Detection of a cellular polypeptide associated with adenovirus-coded VA RNA using in vitro labeling of proteins cross-linked to RNA.

Ultraviolet light induced RNA-protein cross-linking for identification of polypeptides interacting with RNA in intact cells (Wagenmakers et al. 1980), is limited by the intensity of the label in the proteins or in residual nucleotides remaining attached to the proteins after RNase treatment of the RNA-protein complexes. Here we report a method, where th cross-linked RNA-protein complexes are treated with RNase T1 and the T1-oligonucleotides covalently linked to the proteins are labeled in the 5' terminus using gamma-32P-ATP and T4 polynucleotide kinase. The cross-linked proteins can then readily be identified owing to the incorporated 32P label. As examples, proteins associated with polyadenylated mRNA, hnRNA and adenoviral VA RNA were identified. A protein with a molecular weight of approximately 50,000 is found associated with adenovirus-coded VA RNA. This was confirmed by binding assays, in which labeled VAI RNA is incubated with proteins from uninfected and adenovirus infected HeLa cells immobilized on nitrocellulose sheets.     

Analyzing mRNA-protein complexes using a yeast three-hybrid system

RNA-protein interactions are essential for the proper execution and regulation of every step in the life of a eukaryotic mRNA. Here we describe a three-hybrid system in which RNA-protein interactions can be analyzed using simple phenotypic or enzymatic assays in Saccharomyces cerevisiae. The system can be used to detect or confirm an RNA-protein interaction, to analyze RNA-protein interactions genetically, and to discover new protein or RNA partners when only one is known. Multicomponent complexes containing more than one protein can be detected, identified, and analyzed. We describe the method and how to use it, and discuss applications that bear particularly on eukaryotic mRNAs.     

Eukaryotic small ribonucleoproteins. Anti-La human autoantibodies react with U1 RNA-protein complexes

Anti-La sera from patients with autoimmune disorders precipitate a set of nuclear and cytoplasmic small RNA-protein complexes. Up to now, it has been thought that the La antigen is associated only with RNAs transcribed by RNA polymerase III, including precursors of tRNA and 5 S ribosomal RNA. Here we report that anti-La sera also react with ribonucleoprotein particles containing small nuclear RNA U1, which is transcribed by RNA polymerase II. Anti-La sera from 12 out of 12 patients tested were found to precipitate U1 RNA-protein complexes from HeLa cell nuclear extracts, under conditions where nonimmune sera do not. Ribonucleoprotein particles containing a second small nuclear RNA, U2, do not react appreciably with anti-La sera although they are present in HeLa cell nuclei at the same concentration as U1 RNA. Anti-La sera also react with U1 RNA-protein complexes in mouse and frog cells, but not in Drosophila or Chironomus, two organisms which lack the La antigen. Hybridization of cloned U1 DNA with anti-La-reactive RNA from HeLa cell nuclear extracts reveals mature U1 RNA, whereas anti-La-reactive cytoplasmic RNA contains a series of hybridizing bands that represent molecules 1-7 nucleotides longer than U1 and which may include precursors of nuclear U1 RNA (Madore, S. J., Wieben, E. D., and Pederson, T. (1984) J. Cell Biol., 188-192). Pulse-chase experiments suggest that the association of La antigenicity with these cytoplasmic U1 RNA molecules is transient. These results are discussed in relation to the presence of uridylate-rich sequences in the 3' termini of U1 RNA precursors and mature U1 RNA, which are similar to La antigen binding sites in several RNAs transcribed by RNA polymerase III.     

Ribosomal and informational ribonucleoprotein complexes of animal cells. Study on rat liver ribonucleic acids as constituents of ribonucleoprotein complexes by chromatography on nucleoprotein-celite columns.

A novel method of RNA fractionation has been developed. Nuclear and cytoplasmic rat liver RNA species were fractionated as constituents of corresponding ribonucleoprotein particles, which were previously adsorbed on a Celite-column by their protein component. The fractionation is based on a dissociation of the particles (linear concentration gradient of LiCl and urea with subsequent temperature gradient), which results in a gradual release of the RNA molecules from ribonucleoprotein complexes. Thus the fractionation is in accordance with the tightness of the RNA-protein bonds. A gradient elution of RNA from a nucleoprotein-Celite column permitted fractionation of both ribosomal and rapidly labelled non-ribosomal RNA. The latter, both nuclear and cytoplasmic, could be separated by chromatography on nucleoprotein-Celite columns into two main fractions (components I and V). In cytoplasmic RNA components I and V presumably correspond to mlRNA (messenger-like RNA of free cytoplasmic particles) and mRNA (template RNA associated with ribosomes) respectively.     

RNA Whole-Mount In situ Hybridisation Proximity Ligation Assay (rISH-PLA), an Assay for Detecting RNA-Protein Complexes in Intact Cells

Techniques for studying RNA-protein interactions have lagged behind those for DNA-protein complexes as a consequence of the complexities associated with working with RNA. Here we present a method for the modification of the existing In Situ Hybridisation–Proximity Ligation Assay (ISH-PLA) protocol to adapt it to the study of RNA regulation (rISH-PLA). As proof of principle we used the well-characterised interaction of the Xenopus laevis Staufen RNA binding protein with Vg1 mRNA, the complex of which co-localises to the vegetal pole of Xenopus oocytes. The applicability of both the Stau1 antibody and the Locked Nucleic Acid probe (LNA) recognising Vg1 mRNA were independently validated by whole-mount Immunohistochemistry and whole-mount in situ hybridisation assays respectively prior to combining them in the rISH-PLA assay. The rISH-PLA assay allows the identification of a given RNA-protein complex at subcellular and single cell resolution, thus avoiding the lack of spatial resolution and sensitivity associated with assaying heterogenous cell populations from which conventional RNA-protein interaction detection techniques suffer. This technique will be particularly usefully for studying the activity of RNA binding proteins (RBPs) in complex mixtures of cells, for example tissue sections or whole embryos.     

Cell proteins bind specifically to West Nile virus minus-strand 3' stem-loop RNA.

The first 96 nucleotides of the 5'noncoding region (NCR) of West Nile virus (WNV) genomic RNA were previously reported to form thermodynamically predicted stem-loop (SL) structures that are conserved among flaviviruses. The complementary minus-strand 3' NCR RNA, which is thought to function as a promoter for the synthesis of plus-strand RNA, forms a corresponding predicted SL structure. RNase probing of the WNV 3' minus-strand stem-loop RNA [WNV (-)3' SL RNA] confirmed the existence of a terminal secondary structure. RNA-protein binding studies were performed with BHK S100 cytoplasmic extracts and in vitro-synthesized WNV (-)3' SL RNA as the probe. Three RNA-protein complexes (complexes 1,2, and 3) were detected by a gel mobility shift assay, and the specificity of the RNA-protein interactions was confirmed by gel mobility shift and UV-induced cross-linking competition assays. Four BHK cell proteins with molecular masses of 108, 60, 50, and 42 kDa were detected by UV-induced cross-linking to the WNV (-)3' SL RNA. A preliminary mapping study indicated that all four proteins bound to the first 75 nucleotides of the WNV 3' minus-strand RNA, the region that contains the terminal SL. A flavivirus resistance phenotype was previously shown to be inherited in mice as a single, autosomal dominant allele. The efficiencies of infection of resistant cells and susceptible cells are similar, but resistant cells (C3H/RV) produce less genomic RNA than congenic, susceptible cells (C3H/He). Three RNA-protein complexes and four UV-induced cross-linked cell proteins with mobilities identical to those detected in BHK cell extracts with the WNV (-)3' SL RNA were found in both C3H/RV and C3H/He cell extracts. However, the half-life of the C3H/RV complex 1 was three times longer than that of the C3H/He complex 1. It is possible that the increased binding activity of one of the resistant cell proteins for the flavivirus minus-strand RNA could result in a reduced synthesis of plus-strand RNA as observed with the flavivirus resistance phenotype.     

A sequence predicted to form a stem-loop is proposed to be required for formation of an RNA-protein complex involving the 3'UTR of beta-subunit F0F1-ATPase mRNA

ATP-synthase assembly requires coordinated control of ATP mRNA translation; this may e.g. occur through the formation of mRNA-protein complexes. In this study we aim to identify sequences in the 3'UTR of the beta-subunit F(1)-ATPase mRNA necessary for RNA-protein complex formation. We examined the interaction between a brain cytoplasmic protein extract and in vitro-synthesized beta-subunit 3'UTR probes containing successive accumulative 5'- and 3'-deletions, as well as single subregion deletions, with or without poly(A) tail. Using electrophoretic mobility shift assays we found that two major RNA-protein complexes (here called RPC1 and RPC2) were formed with the full-length 3'UTR. The RPC2 complex formation was fully dependent on the presence of both the poly(A) tail and one subregion directly adjacent to it. For RPC1 complex formation, a 3'UTR sequence stretch (experimentally divided into three subregions) adjacent to but not including the poly(A) tail was necessary. This sequence stretch includes a conserved 40-nucleotide region that, according to the structure prediction program mfold, is able to fold into a characteristic stem-loop structure. Since the formation of the RPC1 complex was not dependent on a conventional sequence motif in the 3'UTR of the beta-subunit mRNA but rather on the presence of the predicted stem-loop-forming region as such, we hypothetize that this RNA region, by forming a stem-loop in the 3'UTR beta-subunit mRNA, is necessary for formation of the RNA-protein complex.     

Processing bodies and plant development

Processing bodies (P-bodies) contain RNA-protein complexes linked to cytoplasmic RNA decay pathways including mRNA decapping, nonsense-mediated decay (NMD) and small RNA-mediated decay. Plants deficient in P-body components display severe developmental perturbations, suggesting that these cytoplasmic bodies play important roles in regulating gene expression during plant development. Here, we summarize recent progress in the genetic dissection of P-body components and their roles in translational repression and mRNA decapping.     

Three different cellular proteins bind to complementary sites on the 5'-end-positive and 3'-end-negative strands of mouse hepatitis virus RNA.

The termini of viral genomic RNA and its complementary strand are important in the initiation of viral RNA replication, which probably involves both viral and cellular proteins. To detect the possible cellular proteins involved in the replication of mouse hepatitis virus RNA, we performed RNA-protein binding studies with RNAs representing both the 5' and 3' ends of the viral genomic RNA and the 3' end of the negative-strand complementary RNA. Gel-retardation assays showed that both the 5'-end-positive- and 3'-end-negative-strand RNA formed an RNA-protein complex with cellular proteins from the uninfected cells. UV cross-linking experiments further identified a 55-kDa protein bound to the 5' end of the positive-strand viral genomic RNA and two proteins 35 and 38 kDa in size bound to the 3' end of the negative-strand cRNA. The results of the competition assay confirmed the specificity of this RNA-protein binding. No proteins were found to bind to the 3' end of the viral genomic RNA under the same conditions. The binding site of the 55-kDa protein was mapped within the 56-nucleotide region from nucleotides 56 to 112 from the 5' end of the positive-strand RNA, and the 35- and 38-kDa proteins bound to the complementary region on the negative-strand RNA. The 38-kDa protein was detected only in DBT cells but was not detected in HeLa or COS cells, while the 35-kDa protein was found in all three cell types. The juxtaposition of the different cellular proteins on the complementary sites near the ends of the positive- and negative-strand RNAs suggests that these proteins may interact with each other and play a role in mouse hepatitis virus RNA replication.     

Dissecting the key recognition features of the MS2 bacteriophage translational repression complex.

The MS2 RNA operator capsid offers an unparalleled opportunity to study sequence-specific protein-protein and RNA-protein interactions in molecular detail. RNA molecules encompassing the minimal translational operator recognition elements can be soaked into crystals of RNA-free coat protein shells, allowing the RNA to access the interior of the capsids and make contact with the operator binding sites. Correct interpretation of these structural studies depends critically on functional analysis in solution to confirm that the interactions seen in the crystal are not an artefact of the unusual approach used to generate the RNA-protein complexes. Here we present a series of in vivo and in vitro functional assays, using coat proteins carrying single amino acid substitutions at residues which either interact with the operator RNA or are involved in stabilizing the conformation of the FG loop, the site of the major quasi-equivalent conformational change. Variant operator RNAs have been assayed for coat protein affinity in vitro. The results reveal the robustness of the operator-coat protein interaction and the requirement for both halves of a protein dimer to contact RNA in order to achieve tight binding. They also suggest that there may be a direct link between the conformation of the FG loop and RNA binding.     

In vitro identification and characterization of an early complex linking HIV-1 genomic RNA recognition and Pr55Gag multimerization

The minimal protein requirements that drive virus-like particle formation of human immunodeficiency virus type 1 (HIV-1) have been established. The C-terminal domain of capsid (CTD-CA) and nucleocapsid (NC) are the most important domains in a so-called minimal Gag protein (mGag). The CTD is essential for Gag oligomerization. NC is known to bind and encapsidate HIV-1 genomic RNA. The spacer peptide, SP1, located between CA and NC is important for the multimerization process, viral maturation and recognition of HIV-1 genomic RNA by NC. In this study, we show that NC in the context of an mGag protein binds HIV-1 genomic RNA with almost 10-fold higher affinity. The protein region encompassing the 11th alpha-helix of CA and the proposed alpha-helix in the CA/SP1 boundary region play important roles in this increased binding capacity. Furthermore, sequences downstream from stem loop 4 of the HIV-1 genomic RNA are also important for this RNA-protein interaction. In gel shift assays using purified mGag and a model RNA spanning the region from +223 to +506 of HIV-1 genomic RNA, we have identified an early complex (EC) formation between 2 proteins and 1 RNA molecule. This EC was not present in experiments performed with a mutant mGag protein, which contains a CTD dimerization mutation (M318A). These data suggest that the dimerization interface of the CTD plays an important role in EC formation, and, as a consequence, in RNA-protein association and multimerization. We propose a model for the RNA-protein interaction, based on previous results and those presented in this study.     

Specific regions of beta-globin RNA are resistant to nuclease digestion in RNA-protein complexes in chicken reticulocyte nuclei.

The interaction between beta-globin RNA and proteins in chicken reticulocyte nuclei was studied by determining the sequence of nuclease-resistant beta-globin RNA. Two types of nuclease-resistant RNAs were isolated for this study: endogenous nuclease-resistant RNA from 50S heterogeneous nuclear RNA-protein complexes and micrococcal nuclease-resistant nuclear RNA from whole nuclei. The nuclease-resistant regions were identified with the use of a RNA mapping method we recently developed (J.R. Patton and C.-B. Chae, J. Biol. Chem. 258:3991-3995, 1983). We found that beta-globin RNA is assembled into heterogeneous nuclear RNA-protein complexes in a specific manner. There are several regions of nuclease resistance in the first and third exons interrupted at regular intervals by sensitive regions. The second exon has only one nuclease-resistant region. The resistant regions range in size from 20 to 50 nucleotides. This organization may reflect a specific mode of assembly for heterogeneous nuclear RNA-protein complexes.     

Enod40, a short open reading frame-containing mRNA, induces cytoplasmic localization of a nuclear RNA binding protein in Medicago truncatula

In eukaryotes, diverse mRNAs containing only short open reading frames (sORF-mRNAs) are induced at specific stages of development. Their mechanisms of action may involve the RNA itself and/or sORF-encoded oligopeptides. Enod40 genes code for highly structured plant sORF-mRNAs involved in root nodule organogenesis. A novel RNA binding protein interacting with the enod40 RNA, MtRBP1 (for Medicago truncatula RNA Binding Protein 1), was identified using a yeast three-hybrid screening. Immunolocalization studies and use of a MtRBP1-DsRed2 fluorescent protein fusion showed that MtRBP1 localized to nuclear speckles in plant cells but was exported into the cytoplasm during nodule development in enod40-expressing cells. Direct involvement of the enod40 RNA in MtRBP1 relocalization into cytoplasmic granules was shown using a transient expression assay. Using a (green fluorescent protein)/MS2 bacteriophage system to tag the enod40 RNA, we detected in vivo colocalization of the enod40 RNA and MtRBP1 in these granules. This in vivo approach to monitor RNA-protein interactions allowed us to demonstrate that cytoplasmic relocalization of nuclear proteins is an RNA-mediated cellular function of a sORF-mRNA.     

Genome-wide identification of protein binding sites on RNAs in mammalian cells

RNA-binding proteins (RBPs) are proteins that bind to the RNA and participate in forming ribonucleoprotein complexes. They have crucial roles in various biological processes such as RNA splicing, editing, transport, maintenance, degradation, intracellular localization and translation. The RBPs bind RNA with different RNA-sequence specificities and affinities, thus, identification of protein binding sites on RNAs (R-PBSs) will deeper our understanding of RNA-protein interactions. Currently, high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP, also known as CLIP-Seq) is one of the most powerful methods to map RNA-protein binding sites or RNA modification sites. However, this method is only used for identification of single known RBPs and antibodies for RBPs are required. Here we developed a novel method, called capture of protein binding sites on RNAs (RPBS-Cap) to identify genome-wide protein binding sites on RNAs without using antibodies. Double click strategy is used for the RPBS-Cap assay. Proteins and RNAs are UV-crosslinked in vivo first, then the proteins are crosslinked to the magnetic beads. The RNA elements associated with proteins are captured, reverse transcribed and sequenced. Our approach has potential applications for studying genome-wide RNA-protein interactions.