Mugdha Khaladkar

Deep sequencing reveals cell-type-specific patterns of single-cell transcriptome variation

BackgroundDifferentiation of metazoan cells requires execution of different gene expression programs but recent single-cell transcriptome profiling has revealed considerable variation within cells of seeming identical phenotype. This brings into question the relationship between transcriptome states and cell phenotypes. Additionally, single-cell transcriptomics presents unique analysis challenges that need to be addressed to answer this question.ResultsWe present high quality deep read-depth single-cell RNA sequencing for 91 cells from five mouse tissues and 18 cells from two rat tissues, along with 30 control samples of bulk RNA diluted to single-cell levels. We find that transcriptomes differ globally across tissues with regard to the number of genes expressed, the average expression patterns, and within-cell-type variation patterns. We develop methods to filter genes for reliable quantification and to calibrate biological variation. All cell types include genes with high variability in expression, in a tissue-specific manner. We also find evidence that single-cell variability of neuronal genes in mice is correlated with that in rats consistent with the hypothesis that levels of variation may be conserved.ConclusionsSingle-cell RNA-sequencing data provide a unique view of transcriptome function; however, careful analysis is required in order to use single-cell RNA-sequencing measurements for this purpose. Technical variation must be considered in single-cell RNA-sequencing studies of expression variation. For a subset of genes, biological variability within each cell type appears to be regulated in order to perform dynamic functions, rather than solely molecular noise.

Cytoplasmic intron retention, function, splicing, and the sentinel RNA hypothesis

Cytoplasmic splicing represents a newly emerging level of transcriptional regulation adding to the molecular diversity of mammalian cells. As examples of this noncanonical form of transcript processing are discovered, the evidence of its importance to normal cellular function grows. Work from a number of groups using a variety of cell types is steadily identifying a large number of transcripts (and soon to be even larger as genome‐wide analyses of retained introns across a number of cellular phenotypes are currently underway) that undergo some level of regulated endogenous extranuclear splicing as part of their normal biosynthetic pathway. Here, we review the existing data covering cytoplasmic retained intron sequences and suggest that such sequences may be a component of ‘sentinel RNA’ that serves to generate transcript variants within the cytoplasm as well as a source for RNA‐based secondary messages. WIREs RNA 2014, 5:223–230. doi: 10.1002/wrna.1203

The exonuclease Nibbler regulates age‐associated traits and modulates piRNA length in Drosophila

Nibbler (Nbr) is a 3′‐to‐5′ exonuclease that trims the 3′end of microRNAs (miRNAs) to generate different length patterns of miRNAs in Drosophila. Despite its effect on miRNAs, we lack knowledge of its biological significance and whether Nbr affects other classes of small RNAs such as piRNAs and endo‐siRNAs. Here, we characterized the in vivo function of nbr by defining the Nbr protein expression pattern and loss‐of‐function effects. Nbr protein is enriched in the ovary and head. Analysis of nbr null animals reveals adult‐stage defects that progress with age, including held‐up wings, decreased locomotion, and brain vacuoles, indicative of accelerated age‐associated processes upon nbr loss. Importantly, these effects depend on catalytic residues in the Nbr exonuclease domain, indicating that the catalytic activity is responsible for these effects. Given the impact of nbr on miRNAs, we also analyzed the effect of nbr on piRNA and endo‐siRNA lengths by deep‐sequence analysis of libraries from ovaries. As with miRNAs, nbr mutation led to longer length piRNAs – an effect that was dependent on the catalytic residues of the exonuclease domain. These analyses indicate a role of nbr on age‐associated processes and to modulate length of multiple classes of small RNAs including miRNAs and piRNAs in Drosophila.

Subcellular RNA Sequencing Reveals Broad Presence of Cytoplasmic Intron-Sequence Retaining Transcripts in Mouse and Rat Neurons

Recent findings have revealed the complexity of the transcriptional landscape in mammalian cells. One recently described class of novel transcripts are the Cytoplasmic Intron-sequence Retaining Transcripts (CIRTs), hypothesized to confer post-transcriptional regulatory function. For instance, the neuronal CIRT KCNMA1i16 contributes to the firing properties of hippocampal neurons. Intronic sub-sequence retention within IL1-β mRNA in anucleate platelets has been implicated in activity-dependent splicing and translation. In a recent study, we showed CIRTs harbor functional SINE ID elements which are hypothesized to mediate dendritic localization in neurons. Based on these studies and others, we hypothesized that CIRTs may be present in a broad set of transcripts and comprise novel signals for post-transcriptional regulation. We carried out a transcriptome-wide survey of CIRTs by sequencing micro-dissected subcellular RNA fractions. We sequenced two batches of 150-300 individually dissected dendrites from primary cultures of hippocampal neurons in rat and three batches from mouse hippocampal neurons. After statistical processing to minimize artifacts, we found a broad prevalence of CIRTs in the neurons in both species (44-60% of the expressed transcripts). The sequence patterns, including stereotypical length, biased inclusion of specific introns, and intron-intron junctions, suggested CIRT-specific nuclear processing. Our analysis also suggested that these cytoplasmic intron-sequence retaining transcripts may serve as a primary transcript for ncRNAs. Our results show that retaining intronic sequences is not isolated to a few loci but may be a genome-wide phenomenon for embedding functional signals within certain mRNA. The results hypothesize a novel source of cis-sequences for post-transcriptional regulation. Our results hypothesize two potentially novel splicing pathways: one, within the nucleus for CIRT biogenesis; and another, within the cytoplasm for removing CIRT sequences before translation. We also speculate that release of CIRT sequences prior to translation may form RNA-based signals within the cell potentially comprising a novel class of signaling pathways.

Epigenomic and RNA structural correlates of polyadenylation

Polyadenylation (poly(A)) of mRNA plays a critical role in regulating gene expression. Identifying the sequence, structural, and epigenomic determinants of poly(A) site usage is an important long term goal. Several cis elements that mediate poly(A) regulation have been identified. Highly used poly(A) sites are also known to have a greater nucleosome occupancy in the immediate downstream. However, a detailed exploration of additional epigenomic and mRNA structural correlates of poly(A) site usage has not been reported. Importantly, functional interaction between sequence, structure, and the epigenome in determining the poly(A) site usage is not known. We show that highly used poly(A) sites are positively associated with an mRNA structure that is energetically more favorable and one that better exposes a critical polyadenylation cis element. In exploring potential interplay between RNA and chromatin structure, we found that a stronger nucleosome occupancy downstream of poly(A) site strongly correlated with (1) a more favorable mRNA structure, and (2) a greater accumulation of RNA Polymerase II (PolII) at the poly(A) site. Further analysis suggested a causal relationship pointing from PolII accumulation to a stable RNA structure. Additionally, we found that distinct patterns of histone modifications characterize poly(A) sites and these epigenetic patterns alone can distinguish true poly(A) sites with ~76% accuracy and also discriminate between high and low usage poly(A) sites with ~74% accuracy. Our results suggest a causative link between chromatin structure and mRNA structure whereby a compacted chromatin downstream of the poly(A) site slows down the elongating transcript, thus facilitating the folding of nascent mRNA in a favorable structure at poly(A) site during transcription. Additionally we report hitherto unknown epigenomic correlates for poly(A) site usage.

Divergence of RNA localization between rat and mouse neurons reveals the potential for rapid brain evolution

BackgroundNeurons display a highly polarized architecture. Their ability to modify their features under intracellular and extracellular stimuli, known as synaptic plasticity, is a key component of the neurochemical basis of learning and memory. A key feature of synaptic plasticity involves the delivery of mRNAs to distinct sub-cellular domains where they are locally translated. Regulatory coordination of these spatio-temporal events is critical for synaptogenesis and synaptic plasticity as defects in these processes can lead to neurological diseases. In this work, using microdissected dendrites from primary cultures of hippocampal neurons of two mouse strains (C57BL/6 and Balb/c) and one rat strain (Sprague–Dawley), we investigate via microarrays, subcellular localization of mRNAs in dendrites of neurons to assay the evolutionary differences in subcellular dendritic transcripts localization.ResultsOur microarray analysis highlighted significantly greater evolutionary diversification of RNA localization in the dendritic transcriptomes (81% gene identity difference among the top 5% highly expressed genes) compared to the transcriptomes of 11 different central nervous system (CNS) and non-CNS tissues (average of 44% gene identity difference among the top 5% highly expressed genes). Differentially localized genes include many genes involved in CNS function.ConclusionsSpecies differences in sub-cellular localization may reflect non-functional neutral drift. However, the functional categories of mRNA showing differential localization suggest that at least part of the divergence may reflect activity-dependent functional differences of neurons, mediated by species-specific RNA subcellular localization mechanisms.

Functional divergence of gene duplicates – a domain-centric view

BackgroundGene duplicates have been shown to evolve at different rates. Here we further investigate the mechanism and functional underpinning of this phenomenon by assessing asymmetric evolution specifically within functional domains of gene duplicates.ResultsBased on duplicate genes in five teleost fishes resulting from a whole genome duplication event, we first show that a Fisher Exact test based approach to detect asymmetry is more sensitive than the previously used Likelihood Ratio test. Using our Fisher Exact test, we found that the evolutionary rate asymmetry in the overall protein is largely explained by the asymmetric evolution within specific protein domains. Moreover, among cases of asymmetrically evolving domains, for the gene copy containing a fast evolving domain, the non-synonymous substitutions often cluster within the fast evolving domain. We found that rare substitutions were preferred within asymmetrically evolving domains suggestive of functional divergence. While overall ~32 % of the domains tested were found to be evolving asymmetrically, certain protein domains such as the Tyrosine and Ser/Thr Kinase domains had a much greater prevalence of asymmetric evolution. Finally, based on the spatial expression of Zebra fish duplicate proteins during development, we found that protein pairs containing asymmetrically evolving domains had a greater divergence in gene expression as compared to the duplicate proteins that did not exhibit asymmetric evolution.ConclusionsTaken together, our results suggest that the previously observed asymmetry in the overall duplicate protein evolution is largely due to divergence of specific domains of the protein, and coincides with divergence in spatial expression domains.

KimLabIDV: Application for Interactive RNA- Seq Data Analysis and Visualization

Summary: We developed the KimLabIDV package (IDV) to facilitate fast and interactive RNA-Seq data analysis and visualization. IDV supports routine analysis including differential expression analysis, correlation analysis, dimension reduction, clustering and classification. With the graphical user interface IDV provides, users can easily obtain statistical test results and publication-quality graphs with their data. IDV further supports program state saving and report generation, so that all analysis can be saved, shared and reproduced. Availability and implementation: IDV is implemented in R and is distributed as an R package. It is developed based on the Shiny framework, multiple R packages and a collection of scripts written by members of Junhyong Kims Lab at University of Pennsylvania. IDV supports any system that has R and a modern web browser installed. It can be downloaded from Kim Lab Software Repository (http://kim.bio.upenn.edu/software).Many R packages have been developed for transcriptome analysis but their use often requires familiarity with R and integrating results of different packages is difficult. Here we present PIVOT, an R-based application with a uniform user interface and graphical data management that allows non-programmers to conveniently access various bioinformatics tools and interactively explore transcriptomics data. PIVOT supports many popular open source packages for transcriptome analysis and provides an extensive set of tools for statistical data manipulations. A graph-based visual interface is used to represent the links between derived datasets, allowing easy tracking of data versions. PIVOT further supports automatic report generation, publication-quality plots, and program/data state saving, such that all analysis can be saved, shared and reproduced.

PIVOT: platform for interactive analysis and visualization of transcriptomics data

BackgroundMany R packages have been developed for transcriptome analysis but their use often requires familiarity with R and integrating results of different packages requires scripts to wrangle the datatypes. Furthermore, exploratory data analyses often generate multiple derived datasets such as data subsets or data transformations, which can be difficult to track.ResultsHere we present PIVOT, an R-based platform that wraps open source transcriptome analysis packages with a uniform user interface and graphical data management that allows non-programmers to interactively explore transcriptomics data. PIVOT supports more than 40 popular open source packages for transcriptome analysis and provides an extensive set of tools for statistical data manipulations. A graph-based visual interface is used to represent the links between derived datasets, allowing easy tracking of data versions. PIVOT further supports automatic report generation, publication-quality plots, and program/data state saving, such that all analysis can be saved, shared and reproduced.ConclusionsPIVOT will allow researchers with broad background to easily access sophisticated transcriptome analysis tools and interactively explore transcriptome datasets.

ALZHEIMER'S DISEASE SEQUENCING PROJECT: SEARCH FOR ALZHEIMER'S DISEASE RESILIENCE GENES THAT MAY MODIFY DISEASE SUSCEPTIBILITY IN SPECIFIC APOE GENOTYPE BACKGROUNDS

not available. EC-01-04 PHYSIOLOGICAL SUBSTRATES OF BACE1 AND ADAM10: SAFETY ISSUES OR BIOMARKERS? Stefan Lichtenthaler, Technical University of Munich, Munich, Germany; German Center for Neurodegenerative Diseases (DZNE), Munich, Germany.. Contact e-mail: Stefan.Lichtenthaler@dzne.de Background: The beta-secretase BACE1 is a major drug target in Alzheimer’s disease (AD), but also has other functions, e.g. in myelination and axon targeting. BACE1-deficient mice have additional phenotypes in the brain and several BACE1 substrates, referred to as the BACE1 degradome, are emerging in the CNS. This raises the possibility that therapeutic BACE1 inhibition may have mechanism-based side-effects. Additionally, most BACE1 inhibitors also block the homologous protease BACE2 and, thus, may interfere with BACE2 function. Methods: We developed methods for whole proteome analysis of murine tissue, including CSF Results: We used the novel methods to identify BACE1 substrate candidates in murine CSF, but also in neurons and brains. Selected substrate candidates were validated and functionally and mechanistically characterized. Two of the candidates are seizure protein 6 (SEZ6) and SEZ6-like (SEZ6L), which control neurite outgrowth and synapse formation. Both proteins were validated as BACE1 substrates in vitro and in vivo. In CSF, SEZ6 and SEZ6Lwere found to be suitable markers for measuring BACE1 inhibition in vivo. Conclusions: BACE1 has broad functions in the CNS. The presentation will discuss the implication of the substrates a) for the safety of BACE1 inhibitors and b) for the use as companion diagnostics to monitor BACE inhibition in vivo. SUNDAY, JULY 24, 2016 FEATURED RESEARCH SESSIONS F1-01 THE ALZHEIMER’S DISEASE SEQUENCING PROJECT (ADSP): GENE DISCOVERY IN ACTION F1-01-01 STRUCTURALVARIATION (SV) IN HETEROGENOUS WHOLE-GENOME SEQUENCING DATA FROM 111 FAMILIES AT RISK FOR ALZHEIMER DISEASE: ALZHEIMER DISEASE SEQUENCING PROJECT SV STUDY Li Charlie Xia, Stanford University, Stanford, CA, USA; University of Pennsylvania, Philadelphia, PA, USA. Contact e-mail: lixia@stanford.edu Background: The Alzheimer Disease Sequencing Project (ADSP) is a national initiative to identify novel genetic variants involved in determining risk of late-onset Alzheimer disease (AD). Methods: Structural variation was characterized in 111 families of multiple ethnicities comprising 578 individuals diagnosed with or at risk for AD. We developed a statistical framework that leverages pedigree information to assess accuracy and optimize structural variant (SV) calls. The kinship coefficient was used to filter SVs showing excess heterozygosity. We also formulated a metric called the D-score which is an outgroup-based measure of sib-sharing to filter SVs identified by calling algorithms that detect insertion and deletion breakpoints, but do not render genotypes. These metrics also permitted assessment of reliability of individual calling programs for various SV sizes. An in silico procedure was devised to “spike-in” SVs of varying purity into real sequenced libraries, accommodating multi-library designs, and applied it to three samples that were sequenced at all three centers. This comprehensive QC process allowed benchmarking SV callers across the widely differing libraries and combining these callers in a library-specific way for improved specificity and sensitivity. Candidate SV regions were further refined using an ensemble of 12 SV callers with local assembly to provide precise breakpoints for subsequent calling of genotypes. Results:We identified a high-confidence set of deletions, insertions, and complex variants larger than 20 base pairs (bp) genotyped across all 578 individuals. These variants were prioritized based on predicted functional impact and overlap with known AD genes and linkage regions. Thus far, we detected, and confirmed by sequencing, a 44 bp ABCA7 deletion in 11 members (all have AD) of 5 of 67 CaribbeanHispanic families. This finding is consistent with our recent AD association finding of this deletion in a sample containing >2,800 African Americans which results in a frameshift and truncating mutation that could interfere with protein function. Conclusions: A carefully developed pipeline for detecting, genotyping, and filtering SVs from family-based whole genome sequence data permitted discovery of a robust association of AD risk with a rare 44 bp deletion in ABCA7. Amore comprehensive analysis of these data is underway. F1-01-02 ALZHEIMER’S DISEASE SEQUENCING PROJECT: SEARCH FOR ALZHEIMER’S DISEASE RESILIENCE GENES THAT MAY MODIFY DISEASE SUSCEPTIBILITY IN SPECIFIC APOE GENOTYPE BACKGROUNDS Eduardo Marcora, Alan E. Renton, Gary W. Beecham, Eric Boerwinkle, Laura Cantwell, Carlos Cruchaga, Rebecca Cweibel, Adam Felsenfeld, Myriam Fornage, Manav Kapoor, Keoni Kauwe, Mugdha Khaladkar, Dan Kobolt, Yiyi Ma, Richard Mayeux, Marilyn Miller, Adam C. Naj, Amanda B. Partch, Margaret A. Pericak-Vance, Gerard D. Schellenberg, Sudha Seshadri, Badri N. Vardarajan, Li-San Wang, Joshua C. Bis, Lindsay A. Farrer, Alison M. Goate, 1 Icahn School of Medicine at Mount Sinai, New York, NY, USA; 2 University of Miami Miller School of Medicine, Miami, FL, USA; Baylor College of Medicine, Houston, TX, USA; University of Texas Health Science Center at Houston, Houston, TX, USA; 5 University of Pennsylvania, Philadelphia, PA, USA; 6 Washington University in St. Louis, Saint Louis, MO, USA; Washington University School of Medicine, St. Louis, MO, USA; Knight Alzheimer Disease Center, St. Louis, MO, USA; Division of Genome Sciences, National HumanGenome Research Institute, Bethesda,MD, USA; 10 University of Texas Health Science Center at Houston McGovern Medical School, Houston, TX, USA; Brigham Young University, Provo, UT, USA; Washington University in St. Louis, St. Louis, MO, USA; Washington University, St. Louis, MO, USA; 14 The Genome Institute, Washington University in St. Louis, St. Louis, MO, USA; Boston University, Boston, MA, USA; Columbia University, New York, NY, USA; Division of Neuroscience, National Institute on Aging, Bethesda, MD, USA; 18 University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA; The National Heart, Lung, and Blood Institute’s Framingham Heart Study, Framingham, MA, USA; Boston University School of Medicine, Boston, MA, USA; 21 University of Washington, Seattle, WA, USA. Contact e-mail: edoardo.marcora@mssm.edu Background:Common variation at the APOE locus is the strongest known genetic risk factor for Alzheimer’s disease (AD). Podium Presentations: Sunday, July 24, 2016 P162