2004 — 2013 |
Piano, Fabio |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
A Systematic Rnai-Based Map of C. Elegans Embryogenesis
[unreadable] DESCRIPTION (provided by applicant): [unreadable] One of the next major goals of the Human Genome Project is to identify the function of all the genes it encodes. Arguably the greatest challenge lies in determining the in vivo functions for large sets of genes and how they orchestrate the complex processes of the cell, and animal models can play a significant role in guiding in vivo functional annotation of the human genome. C. elegans is well established as an important model system in which to study the molecular genetics of cell biology and animal development and has also become a leading model for functional genomic research; the early embryo in particular is an excellent system in which to study basic cell biological and developmental processes. RNA interference (RNAi) is a rapid reverse genetics approach to identify the in vivo functions of genes, and several large-scale RNAi scans in C. elegans have so far led to the identification of over 1,500 genes required for embryogenesis. By systematically annotating the functions of such genes in detail using time-lapse microscopy of early embryogenesis, specific cellular roles for over 200 genes have been discovered; however, it is estimated that at least an additional 1000 genes essential for embryonic development remain to be identified. The next step now required is to provide a systematic, detailed functional characterization of all the genes that play a role in this system - a "phenotypic map" of in vivo functions to lay the foundation for integrative systems biology approaches. [unreadable] [unreadable] The goal of this project is to use RNAi to produce a high-resolution phenotypic map of early embryogenesis in C. elegans: a detailed functional description of all the genes required for basic cellular, subcellular, and developmental processes in the early embryo. To accomplish this goal, single-gene RNAi of all approximately 12,000 validated ORFs cloned by the ORFeome project will be performed to assay embryonic lethality and carry out systematic detailed phenotypic analysis of early embryogenesis using DIC optics. An online database, RNAiDB, will be used for all aspects of this study: data collection, scoring, analysis, and distribution. Phenotype-based bioinformatic analysis will be performed, including gene clustering based on phenotypic data and integration with other types of functional genomics data, to identify groups of genes with similar functions and to extend functional annotations, for unknown proteins in particular. The results of these analyses will be used to select groups of genes for further study using a set of subcellular markers to assay specific processes (e.g. chromosome segregation, cytoskeletal organization, cell polarity and cell fate). These secondary studies will be used both to test hypotheses generated from earlier phenotypic and bioinformatic analyses, as well as to generate more in-depth data on the functions of particular gene sets. Since many of these basic functions are carried out by highly conserved proteins, the data gathered in this project will be immediately useful to guide the functional annotation of the human and other genomes. [unreadable] [unreadable]
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2006 |
Piano, Fabio |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Function and Evolution of Micrornas
[unreadable] DESCRIPTION (provided by applicant): Recent experiments have shown that the genomes of organisms such as worm, fly, human, and mouse encode hundreds of novel microRNA genes. Many of these small non-coding genes are thought to regulate the translational expression of other genes by binding to partially complementary sites in target messenger RNAs. Initial phenotypic and expression analyses suggest an important role for microRNAs in basic cellular processes such as development, viral response, and apoptosis. Furthermore, many microRNAs are conserved over large evolutionary distances. However, the biological function of most microRNAs is unknown. Although recent computational methods have made progress towards the identification of microRNA targets, no experimental high-throughput method for dissecting microRNA function exists. Our long-term goal is to create a system where bioinformatic and experimental methods are integrated to make possible the high-throughput combinatorial analysis of microRNA function. Here, we propose to setup this system. Specifically, we will (a) employ in silica methods to predict microRNA targets in five different nematode species, (b) use inhibitor oligonucleotides to knock down the function of 20 microRNAs across several nematode species in vivo, and register their loss-of-function phenotypes throughout development, (c) develop a system to validate predicted microRNA targets in vivo and test 25 computationally predicted targets. These data will generate novel insights into the biological function of microRNAs and their potential role in shaping biological diversity. This project is an intensive collaboration between a bioinformatics lab (focused on gene regulatory processes), and an experimental lab (RNAi functional genomics in nematodes) [unreadable] [unreadable]
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2007 — 2011 |
Piano, Fabio |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Encyclopedia of C Elegans 3'Utrs and Their Regulatory Elements
[unreadable] DESCRIPTION (provided by the applicant): We have organized a "3'UTRome Consortium" whose modENCODE goal is to map all 3' untranslated regions (3'UTRs) and their functional sequence elements in C. elegans. 3'UTRs are DNA encoded elements that are co-transcribed along with mRNAs and whose role is to regulate the activity of mRNA. We currently have only a partial and biased view of the global 3'UTRs sequences (3'UTRome) for any metazoan and have even less information on the motifs in the 3'UTRs that are used by trans-acting factors to drive gene regulation. Yet, what is known reveals a high level of complexity where 3'UTRs are often tissue-specific or are subject to alternative splicing events that lead to 3'UTR sequence diversity that parallels the diversity seen within the coding region of the transcript. Small non-coding RNAs (eg. microRNAs) are a class of posttranscriptional regulators that function through motifs found in the 3'UTRs; however only a subset of 3'UTR::microRNA motifs are though to be known. MicroRNAs add to the previously established fundamental role of RNA-binding proteins known to regulate expression; however, even less is known about these protein-binding motifs. C. elegans provides an excellent model to reveal the DNA-encoded functional elements that drive these complex events in a system where the genome is completely mapped and where 3'UTRs are comparatively compact. We propose to build on our preliminary studies and use a combination of in vitro, in vivo and in silico approaches to identify most or all 3'UTRs and functional sequence elements within them. Specifically, we propose to use genome-wide RT-PCR-based strategies to identify all 3'UTRs in C. elegans; to use computational approaches, microarray analysis and deep sequencing to reveal the vast majority of 3'UTR::microRNA binding motifs and use RIP-CHIP, Yeast-3-Hybrid and computational analysis to map the 3'UTR::RNA-binding-Protein motifs. [unreadable] [unreadable] To use genome data in medicine we need to build a map of the DNA elements that could affect every gene's activity. We are proposing to build a critical part of such a map using the model animal C. elegans by identifying all the 3'UTRs (sequence elements that regulate gene expression) as well as dissect the 3'UTRs and identify sub-elements that are responsible for the 3'UTR's functions. [unreadable] [unreadable] [unreadable] [unreadable]
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