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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Identifying mRNA Sequence Elements for Target Recognition by Human Argonaute Proteins

ABSTRACT: It is commonly known that mammalian microRNAs guide the RNA-induced silencing complex (RISC) to target mRNAs through the seed-pairing rule. However, recent experiments by co-immunoprecipitating the argonaute proteins (AGOs), the central catalytic component of RISC, have consistently revealed extensive AGO-associated mRNAs lacking seed complementarity with microRNAs. We herein test the hypothesis that AGO has its own binding preference within target mRNAs, independent of guide microRNAs. By systematically analyzing the data from in vivo cross-linking experiments with human AGOs, we have identified a structurally accessible and evolutionarily conserved region (~10 nucleotides) that alone can accurately predict AGO-mRNA associations, independent of the presence of microRNA binding sites. Within this region, we further identified an enriched motif that is also consistently present in several independent AGO-immunoprecipitation datasets. We used RNAcompete to enumerate the RNA-binding preference of human AGO2 to all possible 7-mer RNA sequences, and validated our motif in vitro. These findings reveal a novel function of AGOs as sequence-specific RNA-binding proteins, which may aid microRNAs in recognizing their targets with high specificity. Here, we analyze the RNA-binding preference of human Argonaute 2 protein using RNAcompete assay

ORGANISM(S): Homo sapiens

SUBMITTER: Zhaolei Zhang

PROVIDER: E-GEOD-55122 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Identifying mRNA Sequence Elements for Target Recognition by Human Argonaute Proteins

Project description:It is commonly known that mammalian microRNAs guide the RNA-induced silencing complex (RISC) to target mRNAs through the seed-pairing rule. However, recent experiments by co-immunoprecipitating the argonaute proteins (AGOs), the central catalytic component of RISC, have consistently revealed extensive AGO-associated mRNAs lacking seed complementarity with microRNAs. We herein test the hypothesis that AGO has its own binding preference within target mRNAs, independent of guide microRNAs. By systematically analyzing the data from in vivo cross-linking experiments with human AGOs, we have identified a structurally accessible and evolutionarily conserved region (~10 nucleotides) that alone can accurately predict AGO-mRNA associations, independent of the presence of microRNA binding sites. Within this region, we further identified an enriched motif that is also consistently present in several independent AGO-immunoprecipitation datasets. We used RNAcompete to enumerate the RNA-binding preference of human AGO2 to all possible 7-mer RNA sequences, and validated our motif in vitro. These findings reveal a novel function of AGOs as sequence-specific RNA-binding proteins, which may aid microRNAs in recognizing their targets with high specificity.

2014-02-19 | GSE55122 | GEO

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. The bound RNA from each RNA binding protein pulldown assay is analyzed on a custom Agilent microarray using a pool RNA control as a reference.

2010-05-18 | E-GEOD-15769 | biostudies-arrayexpress

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

Project description:Here, we analyze the RNA-binding preferences of several NHL containing RNA-binding proteins (BRAT, NCL-1, TRIM71, WECH, LIN-41, TRIM56, and MEI-P26) using RNAcompete. In the RNAcompete assay, purified GST-tagged RNA-binding protein 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.

2015-09-14 | E-GEOD-73000 | biostudies-arrayexpress

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.

2015-03-30 | E-GEOD-60498 | biostudies-arrayexpress

Conserved functional antagonism between CELF and MBNL proteins regulates stem cell-specific alternative splicing and regeneration in planarians

Project description:Here, we analyze the RNA-binding preferences of the planarian smed-mbnl-like-2, smed-mbnl-1 (isoform X1), smed-bruli, smed-mbnl-1 (isoform Xins), and smed-mbnl-like-1 protein using RNAcompete. In the RNAcompete assay, a purified GST-tagged protein 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.

2016-08-01 | E-GEOD-75554 | biostudies-arrayexpress

A compendium of RNA-binding motifs for analysis of gene regulation

Project description:RNA binding proteins are key regulators of gene expression, yet only a small fraction of these proteins has been functionally characterized. Here, we report the first large-scale analysis of the RNA motifs recognised by RNA binding proteins, encompassing 205 distinct proteins from 24 diverse eukaryotes. The sequence specificities of RBPs display deep evolutionary conservation, such that the recognition preferences for a large fraction of metazoan RNA binding proteins can be inferred from the sequences of their binding domains. The motifs we identify in vitro correlate well with in vivo RNA-binding data. Moreover, we can associate them with distinct functional roles in diverse types of post-transcriptional regulation, enabling new insights into the functions of RNA binding proteins in both normal physiology and in human disease. These data provide an unprecedented global view of RBPs and their targets and constitute an invaluable resource for defining post-transcriptional regulatory mechanisms in eukaryotes. Here, we analyze the RNA-binding preferences of 205 distinct RNA-binding proteins from 24 different eukaryotes, using RNAcompete. In this assay, purified GST-tagged RBPs are incubated with an excess of RNA pool and bound RNA from individual pulldowns are directly labeled, hybridized to a custom Agilent 244K microarray, and analyzed computationally to identify RNA-binding motifs.

2013-05-10 | E-GEOD-41235 | biostudies-arrayexpress

Slicing-Independent RISC Activation Requires the Argonaute PAZ Domain

Project description:Small RNAs regulate the genetic networks through a ribonucleoprotein complex called the RNA induced silencing complexes (RISC), which in mammals contains at its center one of four Argonaute proteins (Ago1-4). A key regulatory event in the RNAi and miRNA pathways is Ago loading, where double stranded small RNA duplexes are incorporated into RISC (pre-RISC) and then become single stranded (mature-RISC), a process that is not well understood. The Agos contain an evolutionary conserved PAZ (Piwi/Argonaute/Zwille) domain whose primary function is to bind the 3’-end of small RNAs. We created multiple Paz domain disrupted Ago mutant proteins and studied their biochemical properties and biological functionality in cells. We found that the Paz domain is dispensable for Ago loading of slicing-competent RISC. In contrast, in the absence of slicer activity or slicer substrate duplex RNAs, Paz-disrupted Agos bound duplex siRNAs but were unable to unwind/eject the passenger strand and form functional RISC complexes. We have discovered that the highly conserved Paz domain plays an important role in RISC activation, providing new mechanistic insights into how miRNAs regulate genes, as well as new insights for future design of miRNA and RNAi-based therapeutics. Various Argonautes associated small RNA profiles were generated by deep sequencing the Agos-IP samples in HEK293 Cells transfected with corresponding Argonaute.

2012-07-12 | E-GEOD-39146 | biostudies-arrayexpress

Slicing-Independent RISC Activation Requires the Argonaute PAZ Domain

2012-07-12 | GSE39146 | GEO

Differential RISC association of endogenous human microRNAs predicts their inhibitory potential

Project description:It has previously been assumed that the generally high stability of microRNAs (miRNAs) reflects their tight association with Argonaute (Ago) proteins, essential components of the RNA-induced silencing complex (RISC). However, recent data have suggested that the majority of mature miRNAs are not, in fact, Ago associated. Here, we demonstrate that endogenous human miRNAs vary widely, by >100-fold, in their level of RISC association and show that the level of Ago binding is a better indicator of inhibitory potential than is the total level of miRNA expression. While miRNAs of closely similar sequence showed comparable levels of RISC association in the same cell line, these varied between different cell types. Moreover, the level of RISC association could be modulated by overexpression of complementary target mRNAs. Together, these data indicate that the level of RISC association of a given endogenous miRNA is regulated by the available RNA targetome and predicts miRNA function. This series includes microRNA sequencing data for human cell lines 293, C8166, A549, SH-SY5Y, and an EBV-transformed LCL line. For each cell line, total small RNA isolated by TRIzol was sequenced for comparison to RISC-associated microRNAs as determined by sequencing small RNAs from an Argonaute immunoprecipitation.

2014-05-30 | E-GEOD-58127 | biostudies-arrayexpress

Differential RISC association of endogenous human microRNAs predicts their inhibitory potential

2014-05-30 | GSE58127 | GEO

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