A proline-rich protein binds to the localization element of Xenopus Vg1 mRNA and to ligands involved in actin polymerization

A 340 nucleotide element within the 3' untranslated region of Vg1 mRNA determines its localization to the vegetal cortex of Xenopus oocytes. To identify protein factors that bind to this region, we screened a cDNA expression library with an RNA probe containing this sequence. Five independent isolates encoded a protein (designated Prrp for proline-rich RNA binding protein) having two RNP domains followed by multiple polyproline segments. Prrp and Vg1 mRNAs are co-localized to the vegetal cortex of stage IV oocytes, substantiating an interaction between the two in vivo. Prrp also associates with VegT mRNA, which like Vg1 mRNA uses the late localization pathway, but not with Xcat-2 or Xwnt-11 mRNAs, which use the early pathway. The proline-rich domain of Prrp interacts with profilin, a protein that promotes actin polymerization. Prrp can also associate with the EVH1 domain of Mena, another microfilament-associated protein. Since the anchoring of Vg1 mRNA to the vegetal cortex is actin dependent, one function of Prrp may be to facilitate local actin polymerization, representing a novel function for an RNA binding protein.     

Contextual utilization of enzymes in discrete AKAP79/150 signalling complexes

Cellular function involves the concerted action of signal transduction enzymes. Restriction of enzyme location contributes to the fidelity of each cellular response. A kinase-anchoring proteins (AKAPs) target the cAMP-dependent protein kinase and other signalling enzymes to defined subcellular locations. We have developed a new strategy that combines RNA interference of the endogenous protein and replacement with AKAP79/150 forms unable to anchor selected binding partners. Using this approach we show that AKAP79/150 coordinates different enzyme combinations to modulate the activity of two distinct neuronal ion channels: AMPA-type glutamate receptors and M-type potassium channels. Utilization of distinct enzyme combinations in this manner provides a means to expand the repertoire of cellular events that the same AKAP modulates.     

The long noncoding RNA ZFAS1 promotes the progression of glioma by regulating the miR-150-5p/PLP2 axis

Numerous studies have reported that long noncoding RNA (lncRNA) dysregulation is involved in the progression of many malignant tumors, including glioma. The lncRNA ZNFX1 antisense RNA 1 (ZFAS1) plays an oncogenic role in various malignant tumors, such as gastric cancer and hepatocellular carcinoma. However, the underlying molecular mechanism of ZFAS1 in glioma has not been fully clarified. In this study, we found that the expression of ZFAS1 was upregulated in both glioma tissues and cell lines. Functional experiments revealed that ZFAS1 promoted glioma proliferation, migration and invasion, and increased resistance to temozolomide in vitro. By using online databases, RNA pull-down assays and luciferase reporter assays, ZFAS1 was demonstrated to act as a sponge of miR-150-5p. Furthermore, proteolipid protein 2 (PLP2) was shown to be the functional target of miR-150-5p. Rescue experiments revealed that ZFAS1 regulated the expression of PLP2 by sponging miR-150-5p. Finally, a xenograft tumor assay demonstrated that ZFAS1 promoted glioma growth in vivo. Our results showed that ZFAS1 promoted glioma malignant progression by regulating the miR-150-5p/PLP2 axis, which may provide a potential therapeutic target for the treatment of glioma.     

The RNA ligands for mouse proline-rich RNA-binding protein (mouse Prrp) contain two consensus sequences in separate loop structure

Mouse proline-rich RNA-binding protein (mPrrp) is a mouse ortholog of Xenopus Prrp, which binds to a vegetal localization element (VLE) in the 3′-untranslated region (3′-UTR) of Vg1 mRNA and is expected to be involved in the transport and/or localization of Vg1 mRNA to the vegetal cortex of oocytes. In mouse testis, mPrrp protein is abundantly expressed in the nuclei of pachytene spermatocytes and round spermatids, and shifts to the cytoplasm in elongating spermatids. To gain an insight into the function of mPrrp in male germ cells, we performed in vitro RNA selection (SELEX) to determine the RNA ligand sequence of mPrrp. This analysis revealed that many of the selected clones contained both of two conserved elements, AAAUAG and GU_1–3AG. RNA-binding study on deletion mutants and secondary structure analyses of the selected RNA revealed that a two-loop structure containing the conserved elements is required for high-affinity binding to mPrrp. Furthermore, we found that the target mRNAs of Xenopus Prrp contain intact AAAUAG and GU_1–3AG sequences in the 3′-UTR, suggesting that these binding sequences are shared by Prrps of Xenopus and mouse.     

Histone H3K56 Acetylation Is Required for Quelling-induced Small RNA Production through Its Role in Homologous Recombination

Quelling and DNA damage-induced small RNA (qiRNA) production are RNA interference (RNAi)-related phenomenon from repetitive genomic loci in Neurospora. We have recently proposed that homologous recombination from repetitive DNA loci allows the RNAi pathway to recognize repetitive DNA to produce small RNA. However, the mechanistic detail of this pathway remains largely unclear. By systematically screening the Neurospora knock-out library, we identified RTT109 as a novel component required for small RNA production. RTT109 is a histone acetyltransferase for histone H3 lysine 56 (H3K56) and H3K56 acetylation is essential for the small RNA biogenesis pathway. Furthermore, we showed that RTT109 is required for homologous recombination and H3K56Ac is enriched around double strand break, which overlaps with RAD51 binding. Taken together, our results suggest that H3K56 acetylation is required for small RNA production through its role in homologous recombination.     

Characterization of the reactivity determinants of a novel hairpin substrate of yeast RNase III

RNase III enzymes form a conserved family of proteins that specifically cleave double-stranded (dsRNA). These proteins are involved in a variety of cellular functions, including the processing of many non-coding RNAs, mRNA decay, and RNA interference. Yeast RNase III (Rnt1p) selects its substrate by recognizing the structure generated by a conserved NGNN tetraloop (G2-loop). Mutations of the invariant guanosine stringently inhibit binding and cleavage of all known Rnt1p substrates. Surprisingly, we have found that the 5' end of small nucleolar RNA 48 is processed by Rnt1p in the absence of a G2-loop. Instead, biochemical and structural analyses revealed that cleavage, in this case, is directed by a hairpin capped with an AAGU tetraloop, with a preferred adenosine in the first position (A1-loop). Chemical probing indicated that A1-loops adopt a distinct structure that varies at the 3' end where Rnt1p interacts with G2-loops. Consistently, chemical footprinting and chemical interference assays indicate that Rnt1p binds to G2 and A1-loops using different sets of nucleotides. Also, cleavage and binding assays showed that the N-terminal domain of Rnt1p aids selection of A1-capped hairpins. Together, the results suggest that Rnt1p recognizes at least two distinct classes of tetraloops using flexible protein RNA interactions. This underscores the capacity of double-stranded RNA binding proteins to use several recognition motifs for substrate identification.     

Prediction of interacting single-stranded RNA bases by protein-binding patterns

Prediction of protein-RNA interactions at the atomic level of detail is crucial for our ability to understand and interfere with processes such as gene expression and regulation. Here, we investigate protein binding pockets that accommodate extruded nucleotides not involved in RNA base pairing. We observed that most of the protein-interacting nucleotides are part of a consecutive fragment of at least two nucleotides whose rings have significant interactions with the protein. Many of these share the same protein binding cavity and more than 30% of such pairs are π-stacked. Since these local geometries cannot be inferred from the nucleotide identities, we present a novel framework for their prediction from the properties of protein binding sites.First, we present a classification of known RNA nucleotide and dinucleotide protein binding sites and identify the common types of shared 3-D physicochemical binding patterns. These are recognized by a new classification methodology that is based on spatial multiple alignment. The shared patterns reveal novel similarities between dinucleotide binding sites of proteins with different overall sequences, folds and functions. Given a protein structure, we use these patterns for the prediction of its RNA dinucleotide binding sites. Based on the binding modes of these nucleotides, we further predict an RNA fragment that interacts with those protein binding sites. With these knowledge-based predictions, we construct an RNA fragment that can have a previously unknown sequence and structure. In addition, we provide a drug design application in which the database of all known small-molecule binding sites is searched for regions similar to nucleotide and dinucleotide binding patterns, suggesting new fragments and scaffolds that can target them.     

Interactions of 40LoVe within the ribonucleoprotein complex that forms on the localization element of Xenopus Vg1 mRNA

Proline rich RNA-binding protein (Prrp), which associates with mRNAs that employ the late pathway for localization in Xenopus oocytes, was used as bait in a yeast two-hybrid screen of an expression library. Several independent clones were recovered that correspond to a paralog of 40LoVe, a factor required for proper localization of Vg1 mRNA to the vegetal cortex. 40LoVe is present in at least three alternatively spliced isoforms; however, only one, corresponding to the variant identified in the two-hybrid screen, can be crosslinked to Vg1 mRNA. In vitro binding assays revealed that 40LoVe has high affinity for RNA, but exhibits little binding specificity on its own. Nonetheless, it was only found associated with localized mRNAs in oocytes. 40LoVe also interacts directly with VgRBP71 and VgRBP60/hnRNP I; it is the latter factor that likely determines the binding specificity of 40LoVe. Initially, 40LoVe binds to Vg1 mRNA in the nucleus and remains with the RNA in the cytoplasm. Immunohistochemical staining of oocytes shows that the protein is distributed between the nucleus and cytoplasm, consistent with nucleocytoplasmic shuttling activity. 40LoVe is excluded from the mitochondrial cloud, which is used by RNAs that localize through the early (METRO) pathway in stage I oocytes; nonetheless, it is associated with at least some early pathway RNAs during later stages of oogenesis. A phylogenetic analysis of 2×RBD hnRNP proteins combined with other experimental evidence suggests that 40LoVe is a distant homolog of Drosophila Squid.     

The three-dimensional arcitecture of the EJC core

The exon junction complex (EJC) is a macromolecular complex deposited at splice junctions on mRNAs as a consequence of splicing. At the core of the EJC are four proteins: eIF4AIII, a member of the DExH/D-box family of NTP-dependent RNA binding proteins, Y14, Magoh, and MLN51. These proteins form a stable heterotetramer that remains bound to the mRNA throughout many different cellular environments. We have determined the three-dimensional (3D) structure of this EJC core using negative-stain random-conical tilt electron microscopy. This structure represents the first structure of a DExH/D-box protein in complex with its binding partners. The EJC core is a four-lobed complex with a central channel and dimensions consistent with its known RNA footprint of about ten nucleotides. Using known X-ray crystallographic structures and a model of three of the four components, we propose a model for complex assembly on RNA and explain how Y14:Magoh may influence eIF4AIII's RNA binding.     

Effects of pH and salt concentration on the siRNA binding activity of the RNA silencing suppressor protein p19

The RNA silencing pathway is an important component of the anti-viral immune response in eukaryotes, particularly in plants. In turn, many viruses have evolved mechanisms to evade or suppress this pathway. Tombusviruses such as the Carnation Italian ringspot virus (CIRV) express a 19kDa protein (p19) that is a suppressor of RNA silencing in infected plants. This protein acts as a dimer and binds specifically to short-interfering RNA (siRNA) through electrostatic interactions between charged residues in the binding cleft. Since pH and salt concentrations can vary widely from host to host, we have investigated the influence of these parameters on the siRNA binding activity of CIRV p19. Previously, we established a convenient fluorescence-based method for assaying CIRV p19:siRNA binding using Ni(2+)-NTA coated 96-well plates. Using this method, we observe that the CIRV p19 protein binds to siRNA with nanomolar affinity and that this binding is sensitive to pH and salt concentration. The pH-dissociation constant profile shows that CIRV p19:siRNA binding is dependent on three different apparent pK(a) values. The values extrapolated from the curve are 7.1, 8.0 and 10.6 that we interpret as the ionization of one or more histidine, cysteine and lysine residues, respectively. We find that the optimal suppression of RNA silencing by CIRV p19 occurs in the pH range from 6.2 to 7.6.     

Steric Block High Affinity Oligonucleotide Analogues: A New Tool for Mapping RNA-Protein Binding Sites

Steric-block ON analogues are efficient inhibitors of RNA-protein interaction and therefore have potential to probe RNA sequences for putative protein binding sites and to investigate mechanisms of protein binding. The packaging process of HIV-1 is highly specific involving an interaction between the Gag protein and a conserved sequence that is only present on genomic viral RNA. Using oligonucleotide probes we have confirmed that the terminal purine loop is the major Gag binding site on SL3 and that a secondary Gag binding site exists at an internal purine bulge. We also demonstrate direct binding of oligonucleotide to their binding sites and confirm this interaction does not alter global RNA conformation, making them highly specific, nondisruptive probes of RNA protein interactions.