NIH Intramural MOSAIC Scholars Project Summaries

Below are summaries of the projects of NIH intramural scholars funded as part of the Maximizing Opportunities for Scientific and Academic Independent Careers (MOSAIC) Program.

Cassandra Hayne, Ph.D.

Project Title: Structural and Functional Characterization of Pontocerebellar Hypoplasia Associated Nucleases
Institution: National Institute of Environmental Health Sciences
Grant ID: K99GM143534

Abstract Text: RNA processing is an essential cellular process that when dysregulated underlies the development of neurological diseases. Several mutations in nuclease containing complexes cause pontocerebellar hypoplasia (PCH), a severe neurological disorder that often leads to prenatal death. Most cases of PCH are linked to mutations in the tRNA Splicing Endonuclease (TSEN) Complex, which is responsible for the cleavage of tRNA introns prior to tRNA maturation, and its accessory protein, CLP1, which is a critical negative regulator of tRNA splicing. Genetic deletion of any single TSEN protein in yeast, engineered to have tRNAs without introns, was shown to be lethal, suggesting that the TSEN complex likely has substrates beyond the tRNAs, which may underlie the development of PCH. Likewise, mutations in another nuclease, target of Egr 1 (TOE1), a deadenylase and 3’-exonuclase, are also are linked to PCH. TOE1 is the only deadenylase believed to mature snRNAs, but it also moonlights in other cellular pathways, highlighting how much is yet to understand about its role in protein complexes. To determine how mutations in TOE1, CLP1, and TSEN proteins lead to PCH, there remains a critical need to understand how these complexes assemble, recognize and process RNAs, and how their enzymatic activity is regulated. Characterizing healthy cellular roles of these proteins is essential to determining how their dysfunction causes PCH. We aim to address these critical questions through the following proposed Aims. In Aim 1, Structural and molecular techniques will be used to identify how the TSEN complex recognizes and processes tRNAs and other RNA substrates. In Aim 2, we will determine how the CLP1/TSEN complex are regulated at the molecular and cellular level and how PCH mutations disrupt their regulation. Further, in Aim 3, we will identify how PCH mutations alter TOE1 function and regulation, using proteomics and molecular biology approaches. The proposed work is significant because it will provide a structural description for how known PCH mutations may interfere with complex formation, stability, or function for a range of PCH-linked proteins. This work will further provide insight into shared mechanisms by which these protein complexes cause PCH. Furthermore, the work here will characterize new RNA processing roles for these nucleases.

Public Health Relevance Statement: Pontocerebellar hypoplasia (PCH), a severe neurological disorder for which there is no cure that is caused by mutations in several RNA processing complexes. The aims of this proposal will determine how several PCH-linked complexes assemble, recognize and process RNA, how they are enzymatically and spatially regulated, and how PCH mutations disrupt their function. This work is essential to better understand how these complexes function normally in healthy states and how their dysfunction leads to PCH.

John R. Jimah, Ph.D.

Project Title: Structural basis of dynamin-mediated membrane fission, actin bundling and interaction with binding partners
Institution: National Institute of Diabetes and Digestive and Kidney Diseases
Grant ID: K99GM140220

Abstract Text: Dynamin GTPases have critical roles in mediating endocytosis by wrapping around the neck of budding vesicles to catalyze membrane fission necessary for the release of nascent vesicles from the plasma membrane. Recently, we discovered a novel role for dynamin in bundling numerous actin filaments, which has implications for actin-mediated processes, such as cell-cell fusion and migration. While structural and biophysical studies have elucidated the mechanism of dynamin assembly and constriction of membranes, several unanswered questions remain including how dynamin is actually organized and mediates fission within cells, how dynamin forms the final pre-fission state where it wraps around lipid tubules in a superconstricted state, how dynamin binds substrates via the proline rich domain (PRD), and how substrate binding regulates dynamin activity. The long-term objective of this application is to elucidate the mechanism of dynamin-mediated membrane fission from in vitro and in vivo studies, and to define the mechanism of dynamin interaction with actin and SH3 domaincontaining proteins that recruit dynamin to sites of endocytosis. These objectives will be addressed by the following specific aims: (1) determine the atomic model of dynamin in the superconstricted prefission state and define the structure of the PRD; (2) investigate the assembly of the dynamin helical polymer on membranes within cells; and (3) Elucidate the mechanism of PRD interaction with actin filaments and SH3 domain-containing binding partners. The rationale for these aims is that: (1) there is no atomic model describing the structural basis by which dynamin constricts membranes to 3.4 nm in the superconstricted state where spontaneous hemifission and membrane fission occurs; (2) how dynamin is actually organized in cells has not been reported; and (3) the structure of the critical PRD and how it binds dynamin substrates including actin, amphiphysin and intersectin is unknown. The research design and methods are as follows : Aim 1, Apply cryo-electron microscopy (cryo-EM) to determine the structure of full-length dynamin (containing the PRD) organized around lipid tubules in the superconstricted state; Aim 2, obtain cryo-electron tomograms and subtomogram averages of dynamin within cells transfected with a GTPase-deficient dynamin mutant which delays membrane fission and extends the lifetime of dynamin helices on cellular membranes; Aim 3, obtain the cryo-EM structure of actin filaments decorated with PRD from dynamin, as well as complexes of dynamin/amphiphysin and dynamin/intersectin. This work is of public health relevance because dysregulation of dynamin causes neuropathies, cancer and defects in organism development due to dynamin’s critical role in endocytosis needed during cell signaling, and for cell-cell fusion and migration which require actin bundling by dynamin.

Public Health Relevance Statement: This project investigates the mechanism by which dynamin enables membrane fission during endocytosis, how SH3 domain-containing proteins interact and regulate dynamin during endocytosis, and how dynamin interacts with actin filaments during various cell processes that rely on the actin cytoskeleton. This work is of public health relevance because dynamin has critical roles in endocytosis necessary for cell signaling, and for cell-cell fusion and migration which require actin bundling by dynamin. Furthermore, dysregulation of dynamin causes neuropathies, cancer and defects in organism development.

Agnes Karasik, Ph.D.

Project Title: The role of host mRNA cleavage by RNase L in viral infections
Institution: National Institute of Diabetes and Digestive and Kidney Diseases
Grant ID: K99GM143484

Abstract Text: Viral infections remain a challenging public health issue worldwide. The presence of many viruses, such as Influenza A and Hepatitis C, activate a latent Ribonuclease (RNase L) in human cells. Activated RNase L cleaves the single stranded regions of viral and host mRNAs. Cleavage of these RNAs leads to physiological changes in the cell, such as autophagy, senescence, decreased cell motility, interferon production and cell death. However, many aspects of RNase L activation in cells are not yet understood. Recent in vivo and in vitro kinetic studies suggested that RNA cleavage by RNase L is modulated in the cell by an unidentified factor. RNase L can directly interact with the ribosome and with several translation factors involved in different steps of protein synthesis. Due to these interactions it was proposed that RNase L’s cleavage activity is modulated by the translation of the host messenger RNAs (mRNA). Therefore, in specific aim 1, I will investigate this relationship between host mRNA cleavage by RNase L and translation at the global and individual gene level by combining two high-throughput sequencing methods, ribosome footprint profiling and RNA sequencing in RNase L activated human cells. In addition, ribosome-mediated RNase L cleavage activity will be also directly observed at the individual gene level by a novel technique, the real-time fluorescent single molecule detection of translating nascent peptides (SINAPS) in living cells. In aim 2, I will also explore the potential involvement of translation factors in translationmediated RNA cleavage by RNase L by first mapping the details of interactions of RNase L and translation factors by mutagenesis studies and Cryo-Electron Microscopy. Then these interactions will be disrupted in living cells to probe their potential involvement in mediating RNA cleavage by RNase L. Furthermore, RNase L activation can lead to many physiological changes in the cell, such as autophagy and apoptosis. It is plausible that the level of active RNase L is the determinant of which physiological processes will occur. To investigate the correlation between active RNase L levels and RNase L mediated changes in the cell, I will develop a fluorescent RNase L activation indicator that will allow us to sort cells into homogenously activated populations in aim 3. Subsequently, in these sorted cells I will detect changes in the transcriptome and translatome and assess signatures of physiological changes of autophagy and apoptosis in the context of RNase L activation levels. In addition, I will also study how RNase L cleaved host RNA fragments contribute to activation of innate immune response pathways in cells by using Cross-linking Co-Immunoprecipitation and sequencing (CLIP-seq). In summary, the proposed project will investigate unexplored aspects and outcomes of RNase L activation. Uncovering new aspects of the defense against viral infections will enable further studies and potentially contribute to the development of new therapeutic strategies.

Public Health Relevance Statement: The persistent presence and continuing emergence of viral infections is one of the major obstacles to improved human health. The majority of viruses, such as human immunodeficiency virus 1 and potentially SARS-CoV2, activate an endoribonuclease, RNase L, that consequently induces several physiological changes in the cell from autophagy to apoptosis. In the present proposal I will investigate how the activity of RNase L is modulated and its role in determining cell fate by using a combination of new innovative and classical molecular biology techniques.

Velencia J. Witherspoon, Ph.D.

Project Title: Quantitative Characterization of the Extracellular Matrix Components of Connective Tissue: Fingerprinting Macromolecular Components through Low-Field Magnetic Resonance
Institution: Eunice Kennedy Shriver National Institute of Child Health and Human Development
Grant ID: K99GM140338

Abstract Text: Fibrotic activity, the accumulation of macromolecules, alters the composition and microstructural organization musculoskeletal connective tissue. The ability to non-invasively quantify and characterize the significant extracellular matrix components such as proteoglycan content and collagen fibrils organization is important clinically since fibrosis is a common inflammatory response that plays a role in many pathologies. Recent advances in instrumentation for low field magnetic resonance (MR) has enabled its adoption in the field of macromolecular characterization, porous media, and recently biological tissues. LF MR is advantageous as an affordable non-cryogen alternative to high-field MR imaging with a greater detectable dynamic range of quantitative MR parameters. This adaptation for this imaging modality is limited by the lack of appropriate phantoms of connective tissue and the identification of biomarkers for healthy and diseased tissue. Composite gels that replicate the salient structural and compositional features of connective tissue will be developed and used to optimize low field MR methods and identify LF biomarkers. These methods will be applied to articular cartilage and lumbodorsal fascia connective tissues. The project is anticipated to have a significant positive impact on the clinical capability and utility of LF MR as an affordable point of care diagnostic application.

Public Health Relevance Statement: Musculoskeletal (MSK) conditions are the leading contributor to disability worldwide, between one and three, and one and five people live with a musculoskeletal pain condition. Regular assessment of the state of MSK connective tissue is needed to better identify the pathophysiological origin of the inflammatory and fibrotic response. This research will advance the adaptation of low field magnetic resonance imaging (MRI) as a novel, noninvasive, and affordable point of care diagnostic imaging method capable of assessing the state of MSK connective tissue and effective of regenerative medicine.