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Genome-wide identification of BRAT- and PUM-associated mRNAs reveals PUM-independent functions for BRAT in translational repression and mRNA degradation during the Drosophila maternal-to-zygotic transition

ABSTRACT: Here, we analyze the RNA-binding preferences of the Drosophila melanogaster BRAT protein using RNAcompete.

ORGANISM(S): synthetic construct

PROVIDER: GSE60498 | GEO | 2015/03/30

SECONDARY ACCESSION(S): PRJNA258416

REPOSITORIES: GEO

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Genome-wide identification of BRAT- and PUM-associated mRNAs reveals PUM-independent functions for BRAT in translational repression and mRNA degradation during the Drosophila maternal-to-zygotic transition

Project description:Here, we analyze the RNA-binding preferences of the Drosophila melanogaster BRAT protein using RNAcompete. In the RNAcompete assay, purified GST-tagged BRAT is incubated with an excess of RNA pool and bound RNA from individual pulldown experiments are directly labeled and hybridized to a custom Agilent 244K microarray.

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The crystal structure of the NHL domain in complex with RNA reveals the molecular basis of Drosophila Brain tumor-mediated gene regulation

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RNAcompete: a method for rapid and systematic analysis of the RNA recognition specificities of RNA-binding proteins

Project description:Metazoan genomes encode hundreds of RNA binding proteins (RBPs) but relatively few have well-defined RNA-binding preferences. Current techniques for determining RNA targets, including those involving in vitro selection and RNA co-immunoprecipitation, require significant time and labour investment. Here we introduce RNAcompete, a new method for the systematic analysis of RNA-binding specificities that employs a single binding reaction to determine the relative preferences of RBPs for short RNAs that containing a complete range of k-mers in structured and unstructured RNA contexts. We tested RNAcompete by analyzing nine diverse RBPs (HuR, Vts1, FUSIP1, PTB, U1A, SF2/ASF, SLM2, RBM4, and YB1). RNAcompete identified both expected and previously unknown RNA binding preferences. Using in vitro and in vivo binding data, we demonstrate that preferences for individual 7-mers identified by RNAcompete are a more accurate representation of binding activity than conventional motif models. We anticipate that RNAcompete will be a valuable tool for the large-scale study of RNA-protein interactions.

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RIP-Chip of BRAT and PUM from 0-3 hour Drosophila melanogaster embryos

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