miRNAs and Other Non-coding RNAs in Nervous System Development and Function

Agenda

Abstracts

miRNAs in the Drosophila Nervous System

Eric Lai, Memorial Sloan-Kettering Cancer Center

miRNAs are ~21-24 nucleotide RNAs with pervasive and fundamental roles in post-transcriptional gene regulation. Neurons have especial requirements for post-transcriptional regulation, and are known to express a diversity of miRNAs. Still, relatively little is known about the functional impact of neural miRNAs. We are using molecular and genetic techniques to study this topic in the Drosophila system. I will present our studies on the function of individual miRNAs and targets during neural development and physiology.

RNA Regulatory Circuitry in Nervous System Development

Mark F. Mehler, Albert Einstein College of Medicine

RNA plays a central role in mediating the explosive and asymmetric evolutionary advances in mammalian nervous system form and function. It serves as an exquisitely environmentally responsive dual interface between the sequence-specific digital-mediated genome and analogue-based protein signaling networks, safeguards CNS bioenergetic reserves, actively modulates other components of the epigenetic hierarchy, participates in dynamic intracellular as well as intercellular transport and local, systemic and germ line targeting, epigenetic memory states, accelerated evolution and multigenerational heritability. These unique properties of RNA molecules mediate multifaceted aspects of genome regulation, essential cellular functions and a diverse array of neural developmental and adult brain functions. In this talk, we will focus on the evolving roles of long non-coding RNAs in orchestrating seminal neural developmental fate decisions and adult homeostatic processes as well as interactions with other site-specific epigenetic remodeling complexes. This will allow us to begin to generate a series of testable hypotheses regarding the roles of non-coding RNAs and their complex post-transcriptional processing in promoting neural cell identity, plasticity, connectivity and higher-order cognitive and behavioral functions in health and in neuropsychiatric disease states.

A Polyq-repeat Protein Promotes The Novel, Morphologically Conserved Death of the Linker Cell in C. elegans

Shai Shaham, The Rockefeller University

Programmed cell death is an essential process during metazoan development. During wild type C. elegans development, nearly all cells slated to die activate caspases and undergo stereotypical morphological changes including chromatin compaction and cell shrinkage. The male-specific linker cell (LC), however, is an exception. The LC leads the migration of the developing gonad, and once migration is complete at the L4-Adult transition, the LC dies. LC death is independent of ced-3 caspase, ced-4/Apaf-1, ced-9/Bcl2, and egl-1/BH3-only protein, indicating that LC death is controlled by a novel program. Indeed, electron micrographs of dying LCs reveal non-apoptotic features, including nuclear crenellation and organelle swelling. Remarkably similar features are also seen in normally dying cells of the vertebrate spinal cord and ciliary ganglion, suggesting that LC death is morphologically conserved. To understand the molecular mechanism of LC death, we performed a genome-wide RNAi screen to identify genes whose loss prevents LC death. RNAi against pqn-41, a gene predicted to encode a polyQ-repeat protein, blocks LC death in 20% of animals examined. Similar defects are seen in pqn-41(ns294) mutants we generated. A transgene containing pqn-41 genomic DNA fused to GFP is expressed in many cells in the animal. Strikingly, expression in the LC is only visible as the cell begins to die, and we identified a pqn-41 promoter region required for LC expression. PQN-41 protein is localized to nuclei and nuclear puncta in all cells, except for the dying LC, where PQN-41 is also cytoplasmic. Previous studies revealed that LC death requires the heterochronic genes let-7, a microRNA, and lin-29, a Zn-finger transcription factor, and that these genes act within the LC to promote its death. We have now shown that a MAPK module containing the TIR-1 adapter protein and the SEK-1 MAPKK also regulate LC death: 30% of tir-1(RNAi) and 49% sek-1(ag1) adult males exhibit LC survival. Epistasis analysis suggests that the heterochronic and MAPK pathways likely function in parallel to regulate LC death. Since both pathways impinge on transcriptional regulators, it is possible that both pathways converge on the promoters of key LC death genes. We are testing this hypothesis with regard to pqn-41. Several human neurodegenerative disorders result from polyQ expansions within endogenous proteins, however, the mechanisms promoting cell death in these diseases is not known. Our studies raise the intriguing possibility that these aberrant proteins activate an endogenous cell death program similar to that driving the death of the LC.

The Role of Small RNAs in Synaptic Plasticity

Priya Rajasethupathy, College of Physicians and Surgeons of Columbia University

To explore the role of small RNAs in learning-related synaptic plasticity, we profiled the small RNAs of Aplysia Californica by massive parallel sequencing. We identified two classes of neuronally expressed small RNAs that are bi-directionally regulated, miRNAs and piRNAs. We here present our progress in understanding the function of these small RNAs during learning-related synaptic-plasticity.

Epigenetic Control of Transcriptional Homeostasis in the Brain

Anne Schaefer, The Rockefeller University

The maintenance of stable patterns and levels of gene expression (transcriptional homeostasis) in functionally distinct neurons is essential for normal brain function. Alteration of the transcriptional homeostasis is associated with numerous neurological diseases such as mental retardation or autism. We will discuss the role of epigenetic regulators, such as histone modifying enzymes and miRNAs, in regulation of gene expression in adult neurons. We will show how changes in the neuronal transcriptional homeostasis in the postnatal brain lead to diseases such a neurodegeneration, mental retardation or epilepsy.

miRNA Expression and Target Site Variation in the Human Central Nervous System

Neil Renwick, The Rockefeller University

Defective post-transcriptional regulation of gene expression, mediated through the actions of RNA-binding proteins (RBPs) and microRNAs (miRNAs), causes or underlies several neurodegenerative and other neurological disorders. To characterize these disorders, we have developed several methods, including small RNA sequencing, miRNA in situ hybridization, photoreactive-uridine enhanced cross-linking and immunoprecipitation (PURE-CLIP) and nested, multiplexed PCR in the Tuschl Lab. Here, I will focus on (1) small RNA profiling of brain regions from persons with neurodegenerative disorders and unaffected controls and (2) genetic variation in RBP and miRNA target sites in the NF1 gene in persons with autism. Our goal is to find disease-related molecular alterations that can be used for diagnostics and/or therapeutic intervention.

Transcriptome-wide Determination of miRNA-binding Sites by PURE-CLIP

Markus Hafner, The Rockefeller University

miRNAs are key mediators of post-transcriptional gene regulation. Their function as parts of larger miRNA-containing ribonucleoproteins (miRNPs), as well as their biogenesis requires a number of different RNA binding proteins (RBPs). We have used PURE-CLIP to map at nucleotide resolution and on a transcriptome-wide scale the RNA-binding sites of the Argonaute proteins (AGO1-4) and TNRC6A-C, RBPs making up the effector miRNPs. PURE-CLIP relies on feeding the nucleoside analog 4-thiouridine (4SU) to cultured cells. 4SU is readily incorporated into newly synthesized RNAs and efficiently crosslinked to RNA-binding proteins (RBPs) by long-wavelength UV irradiation of live cells. The precise position of the crosslink and the RNA recognition site of the protein can be deduced from a characteristic T to C transition in the reverse transcribed cDNA from the crosslinked segments, separating these sequences efficiently from the always present background sequences derived from fragments of abundant cellular RNAs. We could show by AGO1-4-PURE-CLIP that the endogenous miRNAs target approximately 20% of the 22,000 transcripts expressed in HEK293-cells. Surprisingly, more than 50% of the identified miRNA-binding sites were located in the coding sequences (CDS). miRNA-binding at these sites led to a much smaller destabilization effect on the target RNA compared to binding in the 3’UTR. In general, our data are in good agreement with the current knowledge on mRNA-regulation by miRNAs gained by overexpression and inhibition of individual miRNAs. TNRC6A-C PURE-CLIP revealed that the TNRC6 proteins not only bind a closely related set of transcripts as the AGO proteins, but that they also share same binding site – more than 50% of the TNRC6 binding sites were found within 25 nt of an AGO-binding site.

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