University Of California Santa Cruz

NIH Research Projects · FY 2025 · 2019-01

Project Summary This renewal application focuses on studying the roles of messenger RNA binding proteins (mRBPs) in the regulation of alternative splicing and mRNA translation. Our approach uses a combination of genomics, molecular biology and biochemistry to gain fundamental insights to mRBP function and targets in human cells. During the previous funding period we discovered functional RNA elements that control pre-mRNA splicing and mRNA translation. The goal of the next funding period is to determine how these sequences regulate gene expression. Another goal of this project is to determine how the process of alternative splicing impacts protein synthesis. It is well established that alternative splicing expands the coding potential of protein coding genes, but we and others have also discovered that mRNA isoforms can exhibit different patterns of translational control and mRNA stability. We will elucidate the mechanisms through which alternative splicing influences translation by discovering isoform-specific cis-acting RNA elements that influence polyribosome association. To accomplish this goal, we will use composite cell lines that contain a full complement of human and chimpanzee chromosomes. These novel models will enable the discovery of single nucleotide changes that cause allele- specific gene expression within the identical trans-regulatory environment. We can then determine the mechanisms of action for these regulatory elements using biochemical and molecular approaches, such as RNA affinity chromatography and mRNA reporter assays. Together, this project will significantly advance the understanding ofr how mRBPs function in post-transcriptional control of gene expression in human cells and provide key information for novel strategies to treat inherited diseases.

NIH Research Projects · FY 2025 · 2018-09

Project Summary/Abstract A substantial fraction of human inherited disease-causing mutations introduce an early stop codon that truncates protein production and elicits mRNA decay in a process called Nonsense-Mediated mRNA Decay (NMD). Despite much work, it is still unclear how early stop codons are recognized and how they bring about mRNA decay. The long-term goal of the work is to illuminate how cells recognize and repress mRNAs with early stop codons in animals. Work over the last several decades has highlighted many of the factors involved as well as some of their biochemical capabilities, but the steps and structure/function of the molecules involved remains unclear. In this proposal, the PI and his lab will dissect the pathway of protein synthesis and degradation of early stop codon-containing mRNAs in vivo. The specific aims of the proposed work are to: [Aim 1]: characterize the mRNA cleavage reaction, its products, and its dependencies on protein factors. Results from this aim will provide information about the biochemistry of the RNA decay reaction underlying NMD. [Aim 2]: study the role the factor UPF1 has in licensing mRNAs for decay. Results from this aim will illuminate the factors underlying the timing, recruitment, and commitment of mRNAs to decay. [Aim 3]: characterize the role ribosomes have in the NMD pathway. Results from this aim will showcase how ribosomes signal to cellular machinery to bring about RNA decay during NMD. Experiments will: (a) analyze the phenotype of NMD mutant C. elegans strains, (b) profile the RNA species produced during NMD and in particular mutant backgrounds via both short (Illumina) and long-read (Oxford Nanopore) sequencing, and (c) biochemically analyze purified NMD complexes. Results from this work will illuminate the molecular details of the pathway by which cells recognize and repress early stop codon mutations, relevant to many human disease-causing alleles.

NIH Research Projects · FY 2026 · 2018-08

Project Summary Genetic variation is the central component of all major evolutionary processes. Differences among individuals’ genomes contribute to phenotypic variation including disease susceptibility and the evolution of adaptation. The exceptional growth in genome sequencing technologies and concomitant acceleration in the development of bioinformatic software has the potential to address longstanding questions about the evolution of genomes. Our lab will continue to lead this scientific endeavor by developing a range of statistical, computational, and experimental techniques. In particular, we will develop uniquely scalable computational methods to infer and to understand the relationships among densely sampled populations such as SARS-CoV-2. We will develop computational techniques to study adaptation and the underpinning of genetic isolation using genomes from admixed populations. Our group will leverage an established genome engineering technique to rearrange parts of genomes that have remained structurally static for more than 100 million years and to determine what molecular and fitness effects underlie this maintenance. Finally, our team will leverage natural variation in intron abundance to determine the molecular consequences and fitness impacts of massive intron gain. Results from this work have profound implications for understanding the origins, effects, and ultimate consequences of genetic variation on evolutionary outcomes in natural populations.

NIH Research Projects · FY 2025 · 2016-04

Project Summary This project focuses on understanding the molecular mechanisms underlying the coupled translocation of mRNA and tRNAs during protein synthesis. It includes the important related problems of how the translational reading frame is preserved (or shifted) and how the ribosomal helicase unwinds structured mRNAs. Our laboratory uniquely uses a combination of biochemistry, structural biology, genetics, FRET and computational methods to address these challenging problems. We are also extending our approaches to include single-molecule optical tweezer methods, in collaboration with the Bustamante laboratory (UC Berkeley) and single- molecule FRET, in collaboration with the Ermolenko laboratory (Univ. of Rochester), as well as cryo-electron microscopy, in collaboration with the Chiu laboratory (Stanford/SLAC). In previous studies, we have determined the structures of trapped translocation intermediates, which have provided unexpected insights into how the movements of mRNA and tRNA through the ribosome are coupled to large- and small-scale conformational changes in the structure of the ribosome itself. We then created FRET pairs that allowed us to correlate intersubunit rotation, movement of the L1 stalk and rotation of the 30S subunit head domain with movements of mRNA and tRNA. We plan to extend this search to discover new intermediate states. Having exhausted previous strategies for trapping translocation intermediates, we will use a new approach which exploits a set of dominant-lethal mutations in all five structural domains of elongation factor EF-G that we expect will block translocation at different steps. Development of a novel fluorescent labeling approach that will allow site-specific labeling of FRET pairs directly to ribosomal RNA will overcome technical barriers to single-molecule studies of ribosome dynamics, including studies using simultaneous measurement of molecular forces and FRET changes in the ribosome, in collaboration with the Bustamante group. Finally, we have designed model structured mRNAs that will provide the basis for studying the mechanism of the mRNA helicase and for determination of the structures of translocation complexes stalled in the act of encountering and unwinding an mRNA helix.

NIH Research Projects · FY 2025 · 2015-12

Our proposed research focuses on defining the mechanism of action of the TlpC chemoreceptor in modulating colonization and pathogenesis of the ulcer-causing bacterium Helicobacter pylori. TlpC plays important and exciting roles in H. pylori pathogenesis, sensing host lactate and allowing H. pylori to use it to resist a previ- ously-unappreciated innate immune challenge, complement. The central tenet that guides this work is that un- derstanding chemotaxis signals will lend new insight into the nature of host-pathogen interactions. A gap re- mains, however in our understanding of how H. pylori uses lactate to promote complement resistance, how TlpC integrates lactate and other ligand sensing, and how and where TlpC-mediated sensing promotes in vivo growth. Continued existence of this gap prevents us from gaining a full understanding of H. pylori’s pathogenic mechanisms and, in the long term, creating new drugs to thwart these processes. Millions of people worldwide and in the U.S. are infected by H. pylori and suffer from its associated diseases—ulcers and gastric cancer. Gastric cancer is the fourth highest cause of cancer deaths worldwide. H. pylori is here to stay based on recent studies that show H. pylori incidence has stabilized in the developed world. Furthermore, current therapies to cure H. pylori infection fail with unacceptable frequency, e.g., recent estimates in the United States have found that 20-25% of infected individuals are not cured by the current therapeutic regime. New drug targets are des- perately needed. The specific objective of this application is to dissect TlpC signal transduction and the role of it and its sensed compounds in gastric colonization and disease. Our central hypothesis is that the TlpC- sensed compounds play fundamental roles in H. pylori colonization, and are sensed cooperatively using both subdomains of TlpC’s dCACHE ligand binding domain structure. Our hypothesis has been formulated from preliminary data using crystallization of TlpC, analyzing H. pylori ’s response to lactate and host complement, and determining the role of chemotaxis and TlpC in vivo. Our approach has three Aims, which combine H. py- lori molecular biology, mouse models, and high resolution protein crystallography. In Aim 1, we dissect the role of lactate in H. pylori colonization including how it promotes growth and complement resistance. In Aim 2, we determine how H. pylori TlpC integrates information from multiple ligands into a chemotaxis response. In Aim 3, we define how chemotaxis underlies in vivo population control. The proposed research is innovative in that it will create new knowledge about the functions of chemotaxis during bacterial pathogenesis, the role of comple- ment in the stomach, and high resolution information about how dCACHE ligand binding domains bind ligands. The proposed research is significant because both in our understanding of H. pylori pathogenesis but also for advancing our understanding of dCACHE ligand binding domains, the most common bacterial sensing module. The long-term outcomes generated by this research are likely to provide insights that will enable creation of new drugs against H. pylori-related disease.

NIH Research Projects · FY 2026 · 2015-09

Project Summary Prostatitis is a common urinary tract problem for men. Although pathological and epidemiological studies have linked prostatitis to the development of prostate cancer, its molecular mechanism remains unclear. Answering this question will have clinical benefits in prostate cancer prevention and early intervention. It has been proposed that inflammation can induce tissue stem cell expansion, thereby enlarging the cellular pool for oncogenic transformation. In the prostate, epithelial basal cells serve as stem cells during organogenesis and injury repair, but are quiescent in adult homeostasis. Preliminary data in mouse models suggest that prostatitis reactivates adult basal cells to promote basal-to-luminal differentiation, which facilitates the initiation and progression of prostate cancer. The goal of this project is to elucidate the mechanisms regulating basal cell plasticity in prostatitis and apply the knowledge for prostate cancer prevention. Our central hypothesis is that dysregulation of the androgen receptor (AR) and Wnt signaling crosstalk through an increase of Wnt/β-catenin activity and insufficient AR sequestration is a major factor underlying basal cell reactivation in prostatitis. In three specific aims, we will use an integrated approach of mouse genetics, in vivo lineage tracing, prostate organoid culture, ChIP-seq, biochemistry, single cell RNA-seq, and bioinformatics to determine the AR and β-catenin cistromes in basal cells in prostatitis, characterize the inflammatory stromal environment essential for the enhancement of basal Wnt activities, and assess the effectiveness of Wnt inhibition in delaying prostatitis-stimulated cancer. This project will provide novel insights into the mechanistic link between prostatitis and prostate cancer, a long-standing question in the field. The research outcomes will positively impact human health since Wnt signaling is a druggable target and its inhibition is a potentially promising treatment for ameliorating prostatitis-induced cancer progression.

NIH Research Projects · FY 2024 · 2015-09

It has been well documented that the outcomes of success for botanical clinical trials have been poor, leading to greater effort into understanding the basic mechanisms underlying their activity. While it is often hypothesized that botanicals or other complex mixtures work through synergistic or additive processes, there are few proven examples. There is also growing belief to support the role of gut microbiome metabolism for generation of active metabolites, however, relatively few experimental results back this up. This is driven by limitations in obtaining clear biological signatures in relevant biological assays, in accurately defining the chemical constitution of complex mixtures, and in the informatics approaches to bring these disparate data types together. This proposal aims to address these important questions by the development and implementation of technology platforms. In project 1, we will employ highly innovative orthogonal cell-based high content phenotypic screening approaches in primary macrophages, epithelial cells, primary neurons, which will give us comprehensive coverage of signaling pathway and receptor that are believed to be relevant to botanicals. These platforms aim to link botanicals/bioactive molecules of interest together with information about their molecular targets. These platforms have been demonstrated to work with complex mixtures as well as pure compounds and are supported by the development of bioinformatic approaches that allow integration of orthogonal biological activity. Our second project will take advantage of developments in untargeted metabolomics, along with feature reduction, to have a robust pipeline to clearly define the constitution of complex mixtures. Our third project specifically addresses the question of synergy and additivity by the development of informatics approaches that use the comprehensive biological and chemical signatures generated in the other projects. In this project, we will develop universal tools that will allow the community to probe their own biological and chemical assay results to generate compound/activity maps. This program will deliver critical technology platforms for the in-depth study of botanicals and natural products and deliver tools that can be used by the community

NIH Research Projects · FY 2025 · 2015-05

Formation of functional neural circuits depends on the proper generation of different neuronal and glial cell types in the correct numbers and order. In the developing mammalian central nervous system, multipotent neural stem cells initially produce neurons, followed by glia. The cerebral cortex is the brain region that best exemplifies this developmental theme. In the developing cortex, multipotent neural stem cells, known as the radial glial cells (RGCs), sequentially generate the diverse cortical excitatory neuronal subtypes that populate different cortical layers. At the end of cortical neurogenesis, the RGCs switch lineages and generate inhibitory olfactory bulb interneurons, and both types of cortical macroglia. The cellular process and molecular mechanisms that underlie this lineage switch is not known. Lack of this knowledge hinders our effort to understand the etiology of various neurodevelopmental disorders. In this grant application, we propose to determine the lineage segregation patterns among OB interneuron, astrocyte and oligodendrocyte lineages (Aim 1), to investigate whether Shh signaling regulates lineage specification of cortical astrocytes (Aim 2), and to uncover the underlying molecular mechanisms underlying the lineage switch of cortical RGCs (Aims 2 and 3). We will combine mouse genetics, MADM and intersectional lineage analyses, RNA-seq, single-cell RNA-seq, ChIP-seq, CUT&RUN, ATAC-seq, and 4C technologies to achieve these goals.

NIH Research Projects · FY 2024 · 2013-09

PROJECT SUMMARY/ABSTRACT Our long-term research goals are to understand the mechanisms that regulate stem cell fate decisions. In this first renewal application of this award, we propose to pursue the regulation of self-renewal and lineage potential of the developmentally restricted hematopoietic stem cells (drHSCs) that we discovered in the current award period. Although this population fulfills the most stringent criteria for functional HSC, their life-span during normal development is restricted to a limited developmental window. A functional HSC that does not persist into adulthood had never been observed before and therefore defines a novel wave of definitive hematopoiesis with a distinct endpoint. Here, we focus on understanding the contradictory regulation of drHSC self-renewal and multipotency: upon transplantation, drHSC self-renewal is induced, whereas their intrinsic lineage bias is preserved. Amazingly, the latter – lymphoid bias and exceptional B1a reconstitution potential – is maintained over many months even upon the repeated stress of serial transplantation. In contrast, a single, short-term exposure to stress induces the ability of drHSCs to persist long-term. We propose to pursue the epigenetic mechanisms governing this paradox. We will perform comprehensive molecular and cellular comparisons of drHSCs, co-existing fetal liver HSCs, and adult HSCs, and pursue rigorous functional analysis in competitive reconstitution assays. Importantly, we will couple single-cell transcriptional profiles with functional HSC capacity in efficient yet rigorous in vivo assays. Using CRISPRi/a-mediated transcriptional manipulation, we will directly test the requirements for reprogramming HSC self-renewal and lineage potential in vivo. Our transgenic models are uniquely suited for understanding how the core properties of HSCs – self-renewal and multilineage potential - are established during development and maintained for life and we are excited to put these powerful tools to work to pioneer developmental hematopoietic fate decisions.

NIH Research Projects · FY 2025 · 2013-07

ABSTRACT The UCSC Diversity Action Plan at the University of California, Santa Cruz is known as the Research Mentoring Internship Program (RMI), a research education program that improves equity and access to careers in genomic science. Our specific aims are to recruit and retain a full cohort of 14 trainees per year who meet NIH criteria in terms of underrepresentation; to inspire trainees to pursue genomics-focused careers by fostering a community of scholars focused on genomics research and relevant ELSI investigations; to supplement research training with a robust curriculum of professional development activities; and to ensure that 80% or more of our graduating trainees take steps toward the next academic or career level. The RMI program provides mentored research training and financial support for underrepresented minority (URM) undergraduate students, students from low-income households, students with disabilities, first-generation-to-college students, and students who have otherwise been minoritized by systemic discrimination. Our mission is to prepare and advance our students toward successful careers in genomics research or in fields that interrogate the ethical, legal, and social implications (ELSI) of genomics. Students supported by the RMI are assigned to a faculty research mentor with whom they train 10-15 hours per week during the academic year, and up to 40 hours per week during summer. Mentor labs may be in any department, provided that the research focuses on some aspect of genomic sciences. STEM research environments may be wet labs or computational labs; ELSI projects are usually conducted under the aegis of a faculty member from the Division of Social Sciences, and commonly approach a specific aspect of genomics in one of the following areas: bioethics, policy, health care, social implications. The RMI provides financial support in the form of quarterly subsistence payments. In addition to research training, the program offers academic and professional development workshops, one-on-one coaching, career guidance, and near-peer mentoring. The RMI exposes students to the culture and rigors of a research environment under the supervision of a faculty mentor and with the support of an extended mentor network, thus enhancing preparation for and success in graduate school and beyond. In addition to recruiting from the diverse student population of our own campus (designated a Hispanic Serving Institution), we recruit prospective transfer students from regional community colleges that have high percentages of students from BBIPOC (Brown, Black, Indigenous, and People of Color), low-income, and underserved populations. To ensure successful persistence to degree completion, we implement retention strategies based on best practices to create professional support and programming and an expanded mentor network within a cutting-edge research environment that provides our cohort with the knowledge, tools, and confidence needed to advance to meaningful careers in genomics.

NIH Research Projects · FY 2025 · 2013-06

PROJECT SUMMARY The signaling nucleotide cyclic dimeric guanosine monophosphate (c-diGMP) is broadly conserved in bacteria and is a key regulator of central cellular processes including motility, biofilm formation, and virulence. Our understanding of how c-diGMP controls biofilm formation, the environmental signals modulating c-diGMP levels and biofilm formation, and the dynamics and consequences of c-diGMP signaling during infection remains limited. This proposal aims to fill critical gaps in our understanding of how c-diGMP signaling and biofilm formation control the infection cycle of Vibrio cholerae, which causes the disease cholera, an important global public health problem. We will address these information gaps through two specific aims. 1) Determine mechanisms of signal sensing and response in c-diGMP signaling networks; and 2) Analysis of c-diGMP signaling during infection. Under the first aim, the molecular mechanism(s) of activation of key c-diGMP signaling proteins will be determined using structural and ligand-binding studies. The down-stream c-diGMP signaling pathways initiated upon surface attachment will be analyzed by employing microscopy-based community tracking methods to measure motility, division, and c-diGMP levels. The mechanism by which specific key c- diGMP signaling proteins act to regulate surface attachment and biofilm matrix production will be determined using a combination of genetic and biochemical approaches. Under the second aim, we will analyze key c-diGMP signaling pathways that are activated during infection. This part of the proposal will track and examine the mechanisms underlying temporal and spatial dynamics of c-diGMP production and virulence gene regulation during infection using state-of-the-art imaging tools and novel c-diGMP sensors. The proposed work will greatly advance our understanding of how c-diGMP signaling operates, identify the inputs that influence c-diGMP production and degradation, and unveil the biological consequences of c-diGMP signaling in vivo. This research promises molecular and mechanistic insights that will allow us to devise ways to control c-diGMP signal transduction pathways governing motility, biofilm formation, and virulence, ultimately identifying potential therapeutic or preventive targets that can be exploited for preventive measures against cholera.

NIH Research Projects · FY 2026 · 2013-03

Abstract - This is a renewal of our successful 2019-2023 PREP program. UC Santa Cruz PREP is in its 8th year and has supported 50 scholars (43 completed training and 6 currently supported). Of our seven cohorts of PREP scholars that completed training, 3 enrolled in master’s programs, and 32 of 43 (74.4%) have been admitted to top-ranked biomedical Ph.D. programs throughout the country. Of the 32 accepted into Ph. D.s programs, 34% have completed their Ph.D. training, 16% have received their Master’s, and are currently in leadership positions in academia and industry. 50% are at different stages of their doctorate studies. Two honorable mentions and five scholars received prestigious NSF graduate fellowships. Ten additional scholars received coveted graduate fellowships at their universities. 19 co-authored peer-reviewed publications were produced by PREP scholars based on their research at UCSC. The current cohort of 6 scholars has just received numerous graduate school invitations, and we are optimistic that they will be accepted into Biomedical Ph.D. programs. Of our 50 scholars (30 female/20 male), 86% were first-generation college students, 8 were African American, 34 were Hispanic, 3 were Southeast Asian, and 3 were Native American. In this renewal application, we plan to maintain the same class size, admitting yearly cohorts of 6 post-baccalaureate scholars from underserved and underrepresented minority populations. Based on our extensive involvement with this program, we continue to modify and improve the PREP experience for our scholars. Specifically, we propose to increase our state and national recruiting efforts. In addition, we will foster collaborations, retreats, and seminars with the three other Bay Area PREP programs. While great strides have been made over the past 20 years toward increasing the participation of historically marginalized groups in science programs at the undergraduate level, similar increases have not been realized at the doctoral level. The UC Santa Cruz PREP program is well suited to meet this urgent national need to expand diversity and increase participation of historically marginalized students in Biomedical Ph.D. programs. The outstanding research environment at UC Santa Cruz will provide PREP scholars with state-of-the-art resources and faculty who are leaders in their disciplines. Our scholars will also benefit from UCSC’s experience and outstanding success, i.e., training underrepresented and historically marginalized students. PREP faculty and staff will collaborate with campus diversity partners, the Division of Graduate Studies, the Division of Physical and Biological Sciences, the Jack Baskin School of Engineering, and the Office of the Chancellor to ensure the success of the program by creating a welcoming environment for all members of the UCSC PREP community. PREP scholars will participate in intensive mentoring and professional development programs that enhance their ability to gain admission to highly competitive doctoral programs. By training new professionals in cutting-edge research, the UCSC PREP program will help advance the NIH mission of equity and excellence and enhance our workforce with diverse leaders in scientific and technological discovery leaders.

NIH Research Projects · FY 2025 · 2001-07

ABSTRACT The UCSC Genome Browser and associated tools are used by hundreds of thousands of biomedical researchers including clinical geneticists, bioinformaticians, researchers working with model organisms, and wet lab scientists researching human physiology at the molecular level in both healthy and disease states. The browser integrates the results of thousands of biomedical labs – including a wide range of biochemical assays, genetic studies, curations, sequencing projects, and computer analyses into a series of tracks aligned to the underlying genomic sequence. The genome provides a natural integration framework for these diverse data sources, which the browser showcases at a variety of display scales ranging from the single base to individual genes, entire chromosomes, and ultimately to the genome as a whole. The Genome Browser is implemented using robust, fast, high-quality software capable of handling over one million hits per day. This web software provides a window into an exceptionally detailed and well-documented database that can be queried computationally as well as browsed graphically. The database is loaded with a suite of programs, developed both at UCSC and elsewhere, capable of distilling huge genomics data sets into high-quality annotations of the genome. Significant engineering effort is invested to ensure the quality of the software and data sets, including those developed by external contributors. The system is designed to make it easy for users to view their own, unpublished, data sets alongside those that we have fully curated and integrated. Consortia and other resources can make their data visible in our browser via “track hubs.” We plan to extend our resources in significant ways. We will help make genomics cover a larger share of the genetic variants in the US population by moving to a “pangenome” reference. We will enable visualization of individual genomes, not just a single haploid reference genome. We will address the opportunities and challenges of new technologies such as single-cell RNA sequencing and single-molecule long-read DNA sequencing. We will collaborate with others in the increasingly complex ecosystem of biomedical consortia and resources, and will integrate their results into the Genome Browser, and also, through our APIs and our helpful staff, ensure that others can make the best use of data available in their efforts. We will provide tools and data for medical users to understand the significance of sequence variants in the patients they care for and will help characterize regions of greater genomic complexity and medical importance. We will extend our outreach effort to include more online content to help engage a new generation of users

Other NSERC · FY 2024

octopus, ribosome, protein synthesis, translation, adaptation, cephalopod, molecular biology, marine biology