author, Ranjan Perera, Ph.D., director of the Center for RNA Biology at Johns Hopkins All Children’s Hospital in St. Petersburg, Florida, and his colleagues have demonstrated an innovative new approach that shrinks group 3 medulloblastoma tumors in mice. 

    “Group 3 medulloblastoma is very aggressive, and there are currently no targeted therapies,” says Perera, who has a primary affiliation in the Department of Neurosurgery, is a member of the Johns Hopkins Kimmel Cancer Center and is an associate professor of oncology at the Johns Hopkins University School of Medicine. He is also a senior scientist at the Johns Hopkins All Children’s Hospital Cancer and Blood Disorders Institute, and has a secondary affiliation with the hospital’s Institute for Fundamental Biomedical Research. “Our novel therapeutic approach based on noncoding RNA could fill an urgent need for new therapies for this devastating disease in children.” 

    RNA acts as a template for building proteins based on instructions encoded in the DNA. Until recently, scientists thought 97% of RNA was “junk” because only 3% is used to build proteins. However, scientists have realized that RNA’s nonprotein encoding stretches control gene expression. A previous study by Perera and colleagues showed that a long noncoding stretch of RNA called lnc-HLX-2-7 contributes to the growth of group 3 medulloblastoma tumors by attaching to a DNA promoter that increases expression of cancer-causing genes. Promoters are nongene coding stretches of DNA adjacent to genes that act like switches turning them on. 

    The new study provides additional details showing that lnc-HLX-2-7 specifically binds to the HLX promoter region of DNA, increasing HLX gene expression and causing the tumor to grow. HLX triggers tumor growth by binding to promoter regions for several other cancer-causing genes, increasing their expression. One gene that HLX increases expression of is MYC, which also increases the expression of several other cancer-causing genes, causing a cascade of activity that accelerates the growth of group 3 medulloblastoma tumors. 

    Perera and his team developed an intravenous treatment to block lnc-HLX-2-7 from binding to the HLX promoter to stop this cascade of cancer-gene expression. They assembled a sequence of nucleotides (called antisense oligo nucleotides), the building blocks of RNA, that can bind to the corresponding nucleotides that make up lnc-HLX-2-7, preventing it from binding to the HLX promoter in the DNA and leading to its destruction. They coated the sequence with microscopic particles called cerium oxide nanoparticles to protect the lnc-HLX-2-7 until it reaches its target. 

    When the team treated a mouse model of group 3 medulloblastoma with the experimental intravenous therapy, it reduced tumor growth by 40%–50%. Adding cisplatin, a chemotherapy drug currently used to treat medulloblastomas, alongside the new therapy caused the tumors to shrink even more and prolonged the animals’ survival. The combination therapy extended the animals’ lives by about 84 days compared with a 44-day increase in survival on lnc-HLX-2-7 alone. 

    “When you combine the two treatments, you see dramatic effects,” Perera says. 

    Perera and his colleagues will collaborate with Johns Hopkins neurosurgeons to plan studies of the therapy in humans to further test its safety and efficacy. 

    Understanding why MYC is elevated in these tumors is extremely important, and this new link to HLX provides insights that open new therapeutic possibilities.”


    Charles Eberhart, M.D., Ph.D., study co-author, Kimmel Cancer Center researcher, director of neuropathology and ophthalmic pathology and professor of oncology and pathology at the Johns Hopkins University School of Medicine

    The work was supported by the Schamroth Project, funded by Ian’s Friends Foundation, the Hough Family Foundation, the National Institutes of Health (grant P30 CA006973), the National Cancer Institute (grants 5P30CA030199, R01NS124668-01A1, and R35NS122339), and a CPRIT Scholar award from the MD Anderson Cancer Center.

    Study co-authors were Keisuke Katsushima, Kandarp Joshi, Menglang Yuan, Stacie Stapleton and George Jallo from Johns Hopkins. Other authors were from the University of Delaware; the University of Central Florida, Orlando; Institute Curie at PSL University in Paris; Texas Children’s Cancer Center, Houston; Baylor College of Medicine, Houston; and Columbia University Medical Center, New York. 

    Source:

    Journal reference:

    Katsushima, K., et al. (2024). A therapeutically targetable positive feedback loop between lnc-HLX-2-7, HLX, and MYC that promotes group 3 medulloblastoma. Cell Reports. doi.org/10.1016/j.celrep.2024.113938.

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  • European initiative aims to advance the understanding of RNA communication

    European initiative aims to advance the understanding of RNA communication

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    RNA, which stands for ribonucleic acid has become a mainstream word in society due to the COVID-19 RNA vaccine – yet the value of this biomolecule to society could extend to numerous sectors including food security and personalized medicine.

    RNA is one of the oldest molecules on earth that, like DNA, has a relatively simple alphabet of 4 nucleotides. A nucleotide is the basic building block of nucleic acids (RNA and DNA).

    While DNA contains the blueprint for all genes in an organism, RNA instructs the cell which proteins to make and regulates how much and when. The dynamic nature of RNA means that it can help cells to be able to respond to the environment and fight infections.

    Fascinating discoveries show that RNA can also be released from cells and transferred to other cells as a form of cell-to-cell communication. This extracellular RNA (exRNA) is important in a variety of health and disease processes, e.g. in the growth and metastasis of tumors.

    There is increasing interest in using RNA-based therapies in disease and infection control since RNA can be easily designed and exploited to mediate gene regulation. However, there are still gaps in knowledge of how to design and deliver RNA. There is an urgent need to learn from, and account for, the natural RNA communication systems that exist and the different organisms that participate. How exRNAs are selected for export, how they traffic outside the cell, how they integrate into a functional pathway in a recipient, and how pathogens exploit these mechanisms.

    exRNA has also been found across microbe, fungi, plant, and animal systems and could play a much more fundamental role in how organisms communicate with and influence one another. Recent studies have shown that nearly all classes of pathogens can release RNA that might help the pathogen change host cells and survive. This has now led to numerous initiatives to explore how exRNA may be used and exploited for diagnostics, therapies, and pest control.

    Introducing exRNA-PATH COST Action

    Scientists studying exRNA and its carriers across diverse biological models, from honeybees to plants and humans are now gathered through European COST Action, RNA communication across kingdoms: new mechanisms and strategies in pathogen control (exRNA-PATH), which has been established to coordinate basic research as well as healthcare and agricultural initiatives involving exRNA.

    Europe leads the way in RNA communication research, and exRNA-PATH is harnessing this expertise to establish a collaborative platform. The COST Action involves over 180 scientists from 21 countries, to exchange information on how different species use RNA in communication, with a specific focus on host-pathogen interactions. This collaborative network spans diverse biological systems and backgrounds (including biologists, chemists, bioinformaticians, physicists, and medics), and translational applications of RNA delivery. Industry engagement is also a key focus, with three European companies already on board and plans to expand connections.

    The main goal of this initiative is to advance our understanding of RNA communication. The aim is to establish a research agenda focused on targeting RNA in a way that aligns with sustainable development goals, particularly in the realms of infectious disease and pest control.

    There is a huge need for coordination of knowledge and expertise to advance the young field of RNA communication – it is hugely rewarding to help bring together diverse voices and disciplines to do this.”

    Dr. Amy Buck, COST Action Chair

    In a strategic move towards sustained leadership in extracellular RNA (exRNA) research, Europe is set to strengthen its commercial standing and harness exRNA for innovative strategies against emerging infectious diseases, including COVID-19, and sustainable pest control.

    Aligned with European Research Area priorities and the UN Sustainable Development Goals targets, the initiative prioritizes support for exRNA research in International Cooperation. Emphasizing unity and data sharing, the Action aims to foster collaboration among researchers to use diverse models (plants, animals, microbes) in a bottom-up approach to One Health. The establishment of a comprehensive long-term roadmap will support European and national policymakers, as well as funding bodies, in shaping the future of exRNA research and innovation.

    Given the advances in RNA manufacturing during the COVID-19 pandemic, it is an exciting time to understand how to harness this universal language to improve health and the environment safely and sustainably.

    Inter-species RNA communication

    In September 2023, Dr Amy Buck, Professor of RNA and Infection Biology, who chairs the COST Action was awarded the Max Planck-Humboldt Medal, for her research into inter-species communication via RNA.

    Source:

    European Cooperation in Science and Technology (COST)

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  • Novel splicing mechanism for short introns discovered

    Novel splicing mechanism for short introns discovered

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    Researchers confirm that the established pre-mRNA splicing mechanism that appears in textbooks cannot work in a subset of human short introns: A novel SAP30BP–RBM17 complex-dependent splicing has been uncovered.

    The well-known essential pre-mRNA splicing factor U2AF heterodimer (U2AF2–U2AF1) has been considered to mediate early splicing reaction in all introns of different lengths. However, Dr. Kazuhiro Fukumura in the Akila Mayeda lab at Fujita Health University has discovered that a subset of short introns with truncated polypyrimidine tracts are spliced by RBM17–SAP30BP complex instead of U2AF heterodimer. They have proposed a unique mechanism in which SAP30BP guides RBM17 to active early spliceosomes.

    In humans, the length of pre-mRNA varies extensively (from 30 to 1,160,411 nucleotides by recent studies). The fundamental mechanism of splicing has been studied with model pre-mRNAs including 158- and 231-nt introns, for historical instance, that are spliced very efficiently in vitro and in vivo. Such an ideal pre-mRNA contains good splicing signal sequences, i.e., the 5′ splice site, the branch-site (BS) sequence, and the polypyrimidine tract (PPT) followed by the 3′ splice site that are recognized by U1 snRNP, U2 snRNP and U2AF2–U2AF1, respectively. Prof. Mayeda says, “Given the diverse lengths of human introns, it is likely that more than one mechanism exists. This is our motivation to initiate our study of splicing focused on human short introns.”

    Our previous research on the splicing process on short intron revealed that the authentic splicing factor U2AF2 cannot bind to the truncated PPT and then RBM17 is replaced with U2AF to promote splicing. You know, this is reasonable because short introns are often too tight for the sufficient length of PPT. We published this finding in 2021. However, RBM17 cannot bind to the truncated PPT in vitro, so we did not know how the truncated PPT and the following the 3′ splice site are recognized by RRM17. Therefore, we hypothesized that another protein factor is involved in RBM17-dependent splicing.” 


    Dr. Kazuhiro Fukumura, Fujita Health University

    The Mayeda group eventually identified this protein cofactor behind the RBM17-dependent splicing, which is the SAP30BP. Their study was published in Volume 42, Issue 12 of the journal Cell Reports on December 07, 2023.

    Dr. Fukumura states, “It was critical to investigate previous references. From three papers, I was convinced that SAP30BP is the strongest candidate for the cofactor of RBM17.” They showed that the existence of SAP30BP in human early splicing complex, the fruit fly SAP30BP, and RBM17 were detected in fly spliceosome formed on a short intron, and the binding between SAP30BP and RBM17 was indeed detected by yeast two-hybrid analysis. “Nowadays, siRNA-mediated depletion of SAPBP in human cell line is the easy straightforward way to check the repression of RBM17-dependent splicing. And it was bingo!” says Dr. Fukumura.

    The transcripts in SAP30BP-depleted human cells were analyzed by a next-generation sequencer (RNA-Seq analysis), and many RBM17- and SAP30BP-dependent introns were found. These introns were distributed in the shorter range and the truncated PPT was indeed a critical determinant of the RBM17/SAP30BP-dependency. Thus, RBM17 and SAP30BP are the general splicing factors.

    Prof. Mayeda remarks, “It was a lucky coincidence that Prof. Michael Sattler, who is an expert in structural analyses, was keenly interested in our study, and we could start a productive collaboration.” Protein–protein interactions through UHM (U2AF-homology motif)–ULM(UHM-ligand motif) binding play essential roles in general splicing reactions. The Sattler lab found a hidden ULM sequence in SAP30BP, and demonstrated this was critical to interact with UHM in RBM17 by NMR (nuclear magnetic resonance) and ITC (isothermal titration calorimetry) analyses.

    However, the role of RBM17–SAP30BP interaction remained enigmatic. Since RBM17 has only one UHM, the RBM17–SAP30BP binding has to be released before the RBM17 interaction with SF3B1, one component of U2 snRNP, that is essential to promote splicing. So, what is the role of the RBM17–SAP30BP interaction? Prof. Mayeda says, “Fukumura designed a smart binding assay using anti-phospho-SF3B1 antibodies to address this curious question, and we could provide an elegant working model (see IMAGE Figure).” We propose that the intermediary RBM17–SAP30BP complex prevents non-functional RBM17 binding to free unphosphorylated SF3B, which promotes functional RBM17 binding to active phosphorylated SF3B1 on pre-mRNA.

    Source:

    Journal reference:

    Fukumura, K., et al. (2023). SAP30BP interacts with RBM17/SPF45 to promote splicing in a subset of human short introns. Cell Reports. doi.org/10.1016/j.celrep.2023.113534.

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  • Researchers identify a new approach to controlling bacterial infections

    Researchers identify a new approach to controlling bacterial infections

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    Researchers at the Icahn School of Medicine at Mount Sinai have identified a new approach to controlling bacterial infections. The findings were described in the February 6 online issue of Nature Structural & Molecular Biology [DOI # 10.1038/s41594-024-01220-x].

    The team found a way to turn on a vital bacterial defense mechanism to fight and manage bacterial infections. The defense system, called cyclic oligonucleotide-based antiphage signaling system (CBASS), is a natural mechanism used by certain bacteria to protect themselves from viral attacks. Bacteria self-destruct as a means to prevent the spread of virus to other bacterial cells in the population.

    We wanted to see how the bacterial self-killing CBASS system is activated and whether it can be leveraged to limit bacterial infections. This is a fresh approach to tackling bacterial infections, a significant concern in hospitals and other settings. It’s essential to find new tools for fighting antibiotic resistance. In the war against superbugs, we need to constantly innovate and expand our toolkit to stay ahead of evolving drug resistance.”


    Aneel Aggarwal, PhD, Co-Senior Author, Professor of Pharmacological Sciences at Icahn Mount Sinai

    According to a 2019 report by the Centers for Disease Control and Prevention, more than 2.8 million antimicrobial-resistant infections occur in the United States each year, with over 35,000 people dying as a result.

    As part of the experiments, the researchers studied how “Cap5,” or CBASS-associated protein 5, is activated for DNA degradation and how it could be used to control bacterial infections through a combination of structural analysis and various biophysical, biochemical, and cellular assays. Cap5 is a key protein that becomes activated by cyclic nucleotides (small signaling molecules) to destroy the bacterial cell’s own DNA.

    “In our study, we started by identifying which of the many cyclic nucleotides could activate the effector Cap5 of the CBASS system,” says co-senior author Olga Rechkoblit, PhD, Assistant Professor of Pharmacological Sciences at Icahn Mount Sinai. “Once we figured that out, we looked closely at the structure of Cap5 when it’s bound to these small signaling molecules. Then, with expert help from Daniela Sciaky, PhD, a researcher at Icahn Mount Sinai, we showed that by adding these special molecules to the bacteria’s environment, these molecules could potentially be used to eliminate the bacteria.”

    The researchers found that determining the structure of Cap5 with cyclic nucleotides posed a technical challenge, requiring expert help from Dale F. Kreitler, PhD, AMX Beamline Scientist at Brookhaven National Laboratory. It was achieved by using micro-focused synchrotron X-ray radiation at the same facility. Micro-focused synchrotron X-ray radiation is a type of X-ray radiation that is not only produced using a specific type of particle accelerator (synchrotron) but is also carefully concentrated or focused on a tiny area for more detailed imaging or analysis.

    Next, the researchers will explore how their discoveries apply to other types of bacteria and assess whether their method can be used to manage infections caused by various harmful bacteria.

    The paper is titled “Activation of CBASS-Cap5 endonuclease immune effector by cyclic nucleotides.”

    Other authors who contributed to this work are Angeliki Buku, PhD, and Jithesh Kottur, PhD, both with Icahn Mount Sinai.

    The work was funded by National Institutes of Health grants R35-GM131780, P41GM111244, KP1605010, P30 GM124165, S10OD021527, GM103310, and by the Simons Foundation grant SF349247.

    Source:

    Mount Sinai Health System

    Journal reference:

    Rechkoblit, O., et al. (2024). Activation of CBASS Cap5 endonuclease immune effector by cyclic nucleotides. Nature Structural & Molecular Biology. doi.org/10.1038/s41594-024-01220-x.

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