@article {134, title = {The Genetic Control of Stoichiometry Underlying Autism.}, journal = {Annu Rev Neurosci}, volume = {43}, year = {2020}, month = {2020 07 08}, pages = {509-533}, abstract = {

Autism is a common and complex neurologic disorder whose scientific underpinnings have begun to be established in the past decade. The essence of this breakthrough has been a focus on families, where genetic analyses are strongest, versus large-scale, case-control studies. Autism genetics has progressed in parallel with technology, from analyses of copy number variation to whole-exome sequencing (WES) and whole-genome sequencing (WGS). Gene mutations causing complete loss of function account for perhaps one-third of cases, largely detected through WES. This limitation has increased interest in understanding the regulatory variants of genes that contribute in more subtle ways to the disorder. Strategies combining biochemical analysis of gene regulation, WGS analysis of the noncoding genome, and machine learning have begun to succeed. The emerging picture is that careful control of the amounts of transcription, mRNA, and proteins made by key brain genes-stoichiometry-plays a critical role in defining the clinical features of autism.

}, issn = {1545-4126}, doi = {10.1146/annurev-neuro-100119-024851}, author = {Darnell, Robert B} } @article {109, title = {RNA Identification of PRIME Cells Predicting Rheumatoid Arthritis Flares.}, journal = {N Engl J Med}, volume = {383}, year = {2020}, month = {2020 07 16}, pages = {218-228}, abstract = {

BACKGROUND: Rheumatoid arthritis, like many inflammatory diseases, is characterized by episodes of quiescence and exacerbation (flares). The molecular events leading to flares are unknown.

METHODS: We established a clinical and technical protocol for repeated home collection of blood in patients with rheumatoid arthritis to allow for longitudinal RNA sequencing (RNA-seq). Specimens were obtained from 364 time points during eight flares over a period of 4 years in our index patient, as well as from 235 time points during flares in three additional patients. We identified transcripts that were differentially expressed before flares and compared these with data from synovial single-cell RNA-seq. Flow cytometry and sorted-blood-cell RNA-seq in additional patients were used to validate the findings.

RESULTS: Consistent changes were observed in blood transcriptional profiles 1 to 2 weeks before a rheumatoid arthritis flare. B-cell activation was followed by expansion of circulating CD45-CD31-PDPN+ preinflammatory mesenchymal, or PRIME, cells in the blood from patients with rheumatoid arthritis; these cells shared features of inflammatory synovial fibroblasts. Levels of circulating PRIME cells decreased during flares in all 4 patients, and flow cytometry and sorted-cell RNA-seq confirmed the presence of PRIME cells in 19 additional patients with rheumatoid arthritis.

CONCLUSIONS: Longitudinal genomic analysis of rheumatoid arthritis flares revealed PRIME cells in the blood during the period before a flare and suggested a model in which these cells become activated by B cells in the weeks before a flare and subsequently migrate out of the blood into the synovium. (Funded by the National Institutes of Health and others.).

}, keywords = {Adult, Arthritis, Rheumatoid, B-Lymphocytes, Female, Fibroblasts, Flow Cytometry, Gene Expression, Humans, Male, Mesenchymal Stem Cells, Middle Aged, Patient Acuity, Sequence Analysis, RNA, Surveys and Questionnaires, Symptom Flare Up, Synovial Fluid}, issn = {1533-4406}, doi = {10.1056/NEJMoa2004114}, author = {Orange, Dana E and Yao, Vicky and Sawicka, Kirsty and Fak, John and Frank, Mayu O and Parveen, Salina and Blach{\`e}re, Nathalie E and Hale, Caryn and Zhang, Fan and Raychaudhuri, Soumya and Troyanskaya, Olga G and Darnell, Robert B} } @article {53, title = {Differential NOVA2-Mediated Splicing in Excitatory and Inhibitory Neurons Regulates Cortical Development and Cerebellar Function.}, journal = {Neuron}, volume = {101}, year = {2019}, month = {2019 Feb 20}, pages = {707-720.e5}, abstract = {

RNA-binding proteins (RBPs) regulate genetic diversity, but the degree to which they do so in individual cell types in~vivo is unknown. We developed NOVA2 cTag-crosslinking and immunoprecipitation (CLIP) to generate functional RBP-RNA maps from different neuronal populations in the mouse brain. Combining cell type datasets from Nova2-cTag and Nova2 conditional knockout mice revealed differential NOVA2 regulatory actions on alternative splicing (AS) on the same transcripts expressed in different neurons. This includes functional differences in transcripts expressed in cortical and cerebellar excitatory versus inhibitory neurons, where we find NOVA2 is required for, respectively, development of laminar structure, motor coordination, and synapse formation. We also find that NOVA2-regulated AS is coupled to NOVA2 regulation of intron retention in hundreds of transcripts, which can sequester the trans-acting splicing factor PTBP2. In summary, cTag-CLIP complements single-cell RNA sequencing (RNA-seq) studies by providing a means for understanding RNA regulation of functional cell diversity.

}, issn = {1097-4199}, doi = {10.1016/j.neuron.2018.12.019}, author = {Saito, Yuhki and Yuan, Yuan and Zucker-Scharff, Ilana and Fak, John J and Jereb, Sa{\v s}a and Tajima, Yoko and Licatalosi, Donny D and Darnell, Robert B} } @article {47, title = {Cell type-specific CLIP reveals that NOVA regulates cytoskeleton interactions in motoneurons.}, journal = {Genome Biol}, volume = {19}, year = {2018}, month = {2018 08 15}, pages = {117}, abstract = {

BACKGROUND: Alternative RNA processing plays an essential role in shaping cell identity and connectivity in the central nervous system. This is believed to involve differential regulation of RNA processing in various cell types. However, in vivo study of cell type-specific post-transcriptional regulation has been a challenge. Here, we describe a sensitive and stringent method combining genetics and CLIP (crosslinking and immunoprecipitation) to globally identify regulatory interactions between NOVA and RNA in the mouse spinal cord motoneurons.

RESULTS: We developed a means of undertaking motoneuron-specific CLIP to explore motoneuron-specific protein-RNA interactions relative to studies of the whole spinal cord in mouse. This allowed us to pinpoint differential RNA regulation specific to motoneurons, revealing a major role for NOVA in regulating cytoskeleton interactions in motoneurons. In particular, NOVA specifically promotes the palmitoylated isoform of the cytoskeleton protein Septin 8 in motoneurons, which enhances dendritic arborization.

CONCLUSIONS: Our study demonstrates that cell type-specific RNA regulation is important for fine tuning motoneuron physiology and highlights the value of defining RNA processing regulation at single cell type resolution.

}, keywords = {Alternative Splicing, Amino Acid Sequence, Animals, Cercopithecus aethiops, Chromosomes, Artificial, Bacterial, COS Cells, Cross-Linking Reagents, Cytoskeleton, Dendrites, Exons, Immunoprecipitation, Lipoylation, Mice, Mice, Transgenic, Motor Neurons, Nerve Tissue Proteins, NIH 3T3 Cells, Pseudopodia, RNA, RNA-Binding Proteins, Septins, Transcriptome}, issn = {1474-760X}, doi = {10.1186/s13059-018-1493-2}, author = {Yuan, Yuan and Xie, Shirley and Darnell, Jennifer C and Darnell, Andrew J and Saito, Yuhki and Phatnani, Hemali and Murphy, Elisabeth A and Zhang, Chaolin and Maniatis, Tom and Darnell, Robert B} } @article {39, title = {Differential 3{\textquoteright} Processing of Specific Transcripts Expands Regulatory and Protein Diversity Across Neuronal Cell Types.}, journal = {Elife}, volume = {7}, year = {2018}, month = {2018 Mar 26}, abstract = {

Alternative polyadenylation (APA) regulates mRNA translation, stability, and protein localization. However, it is unclear to what extent APA regulates these processes uniquely in specific cell types. Using a new technique, cTag-PAPERCLIP, we discovered significant differences in APA between the principal types of mouse cerebellar neurons, the Purkinje and granule cells, as well as between proliferating and differentiated granule cells. Transcripts that differed in APA in these comparisons were enriched in key neuronal functions and many differed in coding sequence in addition to 3{\textquoteright}UTR length. We characterize , a transcript that shifted from expressing a short 3{\textquoteright}UTR isoform to a longer one during granule cell differentiation. We show that regulates granule cell precursor proliferation and that its long 3{\textquoteright}UTR isoform is targeted by miR-124, contributing to its downregulation during development. Our findings provide insight into roles for APA in specific cell types and establish a platform for further functional studies.

}, issn = {2050-084X}, doi = {10.7554/eLife.34042}, author = {Jereb, Sa{\v s}a and Hwang, Hun-Way and Van Otterloo, Eric and Govek, Eve-Ellen and Fak, John J and Yuan, Yuan and Hatten, Mary E and Darnell, Robert B} } @article {42, title = {Whole-Genome Sequencing of Pharmacogenetic Drug Response in Racially Diverse Children with Asthma.}, journal = {Am J Respir Crit Care Med}, volume = {197}, year = {2018}, month = {2018 Jun 15}, pages = {1552-1564}, abstract = {

RATIONALE: Albuterol, a bronchodilator medication, is the first-line therapy for asthma worldwide. There are significant racial/ethnic differences in albuterol drug response.

OBJECTIVES: To identify genetic variants important for bronchodilator drug response (BDR) in racially diverse children.

METHODS: We performed the first whole-genome sequencing pharmacogenetics study from 1,441 children with asthma from the tails of the BDR distribution to identify genetic association with BDR.

MEASUREMENTS AND MAIN RESULTS: We identified population-specific and shared genetic variants associated with BDR, including genome-wide significant (P < 3.53 {\texttimes} 10) and suggestive (P < 7.06 {\texttimes} 10) loci near genes previously associated with lung capacity (DNAH5), immunity (NFKB1 and PLCB1), and β-adrenergic signaling (ADAMTS3 and COX18). Functional analyses of the BDR-associated SNP in NFKB1 revealed potential regulatory function in bronchial smooth muscle cells. The SNP is also an expression quantitative trait locus for a neighboring gene, SLC39A8. The lack of other asthma study populations with BDR and whole-genome sequencing data on minority children makes it impossible to perform replication of our rare variant associations. Minority underrepresentation also poses significant challenges to identify age-matched and population-matched cohorts of sufficient sample size for replication of our common variant findings.

CONCLUSIONS: The lack of minority data, despite a collaboration of eight universities and 13 individual laboratories, highlights the urgent need for a dedicated national effort to prioritize diversity in research. Our study expands the understanding of pharmacogenetic analyses in racially/ethnically diverse populations and advances the foundation for precision medicine in at-risk and understudied minority populations.

}, issn = {1535-4970}, doi = {10.1164/rccm.201712-2529OC}, author = {Mak, Angel C Y and White, Marquitta J and Eckalbar, Walter L and Szpiech, Zachary A and Oh, Sam S and Pino-Yanes, Maria and Hu, Donglei and Goddard, Pag{\'e} and Huntsman, Scott and Galanter, Joshua and Wu, Ann Chen and Himes, Blanca E and Germer, Soren and Vogel, Julia M and Bunting, Karen L and Eng, Celeste and Salazar, Sandra and Keys, Kevin L and Liberto, Jennifer and Nuckton, Thomas J and Nguyen, Thomas A and Torgerson, Dara G and Kwok, Pui-Yan and Levin, Albert M and Celed{\'o}n, Juan C and Forno, Erick and Hakonarson, Hakon and Sleiman, Patrick M and Dahlin, Amber and Tantisira, Kelan G and Weiss, Scott T and Serebrisky, Denise and Brigino-Buenaventura, Emerita and Farber, Harold J and Meade, Kelley and Lenoir, Michael A and Avila, Pedro C and Sen, Saunak and Thyne, Shannon M and Rodriguez-Cintron, William and Winkler, Cheryl A and Moreno-Estrada, Andr{\'e}s and Sandoval, Karla and Rodriguez-Santana, Jose R and Kumar, Rajesh and Williams, L Keoki and Ahituv, Nadav and Ziv, Elad and Seibold, Max A and Darnell, Robert B and Zaitlen, Noah and Hernandez, Ryan D and Burchard, Esteban G} } @article {24, title = {cTag-PAPERCLIP Reveals Alternative Polyadenylation Promotes Cell-Type Specific Protein Diversity and Shifts Araf Isoforms with Microglia Activation.}, journal = {Neuron}, volume = {95}, year = {2017}, month = {2017 Sep 13}, pages = {1334-1349.e5}, abstract = {

Alternative polyadenylation (APA) is increasingly recognized to regulate gene expression across different cell types, but obtaining APA maps from individual cell types typically requires prior purification, a stressful procedure that can itself alter cellular states. Here, we describe a new platform, cTag-PAPERCLIP,~that generates APA profiles from single cell populations in intact tissues; cTag-PAPERCLIP requires no tissue dissociation and preserves transcripts in native states. Applying cTag-PAPERCLIP to profile four major cell types in the mouse brain revealed common APA preferences between excitatory and inhibitory neurons distinct from astrocytes~and microglia, regulated in part by neuron-specific~RNA-binding proteins NOVA2 and PTBP2. We further~identified a role of APA in switching Araf protein isoforms during microglia activation, impacting production of downstream inflammatory cytokines. Our results demonstrate the broad applicability of cTag-PAPERCLIP and a previously undiscovered role of APA in contributing to protein diversity between different cell types and cellular states within the brain.

}, keywords = {Animals, Antigens, Neoplasm, Astrocytes, Brain, Cells, Cultured, Female, Humans, Male, Mice, Microglia, Nerve Tissue Proteins, Neuro-Oncological Ventral Antigen, Neurons, Organ Specificity, Polyadenylation, Polypyrimidine Tract-Binding Protein, Protein Isoforms, Protein Serine-Threonine Kinases, RNA-Binding Proteins}, issn = {1097-4199}, doi = {10.1016/j.neuron.2017.08.024}, author = {Hwang, Hun-Way and Saito, Yuhki and Park, Christopher Y and Blach{\`e}re, Nathalie E and Tajima, Yoko and Fak, John J and Zucker-Scharff, Ilana and Darnell, Robert B} } @article {23, title = {Genomic Patterns of De Novo Mutation in Simplex Autism.}, journal = {Cell}, volume = {171}, year = {2017}, month = {2017 Oct 19}, pages = {710-722.e12}, abstract = {

To further our understanding of the genetic etiology of autism, we generated and analyzed genome sequence data from 516 idiopathic autism families (2,064 individuals). This resource includes >59 million single-nucleotide variants (SNVs) and 9,212 private copy number variants (CNVs), of which 133,992 and 88 are de novo mutations (DNMs), respectively. We estimate a mutation rate of \~{}1.5~{\texttimes} 10 SNVs per site per generation with a significantly higher mutation rate in repetitive DNA. Comparing probands and unaffected siblings, we observe several DNM trends. Probands carry more gene-disruptive CNVs and SNVs, resulting in severe~missense mutations and mapping to predicted fetal brain promoters and embryonic stem cell enhancers. These differences become more pronounced for autism genes (p~= 1.8~{\texttimes} 10, OR~= 2.2). Patients are more likely to carry multiple coding and noncoding DNMs in different genes, which are enriched for expression in striatal neurons (p~= 3~{\texttimes} 10), suggesting a path forward for genetically characterizing more complex cases of autism.

}, keywords = {Animals, Autistic Disorder, DNA Copy Number Variations, DNA Mutational Analysis, Female, Genome-Wide Association Study, Humans, INDEL Mutation, Male, Mice, Polymorphism, Single Nucleotide}, issn = {1097-4172}, doi = {10.1016/j.cell.2017.08.047}, author = {Turner, Tychele N and Coe, Bradley P and Dickel, Diane E and Hoekzema, Kendra and Nelson, Bradley J and Zody, Michael C and Kronenberg, Zev N and Hormozdiari, Fereydoun and Raja, Archana and Pennacchio, Len A and Darnell, Robert B and Eichler, Evan E} }