Visualization and characterization of RNA–protein interactions in living cells

RNA–protein interactions are the structural and functional basis of significant numbers of RNA molecules. RNA–protein interaction assays though, still mainly depend on biochemical tests in vitro. Here, we establish a convenient and reliable RNA fluorescent three-hybrid (rF3H) method to detect/interrogate the interactions between RNAs and proteins in cells. A GFP tagged highly specific RNA trap is constructed to anchor the RNA of interest to an artificial or natural subcellular structure, and RNA–protein interactions can be detected and visualized by the enrichment of RNA binding proteins (RBPs) at these structures. Different RNA trapping systems are developed and detection of RNA–protein complexes at multiple subcellular structures are assayed. With this new toolset, interactions between proteins and mRNA or noncoding RNAs are characterized, including the interaction between a long noncoding RNA and an epigenetic modulator. Our approach provides a flexible and reliable method for the characterization of RNA–protein interactions in living cells.     

Noncanonical G(syn)-G(anti) base pairs stabilized by sulphate anions in two X-ray structures of the (GUGGUCUGAUGAGGCC) RNA duplex

The structures of two crystal forms of the RNA 16-mer with the sequence GUGGUCUGAUGAGGCC, grown in the presence of a high concentration of sulphate ions, have been determined using synchrotron radiation at 1.4- and 2.0-Å resolution. RNA with this sequence is known as one of the two strands of the noncleavable form of the hammerhead ribozyme. In both crystal structures, two G(syn)–G(anti) noncanonical base pairs are observed in the middle of a 14 base-pair (bp) duplex having 5′-dangling GU residues. Both structures contain sulphate anions interacting with the G–G bp stabilizing G in its syn conformation and bridging the two RNA strands. In both cases the interactions take place in the major groove, although the anions are accommodated within different helix geometries, most pronounced in the changing width of the major groove. In one structure, where a single sulphate spans both G–G pairs, the major groove is closed around the anion, while in the other structure, where each of the two G–G pairs is associated with a separate sulphate, the groove is open. This work provides the first examples of a G–G pair in syn-anti conformation, which minimizes the purine–purine clash in the center of the duplex, while utilizing its residual hydrogen bonding potential in specific interactions with sulphate anions.     

Polypyrimidine Tract Binding Protein Functions as a Negative Regulator of Feline Calicivirus Translation

Background

Positive strand RNA viruses rely heavily on host cell RNA binding proteins for various aspects of their life cycle. Such proteins interact with sequences usually present at the 5′ or 3′ extremities of the viral RNA genome, to regulate viral translation and/or replication. We have previously reported that the well characterized host RNA binding protein polypyrimidine tract binding protein (PTB) interacts with the 5′end of the feline calicivirus (FCV) genomic and subgenomic RNAs, playing a role in the FCV life cycle.

Principal Findings

We have demonstrated that PTB interacts with at least two binding sites within the 5′end of the FCV genome. In vitro translation indicated that PTB may function as a negative regulator of FCV translation and this was subsequently confirmed as the translation of the viral subgenomic RNA in PTB siRNA treated cells was stimulated under conditions in which RNA replication could not occur. We also observed that PTB redistributes from the nucleus to the cytoplasm during FCV infection, partially localizing to viral replication complexes, suggesting that PTB binding may be involved in the switch from translation to replication. Reverse genetics studies demonstrated that synonymous mutations in the PTB binding sites result in a cell-type specific defect in FCV replication.

Conclusions

Our data indicates that PTB may function to negatively regulate FCV translation initiation. To reconcile this with efficient virus replication in cells, we propose a putative model for the function of PTB in the FCV life cycle. It is possible that during the early stages of infection, viral RNA is translated in the absence of PTB, however, as the levels of viral proteins increase, the nuclear-cytoplasmic shuttling of PTB is altered, increasing the cytoplasmic levels of PTB, inhibiting viral translation. Whether PTB acts directly to repress translation initiation or via the recruitment of other factors remains to be determined but this may contribute to the stimulation of viral RNA replication via clearance of ribosomes from viral RNA.     

Kinetic and functional analysis of the small RNA methyltransferase HEN1: The catalytic domain is essential for preferential modification of duplex RNA

The HEN1 RNA methyltransferase from Arabidopsis thaliana catalyzes S-adenosyl-L-methionine (AdoMet)-dependent 2′-O-methylation at the 3′-termini of small double-stranded RNAs and is a crucial factor in the biogenesis of plant small noncoding RNAs, such as miRNAs or siRNAs. We performed functional and kinetic studies of the full-length HEN1 methyltransferase and its truncated form comprising the C-terminal part of the protein (residues 666–942) with a variety of model RNA substrates. Kinetic parameters obtained with natural RNA substrates indicate that HEN1 is highly catalytically efficient in the absence of any supplementary proteins. We find that the enzyme modifies individual strands in succession leading to complete methylation of an RNA duplex. The rates of methyl group transfer to individual strands of hemimethylated substrates under single turnover conditions are comparable with the multiple turnover rate under steady-state conditions, suggesting that release of reaction products is not a rate-limiting event in the reaction cycle. The truncated protein, which includes conserved motifs characteristic for AdoMet binding, efficiently modifies double-stranded RNA substrates in vitro; however, in contrast to the full-length methyltransferase, it shows weaker interactions with both substrates and is sensitive to base mispairing in the first and second positions of the RNA duplex. Our findings suggest an important role for the N-terminal domains in stabilizing the catalytic complex and indicate that major structural determinants required for selective recognition and methylation of RNA duplexes reside in the C-terminal domain.     

Role of Polypyrimidine Tract Binding Protein in Mediating Internal Initiation of Translation of Interferon Regulatory Factor 2 RNA

Background

Earlier we have reported translational control of interferon regulatory factor 2 (IRF2) by internal initiation (Dhar et al, Nucleic Acids Res, 2007). The results implied possible role of IRF2 in controlling the intricate balance of cellular gene expression under stress conditions in general. Here we have investigated the secondary structure of the Internal Ribosome Entry Site of IRF2 RNA and demonstrated the role of PTB protein in ribosome assembly to facilitate internal initiation.

Methodology/Principal Findings

We have probed the putative secondary structure of the IRF2 5′UTR RNA using various enzymatic and chemical modification agents to constrain the secondary structure predicted from RNA folding algorithm Mfold. The IRES activity was found to be influenced by the interaction of trans-acting factor, polypyrimidine tract binding protein (PTB). Deletion of 25 nts from the 3′terminus of the 5′untranslated region resulted in reduced binding with PTB protein and also showed significant decrease in IRES activity compared to the wild type. We have also demonstrated putative contact points of PTB on the IRF2–5′UTR using primer extension inhibition assay. Majority of the PTB toe-prints were found to be restricted to the 3′end of the IRES. Additionally, Circular Dichroism (CD) spectra analysis suggested change in the conformation of the RNA upon PTB binding. Further, binding studies using S10 extract from HeLa cells, partially silenced for PTB gene expression, resulted in reduced binding by other trans-acting factors. Finally, we have demonstrated that addition of recombinant PTB enhances ribosome assembly on IRF2 IRES suggesting possible role of PTB in mediating internal initiation of translation of IRF2 RNA.

Conclusion/Significance

It appears that PTB binding to multiple sites within IRF2 5′UTR leads to a conformational change in the RNA that facilitate binding of other trans-acting factors to mediate internal initiation of translation.     

A Mouse Cytoplasmic Exoribonuclease (mXRN1p) with Preference for G4 Tetraplex Substrates

Exoribonucleases are important enzymes for the turnover of cellular RNA species. We have isolated the first mammalian cDNA from mouse demonstrated to encode a 5′–3′ exoribonuclease. The structural conservation of the predicted protein and complementation data in Saccharomyces cerevisiae suggest a role in cytoplasmic mRNA turnover and pre-rRNA processing similar to that of the major cytoplasmic exoribonuclease Xrn1p in yeast. Therefore, a key component of the mRNA decay system in S. cerevisiae has been conserved in evolution from yeasts to mammals. The purified mouse protein (mXRN1p) exhibited a novel substrate preference for G4 RNA tetraplex–containing substrates demonstrated in binding and hydrolysis experiments. mXRN1p is the first RNA turnover function that has been localized in the cytoplasm of mammalian cells. mXRN1p was distributed in small granules and was highly enriched in discrete, prominent foci. The specificity of mXRN1p suggests that RNAs containing G4 tetraplex structures may occur in vivo and may have a role in RNA turnover.     

The 3′-Untranslated Region of Hepatitis C Virus RNA Enhances Translation from an Internal Ribosomal Entry Site

Translation of most eukaryotic mRNAs and many viral RNAs is enhanced by their poly(A) tails. Hepatitis C virus (HCV) contains a positive-stranded RNA genome which does not have a poly(A) tail but has a stretch of 98 nucleotides (X region) at the 3′-untranslated region (UTR), which assumes a highly conserved stem-loop structure. This X region binds a polypyrimidine tract-binding protein (PTB), which also binds to the internal ribosome entry site (IRES) in HCV 5′-UTR. These RNA-protein interactions may regulate its translation. We generated a set of HCV RNAs differing only in their 3′-UTRs and compared their translation efficiencies. HCV RNA containing the X region was translated three- to fivefold more than the corresponding RNAs without this region. Mutations that abolished PTB binding in the X region reduced, but did not completely abolish, enhancement in translation. The X region also enhanced translation from another unrelated IRES (from encephalomyocarditis virus RNA), but did not affect the 5′-end-dependent translation of globin mRNA in either monocistronic or bicistronic RNAs. It did not appear to affect RNA stability. The free X region added in trans, however, did not enhance translation, indicating that the translational enhancement by the X region occurs only in cis. These results demonstrate that the highly conserved 3′ end of HCV RNA provides a novel mechanism for enhancement of HCV translation and may offer a target for antiviral agents.     

In vivo imaging of protein–protein and RNA–protein interactions using novel far-red fluorescence complementation systems

Imaging of protein–protein and RNA–protein interactions in vivo, especially in live animals, is still challenging. Here we developed far-red mNeptune-based bimolecular fluorescence complementation (BiFC) and trimolecular fluorescence complementation (TriFC) systems with excitation and emission above 600 nm in the ‘tissue optical window’ for imaging of protein–protein and RNA–protein interactions in live cells and mice. The far-red mNeptune BiFC was first built by selecting appropriate split mNeptune fragments, and then the mNeptune-TriFC system was built based on the mNeptune-BiFC system. The newly constructed mNeptune BiFC and TriFC systems were verified as useful tools for imaging protein–protein and mRNA–protein interactions, respectively, in live cells and mice. We then used the new mNeptune-TriFC system to investigate the interactions between human polypyrimidine-tract-binding protein (PTB) and HIV-1 mRNA elements as PTB may participate in HIV mRNA processing in HIV activation from latency. An interaction between PTB and the 3′long terminal repeat region of HIV-1 mRNAs was found and imaged in live cells and mice, implying a role for PTB in regulating HIV-1 mRNA processing. The study provides new tools for in vivo imaging of RNA–protein and protein–protein interactions, and adds new insight into the mechanism of HIV-1 mRNA processing.     

RegA proteins from phage T4 and RB69 have conserved helix-loop groove RNA binding motifs but different RNA binding specificities

The RegA proteins from the bacteriophage T4 and RB69 are translational repressors that control the expression of multiple phage mRNAs. RegA proteins from the two phages share 78% sequence identity; however, in vivo expression studies have suggested that the RB69 RegA protein binds target RNAs with a higher affinity than T4 RegA protein. To study the RNA binding properties of T4 and RB69 RegA proteins more directly, the binding sites of RB69 RegA protein on synthetic RNAs corresponding to the translation initiation region of two RB69 target genes were mapped by RNase protection assays. These assays revealed that RB69 RegA protein protects nucleotides –9 to –3 (relative to the start codon) on RB69 gene 44, which contains the sequence GAAAAUU. On RB69 gene 45, the protected site (nucleotides –8 to –3) contains a similar purine-rich sequence: GAAAUA. Interestingly, T4 RegA protein protected the same nucleotides on these RNAs. To examine the specificity of RNA binding, quantitative RNA gel shift assays were performed with synthetic RNAs corresponding to recognition elements (REs) in three T4 and three RB69 mRNAs. Comparative gel shift assays demonstrated that RB69 RegA protein has an ~7-fold higher affinity for T4 gene 44 RE RNA than T4 RegA protein. RB69 RegA protein also binds RB69 gene 44 RE RNA with a 4-fold higher affinity than T4 RegA protein. On the other hand, T4 RegA exhibited a higher affinity than RB69 RegA protein for RB69 gene 45 RE RNA. With respect to their affinities for cognate RNAs, both RegA proteins exhibited the following hierarchy of affinities: gene 44 > gene 45 > regA. Interestingly, T4 RegA exhibited the highest affinity towards RB69 gene 45 RE RNA, whereas RB69 RegA protein had the highest affinity for T4 gene 44 RE RNA. The helix–loop groove RNA binding motif of T4 RegA protein is fully conserved in RB69 RegA protein. However, homology modeling of the structure of RB69 RegA protein reveals that the divergent residues are clustered in two areas of the surface, and that there are two large areas of high conservation near the helix–loop groove, which may also play a role in RNA binding.     

Flexibility between the Protease and Helicase Domains of the Dengue Virus NS3 Protein Conferred by the Linker Region and Its Functional Implications

The dengue virus (DENV) NS3 protein is essential for viral polyprotein processing and RNA replication. It contains an N-terminal serine protease region (residues 1–168) joined to an RNA helicase (residues 180–618) by an 11-amino acid linker (169–179). The structure at 3.15 Å of the soluble NS3 protein from DENV4 covalently attached to 18 residues of the NS2B cofactor region (NS2B₁₈NS3) revealed an elongated molecule with the protease domain abutting subdomains I and II of the helicase (Luo, D., Xu, T., Hunke, C., Grüber, G., Vasudevan, S. G., and Lescar, J. (2008) J. Virol. 82, 173–183). Unexpectedly, using similar crystal growth conditions, we observed an alternative conformation where the protease domain has rotated by ∼161° with respect to the helicase domain. We report this new crystal structure bound to ADP-Mn²⁺ refined to a resolution of 2.2 Å. The biological significance for interdomain flexibility conferred by the linker region was probed by either inserting a Gly residue between Glu¹⁷³ and Pro¹⁷⁴ or replacing Pro¹⁷⁴ with a Gly residue. Both mutations resulted in significantly lower ATPase and helicase activities. We next increased flexibility in the linker by introducing a Pro¹⁷⁶ to Gly mutation in a DENV2 replicon system. A 70% reduction in luciferase reporter signal and a similar reduction in the level of viral RNA synthesis were observed. Our results indicate that the linker region has evolved to an optimum length to confer flexibility to the NS3 protein that is required both for polyprotein processing and RNA replication.     

Direct observation of the temperature-induced melting process of the Salmonella fourU RNA thermometer at base-pair resolution

In prokaryotes, RNA thermometers regulate a number of heat shock and virulence genes. These temperature sensitive RNA elements are usually located in the 5′-untranslated regions of the regulated genes. They repress translation initiation by base pairing to the Shine–Dalgarno sequence at low temperatures. We investigated the thermodynamic stability of the temperature labile hairpin 2 of the Salmonella fourU RNA thermometer over a broad temperature range and determined free energy, enthalpy and entropy values for the base-pair opening of individual nucleobases by measuring the temperature dependence of the imino proton exchange rates via NMR spectroscopy. Exchange rates were analyzed for the wild-type (wt) RNA and the A8C mutant. The wt RNA was found to be stabilized by the extraordinarily stable G14–C25 base pair. The mismatch base pair in the wt RNA thermometer (A8–G31) is responsible for the smaller cooperativity of the unfolding transition in the wt RNA. Enthalpy and entropy values for the base-pair opening events exhibit linear correlation for both RNAs. The slopes of these correlations coincide with the melting points of the RNAs determined by CD spectroscopy. RNA unfolding occurs at a temperature where all nucleobases have equal thermodynamic stabilities. Our results are in agreement with a consecutive zipper-type unfolding mechanism in which the stacking interaction is responsible for the observed cooperativity. Furthermore, remote effects of the A8C mutation affecting the stability of nucleobase G14 could be identified. According to our analysis we deduce that this effect is most probably transduced via the hydration shell of the RNA.     

Small RNAs derived from snoRNAs

Small nucleolar RNAs (snoRNAs) guide RNA modification and are localized in nucleoli and Cajal bodies in eukaryotic cells. Components of the RNA silencing pathway associate with these structures, and two recent reports have revealed that a human and a protozoan snoRNA can be processed into miRNA-like RNAs. Here we show that small RNAs with evolutionary conservation of size and position are derived from the vast majority of snoRNA loci in animals (human, mouse, chicken, fruit fly), Arabidopsis, and fission yeast. In animals, sno-derived RNAs (sdRNAs) from H/ACA snoRNAs are predominantly 20–24 nucleotides (nt) in length and originate from the 3′ end. Those derived from C/D snoRNAs show a bimodal size distribution at ∼17–19 nt and >27 nt and predominantly originate from the 5′ end. SdRNAs are associated with AGO7 in Arabidopsis and Ago1 in fission yeast with characteristic 5′ nucleotide biases and show altered expression patterns in fly loquacious and Dicer-2 and mouse Dicer1 and Dgcr8 mutants. These findings indicate that there is interplay between the RNA silencing and snoRNA-mediated RNA processing systems, and that sdRNAs comprise a novel and ancient class of small RNAs in eukaryotes.     

Identification of antisense RNA stem–loops that inhibit RNA–protein interactions using a bacterial reporter system

Many well-characterized examples of antisense RNAs from prokaryotic systems involve hybridization of the looped regions of stem–loop RNAs, presumably due to the high thermodynamic stability of the resulting loop–loop and loop–linear interactions. In this study, the identification of RNA stem–loops that inhibit U1A protein binding to the hpII RNA through RNA–RNA interactions was attempted using a bacterial reporter system based on phage λ N-mediated antitermination. As a result, loop sequences possessing 7–8 base complementarity to the 5′ region of the boxA element important for functional antitermination complex formation, but not the U1 hpII loop, were identified. In vitro and in vivo mutational analysis strongly suggested that the selected loop sequences were binding to the boxA region, and that the structure of the antisense stem–loop was important for optimal inhibitory activity. Next, in an attempt to demonstrate the ability to inhibit the interaction between the U1A protein and the hpII RNA, the rational design of an RNA stem–loop that inhibits U1A-binding to a modified hpII was carried out. Moderate inhibitory activity was observed, showing that it is possible to design and select antisense RNA stem–loops that disrupt various types of RNA–protein interactions.