President and Scientific Director, NY Genome Center; Heilbrunn Professor, Rockefeller University; Investigator, HHMI
- Greater New York City Area
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President, Scientific Director and CEO of NY Genome Center;
Professor and Senior Physician at Rockefeller University;
Investigator, Howard Hughes Medical Institute;
Attending Neurologist at Memorial Sloan-Kettering Cancer Center
Functional annotation of genomic data
RNA regulation in the brain
The intersection of autoimmunity and tumor immunity
Professor and Senior Physician
Oxford University PressAugust 2011
The definitive book on Paraneoplastic Neurologic Syndromes and other Paraneoplastic Disorders, for the oncologist and the student. Paraneoplastic syndromes, defined in this book as "disorders caused by cancer, but not a direct result of cancer invasion of the affected organ or tissue", once believed to be rare and esoteric neurologic disorders have assumed increasing importance as an explanation of neurologic and other symptoms in patients suffered from occult or controllable cancers. This book attempts a comprehensive review of paraneoplastic syndromes from considering both clinical and pathophysiologic aspects. The book is divided into 4 sections: The first is an overview, classifying the disorders, describing a clinical approach to the diagnosis and treatment of paraneoplastic syndromes in general and identifying their pathogenesis. The section discusses the clinical findings and treatment of individual paraneoplastic syndromes with chapters dedicated to each of the neurologic syndromes and a chapter discussing nonneurologic syndromes such as endocrine, cutaneous, and rheumatologic paraneoplastic disorders. The third section discusses autoantibodies that characterize individual paraneoplastic syndromes. The final section discusses the paraneoplastic syndromes associated with individual cancers.
- Robert Darnell,
- Jerome B. Posner
This paper describes the development of a breakthrough method in RNA biology termed HITS-CLIP. Protein-RNA interactions have critical roles in all aspects of gene expression. However, applying biochemical methods to understand such interactions in living tissues has been challenging. Here we develop a genome-wide means of mapping protein-RNA binding sites in vivo, by high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). HITS-CLIP analysis of the neuron-specific splicing factor Nova revealed extremely reproducible RNA-binding maps in multiple mouse brains. These maps provide genome-wide in vivo biochemical footprints confirming the previous prediction that the position of Nova binding determines the outcome of alternative splicing; moreover, they are sufficiently powerful to predict Nova action de novo. HITS-CLIP revealed a large number of Nova-RNA interactions in 3' untranslated regions, leading to the discovery that Nova regulates alternative polyadenylation in the brain. HITS-CLIP, therefore, provides a robust, unbiased means to identify functional protein-RNA interactions in vivo.
- Robert Darnell,
- Donny Licatalosi,
- Aldo Mele,
- John Fak,
- Jernej Ule,
- Sung Wook Chi,
- Tyson A. Clark,
- Anthony C. Schweitzer,
- John E. Blume,
- Xuning Wang
This work describes the first application of HITS-CLIP to define miRNA binding sites. MicroRNAs (miRNAs) have critical roles in the regulation of gene expression; however, as miRNA activity requires base pairing with only 6-8 nucleotides of messenger RNA, predicting target mRNAs is a major challenge. Recently, high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) has identified functional protein-RNA interaction sites. Here we use HITS-CLIP to covalently crosslink native argonaute (Ago, also called Eif2c) protein-RNA complexes in mouse brain. This produced two simultaneous data sets-Ago-miRNA and Ago-mRNA binding sites-that were combined with bioinformatic analysis to identify interaction sites between miRNA and target mRNA. We validated genome-wide interaction maps for miR-124, and generated additional maps for the 20 most abundant miRNAs present in P13 mouse brain. Ago HITS-CLIP provides a general platform for exploring the specificity and range of miRNA action in vivo, and identifies precise sequences for targeting clinically relevant miRNA-mRNA interactions.
- Robert Darnell,
- Sung Wook Chi,
- Julie B. Zang,
- Aldo Mele
FMRP loss of function causes Fragile X syndrome (FXS) and autistic features. FMRP is a polyribosome-associated neuronal RNA-binding protein, suggesting that it plays a key role in regulating neuronal translation, but there has been little consensus regarding either its RNA targets or mechanism of action. Here, we use high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) to identify FMRP interactions with mouse brain polyribosomal mRNAs. FMRP interacts with the coding region of transcripts encoding pre- and postsynaptic proteins and transcripts implicated in autism spectrum disorders (ASD). We developed a brain polyribosome-programmed translation system, revealing that FMRP reversibly stalls ribosomes specifically on its target mRNAs. Our results suggest that loss of a translational brake on the synthesis of a subset of synaptic proteins contributes to FXS. In addition, they provide insight into the molecular basis of the cognitive and allied defects in FXS and ASD and suggest multiple targets for clinical intervention.
The control of RNA alternative splicing is critical for generating biological diversity. Despite emerging genome-wide technologies to study RNA complexity, reliable and comprehensive RNA-regulatory networks have not been defined. Here, we used Bayesian networks to probabilistically model diverse data sets and predict the target networks of specific regulators. We applied this strategy to identify approximately 700 alternative splicing events directly regulated by the neuron-specific factor Nova in the mouse brain, integrating RNA-binding data, splicing microarray data, Nova-binding motifs, and evolutionary signatures. The resulting integrative network revealed combinatorial regulation by Nova and the neuronal splicing factor Fox, interplay between phosphorylation and splicing, and potential links to neurologic disease. Thus, we have developed a general approach to understanding mammalian RNA regulation at the systems level.
Exome sequencing of 343 families, each with a single child on the autism spectrum and at least one unaffected sibling, reveal de novo small indels and point substitutions, which come mostly from the paternal line in an age-dependent manner. We do not see significantly greater numbers of de novo missense mutations in affected versus unaffected children, but gene-disrupting mutations (nonsense, splice site, and frame shifts) are twice as frequent, 59 to 28. Based on this differential and the number of recurrent and total targets of gene disruption found in our and similar studies, we estimate between 350 and 400 autism susceptibility genes. Many of the disrupted genes in these studies are associated with the fragile X protein, FMRP, reinforcing links between autism and synaptic plasticity. We find FMRP-associated genes are under greater purifying selection than the remainder of genes and suggest they are especially dosage-sensitive targets of cognitive disorders.
- Robert Darnell,
- Ivan Iossifov,
- Michael Ronemus,
- Dan Levy,
- Jennifer Darnell,
- Elaine R. Mardis,
- Richard K. Wilson,
- Michael C. Schatz,
- W. Richard McCombie,
- Michael WIgler
Nature Structural & Molecular BiologyFebruary 2012
MicroRNAs (miRNAs) regulate mRNA targets through perfect pairing with their seed region (positions 2-7). Recently, a precise genome-wide map of miRNA interaction sites in mouse brain was generated by high-throughput sequencing and analysis of clusters of ~50-nucleotide mRNA tags cross-linked to Argonaute (Ago HITS-CLIP). By analyzing Ago HITS-CLIP 'orphan clusters'-Ago binding regions from HITS-CLIP that cannot be explained by canonical seed matches-we have now identified an alternative binding mode used by miRNAs. Specifically, G-bulge sites (positions 5-6) are often bound and regulated by miR-124 in brain. More generally, bulged sites comprise ≥15% of all Ago-miRNA interactions in mouse brain and are evolutionarily conserved. We call position 6 the 'pivot' nucleotide and suggest a model in which a transitional 'nucleation bulge' leads to functional bulge mRNA-miRNA interactions, expanding the number of potential miRNA regulatory sites.
- Robert Darnell,
- Sung Wook Chi,
- Gregory J. Hannon
Molecular CellNovember 2012
MicroRNAs (miRNAs) are essential components of gene regulation, but identification of miRNA targets remains a major challenge. Most target prediction and discovery relies on perfect complementarity of the miRNA seed to the 3' untranslated region (UTR). However, it is unclear to what extent miRNAs target sites without seed matches. Here, we performed a transcriptome-wide identification of the endogenous targets of a single miRNA-miR-155-in a genetically controlled manner. We found that approximately 40% of miR-155-dependent Argonaute binding occurs at sites without perfect seed matches. The majority of these noncanonical sites feature extensive complementarity to the miRNA seed with one mismatch. These noncanonical sites confer regulation of gene expression, albeit less potently than canonical sites. Thus, noncanonical miRNA binding sites are widespread, often contain seed-like motifs, and can regulate gene expression, generating a continuum of targeting and regulation.
Neuronal Elav-like (Hu) proteins regulate RNA splicing and abundance to control glutamate levels and neuronal excitability.
The paraneoplastic neurologic disorders target several families of neuron-specific RNA binding proteins (RNABPs), revealing that there are unique aspects of gene expression regulation in the mammalian brain. Here, we used HITS-CLIP to determine robust binding sites targeted by the neuronal Elav-like (nElavl) RNABPs. Surprisingly, nElav protein binds preferentially to GU-rich sequences in vivo and in vitro, with secondary binding to AU-rich sequences. nElavl null mice were used to validate the consequence of these binding events in the brain, demonstrating that they bind intronic sequences in a position dependent manner to regulate alternative splicing and to 3'UTR sequences to regulate mRNA levels. These controls converge on the glutamate synthesis pathway in neurons; nElavl proteins are required to maintain neurotransmitter glutamate levels, and the lack of nElavl leads to spontaneous epileptic seizure activity. The genome-wide analysis of nElavl targets reveals that one function of neuron-specific RNABPs is to control excitation-inhibition balance in the brain.
Ptbp2 represses adult-specific splicing to regulate the generation of neuronal precursors in the embryonic brain
Genes and DevelopmentJuly 2012
Two polypyrimidine tract RNA-binding proteins (PTBs), one near-ubiquitously expressed (Ptbp1) and another highly tissue-restricted (Ptbp2), regulate RNA in interrelated but incompletely understood ways. Ptbp1, a splicing regulator, is replaced in the brain and differentiated neuronal cell lines by Ptbp2. To define the roles of Ptbp2 in the nervous system, we generated two independent Ptbp2-null strains, unexpectedly revealing that Ptbp2 is expressed in neuronal progenitors and is essential for postnatal survival. A HITS-CLIP (high-throughput sequencing cross-linking immunoprecipitation)-generated map of reproducible Ptbp2-RNA interactions in the developing mouse neocortex, combined with results from splicing-sensitive microarrays, demonstrated that the major action of Ptbp2 is to inhibit adult-specific alternative exons by binding pyrimidine-rich sequences upstream of and/or within them. These regulated exons are present in mRNAs encoding proteins associated with control of cell fate, proliferation, and the actin cytoskeleton, suggesting a role for Ptbp2 in neurogenesis. Indeed, neuronal progenitors in the Ptbp2-null brain exhibited an aberrant polarity and were associated with regions of premature neurogenesis and reduced progenitor pools. Thus, Ptbp2 inhibition of a discrete set of adult neuronal exons underlies early brain development prior to neuronal differentiation and is essential for postnatal survival.
The US National Institute of Neurological Disorders and Stroke convened major stakeholders in June 2012 to discuss how to improve the methodological reporting of animal studies in grant applications and publications. The main workshop recommendation is that at a minimum studies should report on sample-size estimation, whether and how animals were randomized, whether investigators were blind to the treatment, and the handling of data. We recognize that achieving a meaningful improvement in the quality of reporting will require a concerted effort by investigators, reviewers, funding agencies and journal editors. Requiring better reporting of animal studies will raise awareness of the importance of rigorous study design to accelerate scientific progress.
Comment in: Reporting standards: Rigid guidelines may restrict research. [Nature. 2012]
- Robert Darnell,
- Story Landis,
- Shai Silberberg
- Molecular Biology
- High throughput genomics
- RNA biology
- Translational Research
- Cell Culture
- In Vivo
- In Vitro
- Protein Chemistry
- Protein Expression
- Stem Cells
- Cell Biology
- Cancer Research
- Signal Transduction
- Assay Development
- Western Blotting
- See 17+ See less
- biotechnology; RNA maps; tumor immunity; paraneoplastic neurologic disorders; high throughput RNA profiling,
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Honors & Awards
Elected Member, National Academy of Sciences, 2014
NIH Directors Transformative Research Award, 2012
Cotzias Lecture, American Academy of Neurology, 2012
Elected Member, Institute of Medicine of the National Academies, 2010
Elected Fellow (Neuroscience), American Association for the Advancement of Science, 2010
Neuron Innovator Award, GeneExpression Systems, 2010
Elected Member, Association of American Physicians, 2010
Dana Alliance for Brain Initiatives, 2007
Investigator, Howard Hughes Medical Institute, 2002
Elected Member, Kunkel Society, 2001
Burroughs Wellcome Fund Clinical Scientist Award in Translational Research, 2000
Derek Denny-Brown Young Neurological Scholar Award, 1998
Irma T. Hirschl Career Scientist Award, 1996
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