Publication Highlights

Kristin Koutmou, Ph.D.

Markos Koutmos, Ph.D.

A University of Michigan team of biochemists, led by Kristin Koutmou, Ph.D., and Markos Koutmos, Ph.D., Assistant Professors in the Department of Chemistry and Department of Biophysics, is reframing the understanding of the biology of a class of enzymes called Pseudouridine Synthases (Pus enzymes). These enzymes modify many types of RNAs, and the Koutmou and Koutmos Labs’ research brings new insights into the selection principles that guide modification incorporation. Their results are published in the Proceedings of the National Academy of Sciences (PNAS).

This novel understanding of pseudouridine biology could have important therapeutic implications because the dysregulation of Pus enzymes is linked to inherited diseases impacting muscle and brain function, such as progressive mitochondrial myopathy and sideroblastic anemia (MLASA). Furthermore, these enzymes also catalyze pseudouridine incorporation into RNA viral genomes, including that of SARS-CoV-2. As such, Pus enzymes present a potential new target for the development of therapeutics.

Since RNAs are central to the protein synthesis machinery, its chemical modifications can alter how fast and/or how accurately proteins are made. These modifications can have many consequences for cellular health, overall cellular adaptation, and cellular regulation. [Read more…] about Koutmou and Koutmos results published in the Proceedings of the National Academy of Sciences (PNAS)

Sami Barmada, M.D., Ph.D., Department of Neurology and member of the Center for RNA Biomedicine, with Nathaniel Safren, Ph.D., from Northwestern University, and other UM colleagues, was awarded “Editor’s pick” by the Journal of Biological Chemistry (JBC). This recognition signals “a top-rated paper published in JBC across the field of biological chemistry, as determined by JBC’s Associate Editors, Editorial Board Members and other referees.”

In this article, Barmada and his team are interested in autophagy activity, a pathway responsible for mediating the breakdown of cellular proteins and organelles. Autophagy can be compared to a cellular cleaning service, and its dysfunction is associated with many diseases including cancers and neurodegenerative diseases.

Although autophagy plays an important role in cellular biology, to this day, methods to accurately measure its activity are limited and somewhat unreliable. The Barmada team developed a novel biochemical technique that uses protein fluorescent tags inserted into the genome via CRISPR Cas9. This method enables accurate and sensitive assessments of autophagy in living cells by optical pulse labeling, a noninvasive approach that can be used in living cells

“Autophagy has received a great deal of attention because of its therapeutic implications for neurodegenerative and other age-related conditions,” says lead author Dr. Barmada. “Even so, our ability to accurately track autophagy activity or flux has been severely limited by the available tools. Here, we leveraged CRISPR/Cas9 to label an autophagy-related protein in human cells, allowing us for the first time to actually see autophagy in action, and measure flux in a quantitative and reliable fashion.” [Read more…] about A publication by Dr. Barmada and his team is “Editor’s pick” of the Journal of Biological Chemistry

Exploring the mechanisms involved in sleep-dependent memory storage, a team of University of Michigan (U-M) cellular biologists found that RNAs associated with an understudied cell compartment in hippocampal neurons vary greatly between sleeping and sleep-deprived mice after learning.

Sara Aton, Associate Professor in the Department of Molecular, Cellular, and Developmental Biology, and James Delorme, a recent U-M neuroscience graduate student, hypothesized that both a learning event and subsequent sleep (or sleep loss) would impact mRNA translation. Most prior work on the effects of sleep on mRNAs have focused on transcripts in the neuronal cytosol. However, Drs. Aton and Delorme found that after learning, major changes in RNAs are instead present —almost exclusively— on ribosomes associated with neuronal cell membranes. These results have been published in the Proceedings of the National Academy of Sciences, in November 30, 2021.*

In this representation, the red background is the cytosol, and ribosomes are in light green. tRNAs are the purplish blue blobs. Some tRNAs are in the cytosol and others are bound to the green ribosomes. The mRNAs are represented in yellow. The thin purple strand coming out the other side of the ribosomes (and into the ER lumen) is the protein. The big thick black lines in the bottom left corner represents the lipid bilayer of the ER membrane. (Image credit: Sara Aton)

[Read more…] about Unveiling the hidden cellular logistics of memory storage in neurons

By Morgan Sherburne, Michigan News

Bacteria need to constantly adapt to compete against other species for nutrient sources and to survive against threats such as antibiotics and toxins. In an effort to understand how bacteria control and regulate this adaptation, University of Michigan researchers from the Center for RNA Biomedicine are examining how RNA polymerase—the enzyme that transcribes genetic information from DNA onto RNA—slows during transcription in a process called transcriptional pausing.

They found that a protein called N-utilizing substance A, or NusA, in concert with another control element called a riboswitch, fine-tunes the transcription speed in order to regulate gene expression. Gene expression is the process by which genetic information is converted into the building blocks of the bacterium.

The researchers say their work, published in the Proceedings of the National Academy of Sciences, expands our general understanding of the transcription process in bacteria, and could provide a target for developing new antibiotics.

READ MORE

Long non-coding RNAs (lncRNAs) constitute the new frontier of investigation for molecular biologists. However, lncRNA is inconsistently defined, which fails the research community in several ways. In a scientific review [2], Professor Andrzej Wierzbicki from the University of Michigan, Department of Molecular, Cellular and Developmental Biology, and collaborators challenge the contemporary ways of understanding lncRNAs and propose a definition that is based on function and biogenesis. “Here we propose a definition that is clear and specific,” said Wierzbicki, “and we hope that this operational definition will be widely adopted.”

The authors propose the following definition: “a lncRNA is an RNA that has a function independent of its protein-coding potential and that is produced by a mechanism other than molecular ruler-based dicing or trimming.”

Photo: Arabidopsis thaliana (juvenile, flowering and fruiting). For geneticists, Arabidopsis thaliana, a common plant from the mustard (Brassicaceae) family, is considered the equivalent of the fruit fly (Drosophilia melanogaster). This plant can easily be used as a model system for identifying genes and determining their functions. Arabidopsis thaliana is the first plant for which a complete genome sequence was established in 2000. [1]

[Read more…] about Redefining long non-coding RNAs (lncRNAs) to study transposons in plants

While some students may think it’s a good idea to pull an all-nighter before an exam, conventional wisdom may be correct: a good night’s sleep may actually be more helpful, according to University of Michigan research.

U-M scientists Sara Aton and James Delorme found when mice are sleep deprived, there is an increase in activity in inhibitory neurons in the hippocampus, an area of the brain essential for navigation, as well as for processing and storing new memories.

“Because these neurons limit activity in their neighbors, this physiological response makes it impossible to muster normal neuronal activity in the hippocampal structure,” said Sara Aton, an associate professor in the U-M Department of Molecular, Cellular and Developmental Biology and a member of the U-M Center for RNA Biomedicine executive committee. “I always tell my students that an overnighter is not helping them prepare for an exam.”

The researchers’ results are published in the Proceedings of the National Academy of Sciences, and their findings could have implications for human performance and learning strategies.

Image: Somatostatin-expressing interneurons in the mouse dentate gyrus, labeled with Brainbow 3.0 (which labels each neuron a distinct color). cFos (labeled green) is present in the nuclei of surrounding pyramidal cells which are active during sleep. Image by Frank Raven

[Read more…] about How sleep loss sabotages new memory storage in the hippocampus

by Elisabeth Paymal

Often overlooked because they might not alter the amino acid production, splicing defects can play an important role in rare genetic pituitary gland diseases.

Several clinical endocrinologists and bioinformaticians, led by Sally Camper and Jacob Kitzman, Professors of Human Genetics at the University of Michigan Medical School (UMMS), came together to further investigate splicing defects and their roles in rare genetic hormonal diseases. Cathy Smith, a PhD student, established a catalog of predicted variants that could either be tolerated or disease causing.

The pituitary gland is a very important center for hormonal activity. It produces several different hormones which control growth and the functions of other endocrine glands. Specific to the pituitary gland is POU1F1 (also known as PIT1), a transcription factor critical for the development of three of the hormone-producing cell types: growth hormone, prolactin, and thyroid stimulating hormone. Most of the time, a mutation in POU1F1 is recessive, but there are some cases when the variants have a dominant effect. Either genetic cause usually results in multiple hormone deficiencies.

Figures: POU1F1 acts as a dimer and binds to its own gene and the growth hormone to activate transcription (figure 1). The patient variants shift splicing to producing the repressor isoform, which can block the action of the alpha isoform produced from the normal gene copy of POU1F1 (figure 2). This results in less POU1F1 transcription, fewer hormone producing cells, and less growth hormone.

[Read more…] about An often overlooked cause of genetic disease

by Elisabeth Paymal

In an article published in the journal RNA ^[1] , Karan Bedi, a bioinformatician in Mats Ljungman’s lab, Department of Radiation Oncology at the University of Michigan Medical School, investigated the efficiency of splicing across different human cell types. The results were surprising in that the splicing process appears to be quite inefficient, leaving most intronic sequences untouched as the transcripts are being synthesized. The study also reports variable patterns between the different introns within a gene and across cell lines, and it further highlights the complexity of how newly transcripts are processed into mature mRNAs.

Several processes take place to produce mature mRNAs that then can be exported to the cytoplasm and used as a template for protein synthesis. After initiation of transcription and the go-ahead of elongation to produce the pre-mRNA, introns need to be spliced out and the protein-coding exons connected.   At first, pre-mRNA is made as a complementary sequence of the DNA but with slightly different chemistry and includes all the introns. Then the spliceosome machinery, made up of about 300 proteins, assembles “co-transcriptionally” at each intron junction as the RNA emerges from its synthesis. “Splicing is an incredibly complex process because of the great number of proteins involved that repeatedly need to assemble and disassemble at each junction. Also, the speed at which transcription generates RNA is quite fast so the splicing process has to be well organized. Many steps can go wrong and lead to various pathologies, which is why it is so important to have a better understanding of how splicing happens and how it is regulated,” said Bedi.

Photo: Karan Bedi explains the complex data analysis pipeline. Professor Ljungman is on the left – credit: Elisabeth Paymal

[Read more…] about To splice or not to splice…

Neurons result from a highly complex and unique series of cell divisions. For example, in fruit flies, the process starts with stem cells that divide into mother cells (progenitor cells), that then divide into precursor cells that eventually become neurons.

A team of the University of Michigan (U-M), spearheaded by Nigel Michki, a graduate student, and Associate Professor Dawen Cai in the department of Cell and Developmental Biology in the Medical School and in Biophysics in the College of LS&A, identified many genes that are important in fruit flies’ neuron development, and that had never been described before in that context.

Figure to the left: Neurons in the fruit fly brain are made by passing through various differentiation states, and are segregated into unique subtypes based on the age and cell division number of their mother cell (progenitor). The complexity of this process is modelled in the diagram above. Different RNAs play a role in these neuron formation steps. This study identifies many RNAs that were not previously known to be involved in these processes, and helps us better understand how this complex neuron-generation process works at the molecular level.

Since many genes are conserved across species such as between fruit flies (Drosophila), mice, and humans, what is learnt in flies can also serve as a model to better understand other species, including humans. “Now that we know which genes are involved in this form of neurogenesis in flies, we can look for them in other species and test for them. We work on a multitude of organisms at U-M and we’ve the potential to interrogate across organisms,” explains Michki. “In my opinion, the work we did is one of the many pieces that will inform other work that will inform disease,” adds Michki. “This is why we do foundational research like this one.”

Flies are also commonly used in many different types of research that might benefit from having a more comprehensive list of the fly genes with their associated roles in neuron cell development.

[Read more…] about U-M RNA scientists identify many genes involved in neuron development

ANN ARBOR—To better understand how RNA in bacteria gives rise to protein—and along the way, target these processes in the design of new antibiotics—researchers are turning their attention to the unique way this process happens in bacteria.

In eukaryotic cells, transcription (the process by which information in a DNA strand is copied into messenger RNA) and translation (the process by which a protein is synthesized by the ribosome from the mRNA) are two successive steps. In bacteria, they occur simultaneously: As the RNA is being synthesized by RNA polymerase, the ribosome comes in to make the proteins.

This synchronicity allows for so-called “transcription-translation coupling,” wherein the first ribosome can immediately follow and couple with the transcribing RNA polymerase. It is a new area of research that promises to bring insights into processes unique to bacteria that could be targeted with great specificity in the design of antibiotics.

Now, University of Michigan researchers have directly observed previously hidden RNA regulatory mechanisms within such couplings. The results, spearheaded jointly by postdoctoral fellows Surajit Chatterjee and Adrien Chauvier of the U-M Department of Chemistry and the U-M Center for RNA Biomedicine, are published in the Proceedings of the National Academy of Sciences.

The new results promise to have important implications for the future design of antibiotics that could target the coupling mechanism instead of targeting the transcription or translation processes separately.

Caption: The 30S subunit of the ribosome can dynamically bind to the nascent mRNA as soon as its binding site emerges from the RNAP. Transcription factors and translation initiation factors assist in the initial binding and retention of the 30S subunit on the mRNA, resulting in the stabilization of an early initiation complex. During translation, the ribosome can follow the leading RNAP, establishing transcription-translation coupling and maintaining optimal transcription speed. In the presence of the ligand preQ₁, transcription-translation coupling is disrupted, surprisingly leading to slow transcription.

[Read more…] about RNA holds the reins in bacteria: U-M researchers observe RNA controlling protein synthesis

In our September 2020 issue of RNA Translated, 2020 the year of the RNA virus, we presented how University of Michigan (U-M) Center for RNA Biomedicine’s scientists pivoted their research to address the COVID-19 pandemic. One of them is Dr. Chinnaiyan and his team of prostate cancer researchers who focused on two proteins that are involved in giving access to the virus into a host cell. The production of these two proteins is regulated by male hormones. With J. Sexton from the U-M Center for Drug Repurposing, the collaborators looked at repurposing well-known drugs used in prostate cancer to block a receptor for male hormone, and prevent the coronavirus from entering a host cell.

“We hope that these findings may partly explain why males have higher hospitalization and mortality rates than females, and also suggest that transcriptional inhibition of key host factors may have potential in preventing or treating COVID-19. A number of clinical trials in COVID-19 patients have been initiated with drugs that were previously used to treat prostate cancer,” says Dr. Chinnaiyan.

The results from the current study are published in the January 5, 2021, Proceedings of the National Academy of Sciences.

Referenced article:
Targeting transcriptional regulation of SARS-CoV-2 entry factors ACE2 and TMPRSS2
Yuanyuan Qiao, Xiao-Ming Wang, Rahul Mannan, Sethuramasundaram Pitchiaya, Yuping Zhang, Jesse W. Wotring, Lanbo Xiao, Dan R. Robinson, Yi-Mi Wu, Jean Ching-Yi Tien, Xuhong Cao, Stephanie A. Simko, Ingrid J. Apel, Pushpinder Bawa, Steven Kregel, Sathiya P. Narayanan, Gregory Raskind, Stephanie J. Ellison, Abhijit Parolia, Sylvia Zelenka-Wang, Lisa McMurry, Fengyun Su, Rui Wang, Yunhui Cheng, Andrew D. Delekta, Zejie Mei, Carla D. Pretto, Shaomeng Wang, Rohit Mehra, Jonathan Z. Sexton, and Arul M. Chinnaiyan, PNAS January 5, 2021 118 (1) e2021450118; first published December 11, 2020; https://doi.org/10.1073/pnas.2021450118

The observation of single biomolecules in real-time is crucial for our understanding of the cellular biology that is assembled from these molecules, be they DNA, RNA or protein. The recent development of an array of tools and techniques for single-molecule analysis allows studies at an extremely small scale (nanometers, or 10^-9 meters) over short periods of time (from a few milliseconds to a second).

However, until now, most of such observations required tedious and time-consuming manual data processing of thousands of single molecules.  A team of University of Michigan (U-M) Department of Chemistry and Department of Physics scientists, spearheaded by graduate students Jieming Li, now Ph.D. in Chemistry, and Leyou Zhang, now Ph.D. in Physics, developed a deep learning algorithm to analyze data emerging from a single molecule microscope. The results from this collaboration are published in Nature Communications (November 2020).

Illustration: Deep learning assisted single molecule fluorescence microscopy data analysis [Read more…] about Machine learning expands single-molecule analysis accuracy and accessibility

A cross-disciplinary team of scientists from the University of Michigan (U-M) has developed a biochemical technique that successfully measures the number of individual protein molecules present in blood at low concentrations. These findings are published in the Proceedings of the National Academy of Sciences (PNAS), September 2020.

The scientists developed special antibody probes that repeatedly bind to and then release the target protein. With a single-molecule fluorescence microscope, they measured the number of times and for how long the repeated binding events occurred.

[Read more…] about “Kinetic fingerprinting” of single protein molecules to find the biomarker needle in the haystack

By Elisabeth Paymal

A team of scientists associated with the University of Michigan Center for RNA Biomedicine discovered unexpected cellular adaptation mechanisms in response to dehydration. The observed protein reaction has never been reported before.

The research began while observing processing bodies (P-Bodies), which are membrane-less organelles (MLO) involved in RNA degradation in human cells. The team found that, in response to osmotic changes provoked by high saline or sugar concentrations and subsequent dehydration, many proteins within the cell form aggregates similar to P-Bodies. The reaction takes only a few seconds and is reversible.

This phenomenon is distinct from the well-known integrated stress response that leads to the formation of stress granules, another type of membrane-less organelle that aggregates over several minutes.

“At the fundamental level, our work has unraveled a new paradigm for subcellular (membrane-less) organization and rapid stress response,” says Sethu Pitchiaya, a biophysical chemist, one of two shared first authors and a co-senior author on this publication.

Illustration: A pink cell experiences a gradient of salt levels, depicted as crystals. In response to changes in external salt levels, “clouds” of protein form by phase separation. These protect against the dehydrating effects of salt. (Illustration, Elisabeth Paymal) [Read more…] about Stressed cellular proteins break social distancing rules

by Elisabeth Paymal

One of the hallmarks of ovarian cancer is genomic instability resulting in gain and loss of DNA throughout the entire genome, including many microRNAs (miRNA). Dr. DiFeo, Ph.D., Associate Professor of Pathology and of Obstetrics and Gynecology at Michigan Medicine, and her team, research which miRNAs are involved in the early stages of ovarian cancer. “A lot of research has looked at microRNAs in late stages of cancer, and we’re interested in what happens at the early stages of ovarian cancer,” explains DiFeo.

Their results, published in Nature Communications, show hope that microRNAs could be used as biomarkers for tumor development. If a miRNA regulates several significant pathways involved in ovarian cancer, it could make a great biomarker to detect early stages of ovarian cancers as well as a therapeutic target to treat recurrent disease. Currently, because the symptoms are vague, 85% of ovarian cancers are detected at stage 3 and 4 while early detection is essential to cure success.

 This photograph shows the nuclei of normal fallopian tube cell (left) and miR-181a expressing fallopian tube cell (right) highlighting that the expression of miR-181a converts the normal circular nuclei to a abnormal non-circular nuclei that is prone to nuclear rupture and hallmark of cancer.

[Read more…] about miR-181a, a microRNA that regulates ovarian cancer cells

Before and after treatment

Over half of our genomes are made of repeating elements within DNA. In rare cases, these repeats can become unstable and grow in size. These repeat “expansions” cause neurodegenerative diseases such as ALS and Dementia as well as learning disorders and autism in Fragile X syndrome. Research to date has focused on how these expanded repeats cause disease, but little attention has been given to the repeats themselves and whether they might have normal functions in genes.

By focusing on the biology of healthy nerve cells, a team from the University of Michigan Department of Neurology found that repeats in the gene that causes Fragile X Syndrome normally regulate how and when proteins are made in neurons. This process may be important for learning and memory in these nerve cells and potentially in people. “The repeats function like a switch, slowing down protein production and then quickly turning things back on,” explains Peter Todd, MD., Ph.D., and Principal Investigator of this research.

[Read more…] about Publication in Nature Neuroscience: Disease-causing Repeats Help Human Neurons Function