Identification of proteins interacting with lncRNA Braveheart in mouse using protein microarray
ABSTRACT: The mouse lncRNA Braveheart (Bvht) as a non-coding transcript has been found to act in trans to regulate cardiovascular lineage commitment. However, the mechanism of Bvht action is still not clear. lncRNAs have been shown to regulate gene expression though cooperating with protein partners. Recently, we experimentally determine the secondary structure of Bvht containing a novel structural motif AGIL. AGIL motif deletion (BvhtdAGIL) in mouse embryonic stem cells prevents the transition from mesoderm cells to cardiac progenitors. To identify proteins that interact with the Bvht AGIL motif, we used a human protein microarray platform (Human ProtoArray, Life Technology). Full-length Bvht and BvhtdAGIL transcripts were generated by in vitro transcription and labeled with Cy5. 50pmol Cy5-labeled RNAs were individually incubated with the protein microarray.
Project description:Transcriptional profile of Streptococcus pyogenes stk mutant strain JRS2516 vs its wild type parent strain MGAS2221 The RNA prepared from triplicate cultures for the wild type and the stk mutant strains was each labeled with Cy3 and hybridized to duplicate arrays. Reference RNA consisted of pooled RNA for the wild type and stk mutant, labeled with Cy5.
Project description:HeLa cell extracts with or without GSK3 enzyme inhibition were assayed using protein microarrays in order to detect GSK3-dependent changes in protein polyubiquitination. HeLa lysates in triplicates were supplemented with ubiquitin and incubated on protein microarrays (ProtoArray 5.0; Invitrogen) in the presence or absence of the GSK3 inhibitor SB-216763. Polyubiquitination of the arrayed proteins was detected using specific antibodies. ProtoArray 5.0 contains over 9,000 full-length human proteins purified and arrayed in duplicate under native conditions to maximize functionality.
Project description:APECED is a monogenic autoimmune disease caused by defects in Autoimmune Regulator (AIRE) gene that promotes the expression of self-antigens in the thymus. The autoimmune phenotype in APECED is variable, and multiple proteins are targeted by autoantibodies. This study used human protein arrays to screen autoantibody targets in APECED and to identify novel autoimmune targets. Altogether 100 sera samples from 82 individual APECED patients were profiled using Protoarray antibody specificity service at Invitrogen as well as samples from 12 healthy controls and 8 healthy relatives. The protein arrays (ProtoArray v5.1) were probed as described in Invitrogen’s protocol for Immune Response BioMarker Profiling using detection reagent (Alexa Fluor 647 Goat Anti-Human IgG A21445, Invitrogen) and blocking buffer (Blocking Buffer Kit PA055, Invitrogen). Arrays were scanned using a GenePix 4000B fluorescent scanner, and the data was acquired with GenePix® Pro software. The arrays were probed with sera at a dilution of 1:500.
Project description:Putative RNA-protein interactions with selected snoRNAs were screened using labaled RNA hybridized to a human protein microarray snoRNAs SNORD50A and SNORD50B were in vitro transcribed, labeled with Cy5 and independently hybridized on human protein microarrays. The labeling process was optimized in order to achieve ~ 3 pmol dye per every microgram RNA while maintaining signal intensities that were readily visualized.
Project description:Glucocorticoid receptor (GR) was demonstrated to undergo an N/C interaction between the 26VMDFY30 motif in the N-terminus and C-terminal LBD in the presence of its co-chaperone cytoplasmic constitutive active/androstane receptor retention protein (CCRP). Utilizing the fact that phosphor-mimic mutation of tyrosine 30 to glutamic acid abolished this N/C interaction, CCRP and either GR Y30F or GR Y30E mutant was ectopically expressed in COS-1 cells for subsequent cDNA microarray analysis. Dexamethasone, a potent GR ligand, significantly induced or repressed 673 and 689 genes in Y30F and Y30E, respectively. Among them, only 166 genes were commonly regulated. This analysis strongly suggested that the GR N/C interaction serves to regulate distinct gene sets. Overall design: Gene expressions induced by dexametahsone treatment for 6 hours were measured in COS-1 cells were transiently transfected with mCCRP-V5 and FLAG-hGR (Y30F or Y30E). Three repricates were used in each group. As negative controls, samples prepared from 0.1% DMSO treated cell were used.
Project description:The animal replication-dependent (RD) histone mRNAs are coordinately regulated with chromosome replication. They are the only known cellular mRNAs that are not polyadenylated. Instead, the mature transcripts end in at a conserved stem-loop (SL) structure. This SL structure interacts stably with the stem-loop binding protein (SLBP), which is involved in all aspects of RD-histone mRNA metabolism. We used several genomic methods, including RNA Binding Protein IP followed by microarray analysis (RIP-ChIP) to analyze the RNA binding landscape of SLBP. Reference based design on two color Agilent DNA microarrays. Experimental sample is labeled in Ch1 (Cy3), which are mRNA bound by the SLBP RNA-binding protein. The reference sample is labeled in Ch2 (Cy5) which are the polyribosomal mRNAs from the supernatant that were not bound by anti-SLBP. Controls included no antibody in the IP (which tends to have non-specific background binding to the beads) and pre-incubation of the SLBP with the antibodies antigenic peptide, which blocks antibody binding to the SLBP.
Project description:We describe a refined approach to identify new human RNA-protein interactions. In vitro transcribed labeled RNA is bound to ~9,400 human recombinant proteins spotted on protein microarrays. This approach identified 137 RNA-protein interactions for 10 human coding and non-coding RNAs, including an interaction between Staufen 1 protein and TP53 mRNA that promoted the latter’s stability. RNA hybridization to protein microarrays allows rapid identification of human RNA-protein interactions on a large scale. Sense and antisense strands for 10 RNA transcripts representing protein coding RNAs TP53, HRAS, MYC, BCL2 and non-coding sequences PWRN1, SOX2OT, OCC1, IGF2RNC, lncRBM26 and DLEU1 were in vitro transcribed, labeled with Cy5 and independently hybridized on human protein microarrays. The labeling process was optimized in order to achieve ~ 3 pmol dye per every microgram RNA with average efficacy of 1 dye molecule for approximately every 850 bp RNA to minimally influence RNA native structure and at the same time yield in signal intensities that were readily visualized.
Project description:We have developed an algorithm (“Lever”) that systematically maps metazoan DNA regulatory motifs or motif combinations to the sets of genes that they likely regulate. Lever accomplishes this by assessing whether the motifs are enriched within cis regulatory modules (CRMs), predicted by our “PhylCRM” algorithm, in the noncoding sequences surrounding genes in a collection of gene sets. When these gene sets correspond to Gene Ontology (GO) categories, the results of Lever analysis allow the unbiased assignment of functional annotations to the regulatory motifs and also to the candidate CRMs that comprise the genomic motif occurrences. We demonstrate these methods using human myogenic differentiation as a model system, for which we statistically assessed greater than 25,000 pairings of gene sets and motifs / motif combinations. These results allowed us to assign functional annotations to candidate regulatory motifs predicted previously, and to identify gene sets that are likely to be co-regulated via shared regulatory motifs. Lever allows moving beyond the identification of putative regulatory motifs in mammalian genomes, towards understanding their biological roles. This approach is general and can be applied readily to any cell type, gene expression pattern, or organism of interest. Keywords: expression profiling, time course Primary human skeletal muscle cells were grown in proliferating medium (DMEM + 10% FCS) for 48 hours until about 80% confluence. Cells were then switched to differentiation medium (DMEM/F12 + 2% horse serum) for 48 hours. Total RNA was extracted from cells at -48, -24, 0, +12, +24, and +48 hours relative to induction of differentiation, and labeled with either Cy5 or Cy3. Universal total RNA was also labeled with either Cy3 or Cy5 and was hybridized to the array with the experimental sample. Dye-swaps were performed in addition to duplicate hybridizations for a total of 4 hybridizations per timepoint.
Project description:To investigate the DNA binding specificity of the CLAMP protein, we have designed a custom PBM to interrogate the binding of CLAMP to DNA sequences extracted from the Drosophila melanogaster genome. Specific regions were extracted based on ChIP-seq data, motif occurence and proximity to gene transcription start sites. N-terminal GST-tagged protein samples were made for the C-terminal four and six zinc finger portions of the protein CLAMP; samples were made by in vitro transcription translation (IVT). IVT reaction mixtures for the two CLAMP constructs were applied directly to the PBM microarray and incubated for 1hour. Microarray-bound protein was fluorescently labeled using Alexa488-conjugated antibodies targeting GST, and the microarray was scanned in using a standard microarray scanner. Median fluorescence intensity over eight replicate probes was reported for each unique DNA sequence on the microarray.
Project description:Many human monoclonal antibodies that neutralize multiple clades of HIV-1 are polyreactive and bind avidly to mammalian autoantigens. Indeed, the generation of neutralizing antibodies to the 2F5 and 4E10 epitopes of HIV-1 gp41 in man may be proscribed by immune tolerance since mice expressing the VH and VL regions of 2F5 have a block in B-cell development characteristic of central tolerance. This developmental blockade implies the presence of tolerizing autoantigens that are mimicked by the membrane-proximal external region of HIV-1 gp41. Here we identify human kynureninase (KYNU) and splicing factor 3b subunit 3 (SF3B3) as the primary conserved, vertebrate self-antigens recognized by the 2F5 and 4E10 antibodies, respectively. 2F5 binds the H4 domain of KYNU which contains the complete 2F5 linear epitope (ELDKWA). 4E10 recognizes a conformational epitope of SF3B3 that is strongly dependent on hydrophobic interactions. Opossums carry a rare KYNU H4 domain that abolishes 2F5 binding, but retain all SF3B3 4E10 epitopes. Immunization of opossums with HIV-1 gp140 induced extraordinary titers of serum antibody to the 2F5 ELDKWA epitope but little or nothing to the 4E10 determinant. Identification of structural motif shared by vertebrates and HIV-1 provides direct evidence that immunological tolerance can impair humoral responses to HIV-1. The invitrogen protoarray that contains >9,400 recombinant human proteins was used to identify self-ligands that are recognized by broadly neutralizing HIV-1 antibodies 2F5 and 4E10. An isotype-matched human myeloma protein (151K, Southern Biotech) was used as control.