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Open Access Publications from the University of California

Scholarly Works (10 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2019)

Autism spectrum disorders (ASD) encompass neurodevelopmental diseases that share core deficits in verbal and nonverbal language, reciprocal interactions, and stereotyped and repetitive behaviors. Unfortunately, the molecular etiology of ASD remains incompletely understood. Excitingly, the recent advent of next generation RNA sequencing has now enabled whole-genome characterization of RNA regulation, expression, and modification in ASD. One such RNA modification, that is highly prevalent in mammalian synapses yet not studied in ASD, is RNA editing. Thus, in this dissertation, we perform a comprehensive spatiotemporal and first genome-wide study of RNA editing in ASD across multiple implicated brain regions, genetic etiologies, and developmental time points spanning fetal development to adulthood. Collectively, we uncovered general trends and regulatory mechanisms of RNA editing relevant to neuronal tissue.

We first characterized RNA editing in the largest cohort of ASD postmortem brains to date. Strikingly the ASD patients exhibited convergent trends of global downregulated RNA editing (hypoediting) affecting synaptic development and transmission genes. The global hypoediting was observed across multiple brain regions and multiple syndromic forms of ASD including dup15q11.2-13.1 duplication syndrome patients and Fragile X syndrome patients. Network analyses and experimental work demonstrated that Fragile X proteins, FMRP and FXR1P, regulated many of the dysregulated editing sites in human brain.

Since postmortem brains only provide postnatal time windows for studying ASD, we next used organoid models to characterize the landscape of RNA editing over ASD fetal brain neurodevelopment. We generated the first large-scale dataset of hundreds of organoids modelling cerebral cortex development (cortical spheroids) over multiple time periods and encompassing a myriad of penetrant autism-susceptibility mutations. RNA editing gradually increased over cortical spheroid development both in control spheroids and ASD spheroids. However, at all developmental timepoints, the ASD cohort again displayed global trends of hypoediting. Functional enrichment analyses implicated the hypoedited RNA editing in cellular development and proliferation of radial glia, intermediate progenitors, and newborn neurons.

Throughout these ASD studies, we encountered difficulties running common statistical analyses due to distinctive properties of RNA editing data. Thus, we lastly developed a statistical framework to handle RNA editing data based on a beta-binomial distribution. We developed a method, called REDITs (RNA editing tests), to handle important RNA editing analyses including significance of case-control differences and regression associations with covariates. REDITs had demonstrably higher sensitivity and specificity on simulated and real RNA editing datasets when compared to the most alternative methods used in the RNA editing field.

  • 11 supplemental ZIPs
Cover page: Landscape of RNA editing across Autism Spectrum Disorders
  • Article
  • Peer Reviewed
UCLA Previously Published Works (2020)

Motivation

RNA-sequencing (RNA-seq) enables global identification of RNA-editing sites in biological systems and disease. A salient step in many studies is to identify editing sites that statistically associate with treatment (e.g. case versus control) or covary with biological factors, such as age. However, RNA-seq has technical features that incumbent tests (e.g. t-test and linear regression) do not consider, which can lead to false positives and false negatives.

Results

In this study, we demonstrate the limitations of currently used tests and introduce the method, RNA-editing tests (REDITs), a suite of tests that employ beta-binomial models to identify differential RNA editing. The tests in REDITs have higher sensitivity than other tests, while also maintaining the type I error (false positive) rate at the nominal level. Applied to the GTEx dataset, we unveil RNA-editing changes associated with age and gender, and differential recoding profiles between brain regions.

Availability and implementation

REDITs are implemented as functions in R and freely available for download at https://github.com/gxiaolab/REDITs. The repository also provides a code example for leveraging parallelization using multiple cores.
    Cover page: Statistical inference of differential RNA-editing sites from RNA-sequencing data by hierarchical modeling
    • Article
    • Peer Reviewed
    UC Davis Previously Published Works (2015)
    The ability to biosynthetically produce chemicals beyond what is commonly found in Nature requires the discovery of novel enzyme function. Here we utilize two approaches to discover enzymes that enable specific production of longer-chain (C5-C8) alcohols from sugar. The first approach combines bioinformatics and molecular modelling to mine sequence databases, resulting in a diverse panel of enzymes capable of catalysing the targeted reaction. The median catalytic efficiency of the computationally selected enzymes is 75-fold greater than a panel of naively selected homologues. This integrative genomic mining approach establishes a unique avenue for enzyme function discovery in the rapidly expanding sequence databases. The second approach uses computational enzyme design to reprogramme specificity. Both approaches result in enzymes with >100-fold increase in specificity for the targeted reaction. When enzymes from either approach are integrated in vivo, longer-chain alcohol production increases over 10-fold and represents >95% of the total alcohol products.
      Cover page: Integrative genomic mining for enzyme function to enable engineering of a non-natural biosynthetic pathway
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2018)
      In eukaryotes, nascent RNA transcripts undergo an intricate series of RNA processing steps to achieve mRNA maturation. RNA editing and alternative splicing are two major RNA processing steps that can introduce significant modifications to the final gene products. By tackling these processes in isolation, recent studies have enabled substantial progress in understanding their global RNA targets and regulatory pathways. However, the interplay between individual steps of RNA processing, an essential aspect of gene regulation, remains poorly understood. By sequencing the RNA of different subcellular fractions, we examined the timing of adenosine-to-inosine (A-to-I) RNA editing and its impact on alternative splicing. We observed that >95% A-to-I RNA editing events occurred in the chromatin-associated RNA prior to polyadenylation. We report about 500 editing sites in the 3' acceptor sequences that can alter splicing of the associated exons. These exons are highly conserved during evolution and reside in genes with important cellular function. Furthermore, we identified a second class of exons whose splicing is likely modulated by RNA secondary structures that are recognized by the RNA editing machinery. The genome-wide analyses, supported by experimental validations, revealed remarkable interplay between RNA editing and splicing and expanded the repertoire of functional RNA editing sites.
        Cover page: RNA editing in nascent RNA affects pre-mRNA splicing
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2018)
        To determine the role for mutations of MECP2 in Rett syndrome, we generated isogenic lines of human induced pluripotent stem cells, neural progenitor cells, and neurons from patient fibroblasts with and without MECP2 expression in an attempt to recapitulate disease phenotypes in vitro. Molecular profiling uncovered neuronal-specific gene expression changes, including induction of a senescence-associated secretory phenotype (SASP) program. Patient-derived neurons made without MECP2 showed signs of stress, including induction of P53, and senescence. The induction of P53 appeared to affect dendritic branching in Rett neurons, as P53 inhibition restored dendritic complexity. The induction of P53 targets was also detectable in analyses of human Rett patient brain, suggesting that this disease-in-a-dish model can provide relevant insights into the human disorder.
          Cover page: Loss of MECP2 Leads to Activation of P53 and Neuronal Senescence
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2021)
          Human stem-cell-derived models provide the promise of accelerating our understanding of brain disorders, but not knowing whether they possess the ability to mature beyond mid- to late-fetal stages potentially limits their utility. We leveraged a directed differentiation protocol to comprehensively assess maturation in vitro. Based on genome-wide analysis of the epigenetic clock and transcriptomics, as well as RNA editing, we observe that three-dimensional human cortical organoids reach postnatal stages between 250 and 300 days, a timeline paralleling in vivo development. We demonstrate the presence of several known developmental milestones, including switches in the histone deacetylase complex and NMDA receptor subunits, which we confirm at the protein and physiological levels. These results suggest that important components of an intrinsic in vivo developmental program persist in vitro. We further map neurodevelopmental and neurodegenerative disease risk genes onto in vitro gene expression trajectories to provide a resource and webtool (Gene Expression in Cortical Organoids, GECO) to guide disease modeling.
            Cover page: Long-term maturation of human cortical organoids matches key early postnatal transitions
            • Article
            • Peer Reviewed
            UCLA Previously Published Works (2019)
            Adenosine-to-inosine (A-to-I) editing, mediated by the ADAR enzymes, diversifies the transcriptome by altering RNA sequences. Recent studies reported global changes in RNA editing in disease and development. Such widespread editing variations necessitate an improved understanding of the regulatory mechanisms of RNA editing. Here, we study the roles of >200 RNA-binding proteins (RBPs) in mediating RNA editing in two human cell lines. Using RNA-sequencing and global protein-RNA binding data, we identify a number of RBPs as key regulators of A-to-I editing. These RBPs, such as TDP-43, DROSHA, NF45/90 and Ro60, mediate editing through various mechanisms including regulation of ADAR1 expression, interaction with ADAR1, and binding to Alu elements. We highlight that editing regulation by Ro60 is consistent with the global up-regulation of RNA editing in systemic lupus erythematosus. Additionally, most key editing regulators act in a cell type-specific manner. Together, our work provides insights for the regulatory mechanisms of RNA editing.
              Cover page: Regulation of RNA editing by RNA-binding proteins in human cells
              • Article
              • Peer Reviewed
              UCLA Previously Published Works (2015)
              In previous work, we showed that coinoculating Rhizobium leguminosarum bv. viciae 128C53 and Bacillus simplex 30N-5 onto Pisum sativum L. roots resulted in better nodulation and increased plant growth. We now expand this research to include another alpha-rhizobial species as well as a beta-rhizobium, Burkholderia tuberum STM678. We first determined whether the rhizobia were compatible with B. simplex 30N-5 by cross-streaking experiments, and then Medicago truncatula and Melilotus alba were coinoculated with B. simplex 30N-5 and Sinorhizobium (Ensifer) meliloti to determine the effects on plant growth. Similarly, B. simplex 30N-5 and Bu. tuberum STM678 were coinoculated onto Macroptilium atropurpureum. The exact mechanisms whereby coinoculation results in increased plant growth are incompletely understood, but the synthesis of phytohormones and siderophores, the improved solubilization of inorganic nutrients, and the production of antimicrobial compounds are likely possibilities. Because B. simplex 30N-5 is not widely recognized as a Plant Growth Promoting Bacterial (PGPB) species, after sequencing its genome, we searched for genes proposed to promote plant growth, and then compared these sequences with those from several well studied PGPB species. In addition to genes involved in phytohormone synthesis, we detected genes important for the production of volatiles, polyamines, and antimicrobial peptides as well as genes for such plant growth-promoting traits as phosphate solubilization and siderophore production. Experimental evidence is presented to show that some of these traits, such as polyamine synthesis, are functional in B. simplex 30N-5, whereas others, e.g., auxin production, are not.
                Cover page: Mining the phytomicrobiome to understand how bacterial coinoculations enhance plant growth
                • Article
                • Peer Reviewed
                UC Davis Previously Published Works (2019)
                Transcriptomic analyses of postmortem brains have begun to elucidate molecular abnormalities in autism spectrum disorder (ASD). However, a crucial pathway involved in synaptic development, RNA editing, has not yet been studied on a genome-wide scale. Here we profiled global patterns of adenosine-to-inosine (A-to-I) editing in a large cohort of postmortem brains of people with ASD. We observed a global bias for hypoediting in ASD brains, which was shared across brain regions and involved many synaptic genes. We show that the Fragile X proteins FMRP and FXR1P interact with RNA-editing enzymes (ADAR proteins) and modulate A-to-I editing. Furthermore, we observed convergent patterns of RNA-editing alterations in ASD and Fragile X syndrome, establishing this as a molecular link between these related diseases. Our findings, which are corroborated across multiple data sets, including dup15q (genomic duplication of 15q11.2-13.1) cases associated with intellectual disability, highlight RNA-editing dysregulation in ASD and reveal new mechanisms underlying this disorder.
                  Cover page: Widespread RNA editing dysregulation in brains from autistic individuals
                  • Article
                  • Peer Reviewed
                  UCLA Previously Published Works (2013)
                  Micromonospora species live in diverse environments and exhibit a broad range of functions, including antibiotic production, biocontrol, and degradation of complex polysaccharides. To learn more about these versatile actinomycetes, we sequenced the genome of strain L5, originally isolated from root nodules of an actinorhizal plant growing in Mexico.
                    Cover page: Complete Genome Sequence of Micromonospora Strain L5, a Potential Plant-Growth-Regulating Actinomycete, Originally Isolated from Casuarina equisetifolia Root Nodules
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