A peptide derived from RNA recognition motif 2 of human la protein binds to hepatitis C virus internal ribosome entry site, prevents ribosomal assembly, and inhibits internal initiation of translation

Human La protein is known to interact with hepatitis C virus (HCV) internal ribosome entry site (IRES) and stimulate translation. Previously, we demonstrated that mutations within HCV SL IV lead to reduced binding to La-RNA recognition motif 2 (RRM2) and drastically affect HCV IRES-mediated translation. Also, the binding of La protein to SL IV of HCV IRES was shown to impart conformational alterations within the RNA so as to facilitate the formation of functional initiation complex. Here, we report that a synthetic peptide, LaR2C, derived from the C terminus of La-RRM2 competes with the binding of cellular La protein to the HCV IRES and acts as a dominant negative inhibitor of internal initiation of translation of HCV RNA. The peptide binds to the HCV IRES and inhibits the functional initiation complex formation. An Huh7 cell line constitutively expressing a bicistronic RNA in which both cap-dependent and HCV IRES-mediated translation can be easily assayed has been developed. The addition of purified TAT-LaR2C recombinant polypeptide that allows direct delivery of the peptide into the cells showed reduced expression of HCV IRES activity in this cell line. The study reveals valuable insights into the role of La protein in ribosome assembly at the HCV IRES and also provides the basis for targeting ribosome-HCV IRES interaction to design potent antiviral therapy.     

Hepatitis C virus internal ribosome entry site-mediated translation is stimulated by specific interaction of independent regions of human La autoantigen

The human La autoantigen has been shown to interact with the internal ribosome entry site (IRES) of hepatitis C virus (HCV) in vitro. Using a yeast three-hybrid system, we demonstrated that, in addition to full-length La protein, both N- and C-terminal halves were able to interact with HCV IRES in vivo. The exogenous addition of purified full-length and truncated La proteins in rabbit reticulocyte lysate showed dose-dependent stimulation of HCV IRES-mediated translation. However, an additive effect was achieved adding the terminal halves together in the reaction, suggesting that both might play critical roles in achieving full stimulatory activity of the full-length La protein. Using computational analysis, three-dimensional structures of the RNA recognition motifs (RRM) of the La protein were independently modeled. Of the three putative RRMs, RRM2 was predicted to have a good binding pocket for the interaction with the HCV IRES around the GCAC motif near the initiator AUG and RRM3 binds perhaps in a different location. This observation was further investigated by the filter-binding and toe-printing assays. The results presented here strongly suggest that both the N- and C-terminal halves can interact independently with the HCV IRES and are involved in stimulating internal initiation of translation.     

La protein binding at the GCAC site near the initiator AUG facilitates the ribosomal assembly on the hepatitis C virus RNA to influence internal ribosome entry site-mediated translation

Human La autoantigen has been shown to influence internal initiation of translation of hepatitis C virus (HCV) RNA. Previously, we have demonstrated that, among the three RRMs of La protein, the RRM2 interacts with HCV internal ribosome entry site (IRES) around the GCAC motif near the initiator AUG present in the stem region of stem-loop IV (SL IV) (Pudi, R., Abhiman, S., Srinivasan, N., and Das S. (2003) J. Biol. Chem. 278, 12231-12240). Here, we have demonstrated that the mutations in the GCAC motif, which altered the binding to RRM2, had drastic effect on HCV IRES-mediated translation, both in vitro and in vivo. The results indicated that the primary sequence of the stem region of SL IV plays an important role in mediating internal initiation. Furthermore, we have shown that the mutations also altered the ability to bind to ribosomal protein S5 (p25), through which 40 S ribosomal subunit is known to contact the HCV IRES RNA. Interestingly, binding of La protein to SL IV region induced significant changes in the circular dichroism spectra of the HCV RNA indicating conformational alterations that might assist correct positioning of the initiation complex. Finally, the ribosome assembly analysis using sucrose gradient centrifugation implied that the mutations within SL IV of HCV IRES impair the formation of functional ribosomal complexes. These observations strongly support the hypothesis that La protein binding near the initiator AUG facilitates the interactions with ribosomal protein S5 and 48 S ribosomal assembly and influences the formation of functional initiation complex on the HCV IRES RNA to mediate efficient internal initiation of translation.     

Structural determinant of human La protein critical for internal initiation of translation of hepatitis C virus RNA

Human La protein has been implicated in facilitating internal ribosome entry site (IRES)-mediated translation of hepatitis C virus (HCV). Earlier, we demonstrated that the RNA recognition motif (RRM) encompassing residues 112 to 184 of La protein [La (112-184)] interacts with the HCV IRES near the initiator AUG codon. A synthetic peptide, LaR2C (24-mer), derived from La RRM (112-184), retains RNA binding ability, competes with La protein binding to the HCV IRES, and inhibits translation. The peptide interferes with the assembly of 48S complexes, resulting in the accumulation of preinitiation complexes that are incompetent for the 60S ribosomal subunit joining. Here, nuclear magnetic resonance spectroscopy of the HCV IRES-bound peptide complex revealed putative contact points, mutations that showed reduced RNA binding and translation inhibitory activity. The residues responsible for RNA recognition were found to form a turn in the RRM (112-184) structure. A 7-mer peptide comprising this turn showed significant translation inhibitory activity. The bound structure of the peptide inferred from transferred nuclear Overhauser effect experiments suggests that it is a β turn. This conformation is significantly different from that observed in the free RRM (112-184) NMR structure, suggesting paths toward a better-stabilized mimetic peptide. Interestingly, addition of hexa-arginine tag enabled the peptide to enter Huh7 cells and showed inhibition of HCV IRES function. More importantly, the peptide significantly inhibited replication of the HCV monocistronic replicon. Elucidation of the structural determinant of the peptide provides a basis for developing small peptidomimetic structures as potent anti-HCV therapeutics.     

Evidence for an RNA chaperone function of polypyrimidine tract-binding protein in picornavirus translation

The cellular polypyrimidine tract-binding protein (PTB) is recruited by the genomic RNAs of picornaviruses to stimulate translation initiation at their internal ribosome entry site (IRES) elements. We investigated the contribution of the individual RNA recognition motif (RRM) domains of PTB to its interaction with the IRES of foot-and-mouth disease virus (FMDV). Using a native gel system, we found that PTB is a monomer, confirming recent reports that challenged the previous view that PTB is a dimer. Mapping the spatial orientation of PTB relative to the bound IRES RNA, we found that the two C-terminal RRM domains III and IV of PTB bind in an oriented way to the IRES. Domain III contacts the IRES stem-loop 2, while domain IV contacts the separate IRES 3' region. PTB domain I appears not to be involved directly in RNA binding, but domain II stabilizes the RNA binding conferred by domains III and IV. A PTB protein containing only these two C-terminal PTB domains is sufficient to enhance the entry of initiation factor eIF4G to the IRES and stimulate IRES activity, and the long-lived PTB-IRES interaction stabilized by domain II is not a prerequisite for this function. Thus, PTB most likely acts as an RNA chaperone to stabilize IRES structure and, in that way, augment IRES activity.     

Separate RNA-binding surfaces on the multifunctional La protein mediate distinguishable activities in tRNA maturation

By sequence-specific binding to 3' UUU-OH, the La protein shields precursor (pre)-RNAs from 3' end digestion and is required to protect defective pre-transfer RNAs from decay. Although La is comprised of a La motif and an RNA-recognition motif (RRM), a recent structure indicates that the RRM beta-sheet surface is not involved in UUU-OH recognition, raising questions as to its function. Progressively defective suppressor tRNAs in Schizosaccharomyces pombe reveal differential sensitivities to La and Rrp6p, a 3' exonuclease component of pre-tRNA decay. 3' end protection is compromised by mutations to the La motif but not the RRM surface. The most defective pre-tRNAs require a second activity of La, in addition to 3' protection, that requires an intact RRM surface. The two activities of La in tRNA maturation map to its two conserved RNA-binding surfaces and suggest a modular model that has implications for its other ligands.     

Conservation of RNA chaperone activity of the human La-related proteins 4, 6 and 7

The La module is a conserved tandem arrangement of a La motif and RNA recognition motif whose function has been best characterized in genuine La proteins. The best-characterized substrates of La proteins are pre-tRNAs, and previous work using tRNA mediated suppression in Schizosaccharomyces pombe has demonstrated that yeast and human La enhance the maturation of these using two distinguishable activities: UUU-3′OH-dependent trailer binding/protection and a UUU-3′OH independent activity related to RNA chaperone function. The La module has also been identified in several conserved families of La-related proteins (LARPs) that engage other RNAs, but their mode of RNA binding and function(s) are not well understood. We demonstrate that the La modules of the human LARPs 4, 6 and 7 are also active in tRNA-mediated suppression, even in the absence of stable UUU-3′OH trailer protection. Rather, the capacity of these to enhance pre-tRNA maturation is associated with RNA chaperone function, which we demonstrate to be a conserved activity for each hLARP in vitro. Our work reveals insight into the mechanisms by which La module containing proteins discriminate RNA targets and demonstrates that RNA chaperone activity is a conserved function across representative members of the La motif-containing superfamily.     

The La motif and the RNA recognition motifs of human La autoantigen contribute individually to RNA recognition and subcellular localization

The human La autoantigen (hLa) protein is a predominantly nuclear phosphoprotein that contains three potential RNA binding domains referred to as the La motif and the RNA recognition motifs RRMs 1 and 2. With this report, we differentiated the contribution of its three RNA binding domains to RNA binding by combining in vitro and in vivo assays. Also, surface plasmon resonance technology was used to generate a model for the sequential contribution of the RNA binding domains to RNA binding. The results indicated that the La motif may contribute to specificity rather than affinity, whereas RRM1 is indispensable for association with pre-tRNA and hY1 RNA. Furthermore, RRM2 was not crucial for the interaction with various RNAs in vivo, although needed for full-affinity binding in vitro. Moreover, earlier studies suggest that RNA binding by hLa may direct its subcellular localization. As shown previously for RRM1, deletion of RNP2 sequence in RRM1 alters nucleolar distribution of hLa, not observed after deletion of the La motif. Here we discuss a model for precursor RNA binding based on a sequential association process mediated by RRM1 and the La motif.     

Novel RNA recognition motif domain in the cytoplasmic polyadenylation element binding protein 3

The family of cytoplasmic polyadenylation element binding proteins CPEB1, CPEB2, CPEB3, and CPEB4 binds to the 3′‐untranslated region (3′‐UTR) of mRNA, and plays significant roles in mRNA metabolism and translation regulation. They have a common domain organization, involving two consecutive RNA recognition motif (RRM) domains followed by a zinc finger domain in the C‐terminal region. We solved the solution structure of the first RRM domain (RRM1) of human CPEB3, which revealed that CPEB3 RRM1 exhibits structural features distinct from those of the canonical RRM domain. Our structural data provide important information about the RNA binding ability of CPEB3 RRM1. Proteins 2014; 82:2879–2886. © 2014 Wiley Periodicals, Inc.     

Interactions of viral protein 3CD and poly(rC) binding protein with the 5' untranslated region of the poliovirus genome

The poly(rC) binding protein (PCBP) is a cellular protein required for poliovirus replication. PCBP specifically interacts with two domains of the poliovirus 5' untranslated region (5'UTR), the 5' cloverleaf structure, and the stem-loop IV of the internal ribosome entry site (IRES). Using footprinting analysis and site-directed mutagenesis, we have mapped the RNA binding site for this cellular protein within the stem-loop IV domain. A C-rich sequence in a loop at the top of this large domain is required for PCBP binding and is crucial for viral translation. PCBP binds to stem-loop IV RNA with six-times-higher affinity than to the 5' cloverleaf structure. However, the binding of the viral protein 3CD (precursor of the viral protease 3C and the viral polymerase 3D) to the cloverleaf RNA dramatically increases the affinity of PCBP for this RNA element. The viral protein 3CD binds to the cloverleaf RNA but does not interact directly with stem-loop IV nor with other RNA elements of the viral IRES. Our results indicate that the interactions of PCBP with the poliovirus 5'UTR are modulated by the viral protein 3CD.     

Solution structure of the second RNA recognition motif (RRM) domain of murine T cell intracellular antigen-1 (TIA-1) and its RNA recognition mode

T cell intracellular antigen-1 (TIA-1), an apoptosis promoting factor, functions as a splicing regulator for the Fas pre-mRNA. TIA-1 possesses three RNA recognition motifs (RRMs) and a glutamine-rich domain. The second RRM (RRM2) is necessary and sufficient for tight, sequence-specific binding to the uridine-rich sequences buried around the 5' splice sites. In the present study, we solved the solution structure of the murine TIA-1 RRM2 by heteronuclear-nuclear magnetic resonance spectroscopy. The TIA-1 RRM2 adopts the RRM fold (betaalphabetabetaalphabeta) and possesses an extra beta-strand between beta2 and beta3, which forms an additional beta-sheet with the C-terminal part of beta2. We refer to this structure as the beta2-beta2' beta-loop. Interestingly, this characteristic beta-loop structure is conserved among a number of RRMs, including the U2AF65 RRM2 and the Sex-lethal RRM1 and RRM2, which also bind to uridine-rich RNAs. Furthermore, we identified a new sequence motif in the beta2-beta2' beta-loop, the DxxT motif. Chemical shift perturbation analyses of both the main and side chains upon binding to the uridine pentamer RNA revealed that most of the beta-sheet surface, including the beta2-beta2' beta-loop, is involved in the RNA binding. An investigation of the chemical shift perturbation revealed similarity in the RNA recognition modes between the TIA-1 and U2AF65 RRMs.     

RNA Binding of T-cell Intracellular Antigen-1 (TIA-1) C-terminal RNA Recognition Motif Is Modified by pH Conditions

T-cell intracellular antigen-1 (TIA-1) is a DNA/RNA-binding protein that regulates critical events in cell physiology by the regulation of pre-mRNA splicing and mRNA translation. TIA-1 is composed of three RNA recognition motifs (RRMs) and a glutamine-rich domain and binds to uridine-rich RNA sequences through its C-terminal RRM2 and RRM3 domains. Here, we show that RNA binding mediated by either isolated RRM3 or the RRM23 construct is controlled by slight environmental pH changes due to the protonation/deprotonation of TIA-1 RRM3 histidine residues. The auxiliary role of the C-terminal RRM3 domain in TIA-1 RNA recognition is poorly understood, and this work provides insight into its binding mechanisms.     

The domain of the Bacillus subtilis DEAD-box helicase YxiN that is responsible for specific binding of 23S rRNA has an RNA recognition motif fold

The YxiN protein of Bacillus subtilis is a member of the DbpA subfamily of prokaryotic DEAD-box RNA helicases. Like DbpA, it binds with high affinity and specificity to segments of 23S ribosomal RNA as short as 32 nucleotides (nt) that include hairpin 92. Several experiments have shown that the 76-residue carboxy-terminal domain of YxiN is responsible for the high-affinity RNA binding. The domain has been crystallized and its structure has been solved to 1.7 Angstroms resolution. The structure reveals an RNA recognition motif (RRM) fold that is found in many eukaryotic RNA binding proteins; the RRM fold was not apparent from the amino acid sequence. The domain has two solvent exposed aromatic residues at sites that correspond to the aromatic residues of the ribonucleoprotein (RNP) motifs RNP1 and RNP2 that are essential for RNA binding in many RRMs. However, mutagenesis of these residues (Tyr404 and Tyr447) to alanine has little effect on RNA affinity, suggesting that the YxiN domain binds target RNAs in a manner that differs from the binding mode commonly found in many eukaryotic RRMs.     

Solution structure and RNA interactions of the RNA recognition motif from eukaryotic translation initiation factor 4B

Eukaryotic initiation factor 4B (eIF4B) is a multidomain protein with a range of activities that serves primarily to promote association of messenger RNA to the 40S ribosomal subunit during translation initiation. We report here the solution structure of the eIF4B RNA recognition motif (RRM) domain. It adopts a classical RRM fold, with a beta alpha beta beta alpha beta topology. The most striking difference with other RRM structures is in the disposition of loop 3, which connects the beta 2 and beta 3 strands and is implicated in RNA recognition. This loop folds down against the body of the RRM and exhibits restricted motion on a milli- to microsecond time scale. Although it contributes to a large basic patch on the RNA binding surface, it does not protrude out from the domain as observed in other RRM structures, possibly implying a different mode of RNA binding. On its own, the core RRM domain provides only a relative weak interaction with RNA targets and appears to require extensions at the N- and C-terminus for high-affinity binding.