eIF4G, eIFiso4G, and eIF4B bind the poly(A)-binding protein through overlapping sites within the RNA recognition motif domains

The poly(A)-binding protein (PABP), a protein that contains four conserved RNA recognition motifs (RRM1-4) and a C-terminal domain, is expressed throughout the eukaryotic kingdom and promotes translation through physical and functional interactions with eukaryotic initiation factor (eIF) 4G and eIF4B. Two highly divergent isoforms of eIF4G, known as eIF4G and eIFiso4G, are expressed in plants. As little is known about how PABP can interact with RNA and three distinct translation initiation factors in plants, the RNA binding specificity and organization of the protein interaction domains in wheat PABP was investigated. Wheat PABP differs from animal PABP in that its RRM1 does not bind RNA as an individual domain and that RRM 2, 3, and 4 exhibit different RNA binding specificities to non-poly(A) sequences. The PABP interaction domains for eIF4G and eIFiso4G were distinct despite the functional similarity between the eIF4G proteins. A single interaction domain for eIF4G is present in the RRM1 of PABP, whereas eIFiso4G interacts at two sites, i.e. one within RRM1-2 and the second within RRM3-4. The eIFiso4G binding site in RRM1-2 mapped to a 36-amino acid region encompassing the C-terminal end of RRM1, the linker region, and the N-terminal end of RRM2, whereas the second site in RRM3-4 was more complex. A single interaction domain for eIF4B is present within a 32-amino acid region representing the C-terminal end of RRM1 of PABP that overlaps with the N-proximal eIFiso4G interaction domain. eIF4B and eIFiso4G exhibited competitive binding to PABP, supporting the overlapping nature of their interaction domains. These results support the notion that eIF4G, eIFiso4G, and eIF4B interact with distinct molecules of PABP to increase the stability of the interaction between the termini of an mRNA.     

Wheat eukaryotic initiation factor 4B organizes assembly of RNA and eIFiso4G, eIF4A, and poly(A)-binding protein

The eukaryotic translation initiation factor (eIF) 4B promotes the RNA-dependent ATP hydrolysis activity and ATP-dependent RNA helicase activity of eIF4A and eIF4F during translation initiation. Although this function is conserved among plants, animals, and yeast, eIF4B is one of the least conserved of initiation factors at the sequence level. To gain insight into its functional conservation, the organization of the functional domains of eIF4B from wheat has been investigated. Plant eIF4B contains three RNA binding domains, one more than reported for mammalian or yeast eIF4B, and each domain exhibits a preference for purine-rich RNA. In addition to a conserved RNA recognition motif and a C-terminal RNA binding domain, wheat eIF4B contains a novel N-terminal RNA binding domain that requires a short, lysine-rich containing sequence. Both the lysine-rich motif and an adjacent, C-proximal motif are conserved with an N-proximal sequence in human and yeast eIF4B. The C-proximal motif within the N-terminal RNA binding domain in wheat eIF4B is required for interaction with eIFiso4G, an interaction not reported for other eIF4B proteins. Moreover, each RNA binding domain requires dimerization for binding activity. Two binding sites for the poly(A)-binding protein were mapped to a region within each of two conserved 41-amino acid repeat domains on either side of the C-terminal RNA binding domain. eIF4A bound to an adjacent region within each repeat, supporting a central role for these conserved eIF4B domains in facilitating interaction with other components of the translational machinery. These results support the notion that eIF4B functions by organizing multiple components of the translation initiation machinery and RNA.     

RNA Recognition Motif 2 of Yeast Pab1p Is Required for Its Functional Interaction with Eukaryotic Translation Initiation Factor 4G

The eukaryotic mRNA 3′ poly(A) tail and its associated poly(A)-binding protein (Pab1p) are important regulators of gene expression. One role for this complex in the yeast Saccharomyces cerevisiae is in translation initiation through an interaction with a 115-amino-acid region of the translation initiation factor eIF4G. The eIF4G-interacting domain of Pab1p was mapped to its second RNA recognition motif (RRM2) in an in vitro binding assay. Moreover, RRM2 of Pab1p was required for poly(A) tail-dependent translation in yeast extracts. An analysis of a site-directed Pab1p mutation which bound to eIF4G but did not stimulate translation of uncapped, polyadenylated mRNA suggested additional Pab1p-dependent events during translation initiation. These results support the model that the association of RRM2 of yeast Pab1p with eIF4G is a prerequisite for the poly(A) tail to stimulate the translation of mRNA in vitro.     

Related eIF3 subunits TIF32 and HCR1 interact with an RNA recognition motif in PRT1 required for eIF3 integrity and ribosome binding

eIF3 binds to 40S ribosomal subunits and stimulates recruitment of Met-tRNAiMet and mRNA to the pre-initiation complex. Saccharomyces cerevisiae contains an ortholog of human eIF3 subunit p35, HCR1, whose interactions with yeast eIF3 are not well defined. We found that HCR1 has a dual function in translation initiation: it binds to, and stabilizes, the eIF3-eIF5- eIF1-eIF2 multifactor complex and is required for the normal level of 40S ribosomes. The RNA recognition motif (RRM) of eIF3 subunit PRT1 interacted simultaneously with HCR1 and with an internal domain of eIF3 subunit TIF32 that has sequence and functional similarity to HCR1. PRT1, HCR1 and TIF32 were also functionally linked by genetic suppressor analysis. We propose that HCR1 stabilizes or modulates interaction between TIF32 and the PRT1 RRM. Removal of the PRT1 RRM resulted in dissociation of TIF32, NIP1, HCR1 and eIF5 from eIF3 in vivo, and destroyed 40S ribosome binding by the residual PRT1-TIF34-TIF35 subcomplex. Hence, the PRT1 RRM is crucial for the integrity and ribosome-binding activity of eIF3.     

Crystal Structure of the RNA Recognition Motif of Yeast Translation Initiation Factor eIF3b Reveals Differences to Human eIF3b

Background

The multi-subunit eukaryotic initiation factor3 (eIF3) plays a central role in the initiation step of protein synthesis in eukaryotes. One of its large subunits, eIF3b, serves as a scaffold within eIF3 as it interacts with several other subunits. It harbors an RNA Recognition Motif (RRM), which is shown to be a non-canonical RRM in human as it is not capable to interact with oligonucleotides, but rather interacts with eIF3j, a sub-stoichiometric subunit of eIF3.

Principal Finding

We have analyzed the high-resolution crystal structure of the eIF3b RRM domain from yeast. It exhibits the same fold as its human ortholog, with similar charge distribution on the surface interacting with the eIF3j in human. Thermodynamic analysis of the interaction between yeast eIF3b-RRM and eIF3j revealed the same range of enthalpy change and dissociation constant as for the human proteins, providing another line of evidence for the same mode of interaction between eIF3b and eIF3j in both organisms. However, analysis of the surface charge distribution of the putative RNA-binding β-sheet suggested that in contrast to its human ortholog, it potentially could bind oligonucleotides. Three-dimensional positioning of the so called “RNP1” motif in this domain is similar to other canonical RRMs, suggesting that this domain might indeed be a canonical RRM, conferring oligonucleotide binding capability to eIF3 in yeast. Interaction studies with yeast total RNA extract confirmed the proposed RNA binding activity of yeast eIF3b-RRM.

Conclusion

We showed that yeast eIF3b-RRM interacts with eIF3j in a manner similar to its human ortholog. However, it shows similarities in the oligonucleotide binding surface to canonical RRMs and interacts with yeast total RNA. The proposed RNA binding activity of eIF3b-RRM may help eIF3 to either bind to the ribosome or recruit the mRNA to the 43S pre-initiation complex.     

Structure of tandem RNA recognition motifs from polypyrimidine tract binding protein reveals novel features of the RRM fold

Polypyrimidine tract binding protein (PTB), an RNA binding protein containing four RNA recognition motifs (RRMs), is involved in both pre-mRNA splicing and translation initiation directed by picornaviral internal ribosome entry sites. Sequence comparisons previously indicated that PTB is a non-canonical RRM protein. The solution structure of a PTB fragment containing RRMs 3 and 4 shows that the protein consists of two domains connected by a long, flexible linker. The two domains tumble independently in solution, having no fixed relative orientation. In addition to the betaalphabetabetaalphabeta topology, which is characteristic of RRM domains, the C-terminal extension of PTB RRM-3 incorporates an unanticipated fifth beta-strand, which extends the RNA binding surface. The long, disordered polypeptide connecting beta4 and beta5 in RRM-3 is poised above the RNA binding surface and is likely to contribute to RNA recognition. Mutational analyses show that both RRM-3 and RRM-4 contribute to RNA binding specificity and that, despite its unusual sequence, PTB binds RNA in a manner akin to that of other RRM proteins.     

The RNA recognition motif of yeast translation initiation factor Tif3/eIF4B is required but not sufficient for RNA strand-exchange and translational activity.

The Saccharomyces cerevisiae TIF3 gene encodes a 436-amino acid (aa) protein that is the yeast homologue of mammalian translation Initiation factor eIF4B. Tif3p can be divided into three parts, the N-terminal region with an RNA recognition motif (RRM) (aa 1-182), followed in the middle part by a sevenfold repeat of 26 amino acids rich in basic and acidic residues (as 183-350), and a C-terminal region without homology to any known sequence (aa 351-436). We have analyzed several Tif3 proteins with deletions at their N and C termini for their ability (1) to complement a tif3delta strain in vivo, (2) to stimulate Tif3-dependent translation extracts, (3) to bind to single-stranded RNA, and (4) to catalyze RNA strand-exchange in vitro. Here we report that yeast Tif3/eIF4B contains at least two RNA binding domains able to bind to single-stranded RNA. One is located in the N-terminal region of the protein carrying the RRM, the other in the C-terminal two-thirds region of Tif3p. The RRM-containing domain and three of the seven repeat motifs are essential for RNA strand-exchange activity of Tif3p and translation in vitro and for complementation of a tif3delta strain, suggesting an important role for RNA strand-exchange activity in translation.     

Structural and functional analysis of RNA and TAP binding to SF2/ASF

The serine/arginine-rich (SR) protein splicing factor 2/alternative splicing factor (SF2/ASF) has a role in splicing, stability, export and translation of messenger RNA. Here, we present the structure of the RNA recognition motif (RRM) 2 from SF2/ASF, which has an RRM fold with a considerably extended loop 5 region, containing a two-stranded beta-sheet. The loop 5 extension places the previously identified SR protein kinase 1 docking sequence largely within the RRM fold. We show that RRM2 binds to RNA in a new way, by using a tryptophan within a conserved SWQLKD motif that resides on helix alpha1, together with amino acids from strand beta2 and a histidine on loop 5. The linker connecting RRM1 and RRM2 contains arginine residues, which provide a binding site for the mRNA export factor TAP, and when TAP binds to this region it displaces RNA bound to RRM2.     

Concerted activities of the RNA recognition and the glycine-rich C-terminal domains of nucleolin are required for efficient complex formation with pre-ribosomal RNA.

Nucleolin is an abundant nucleolar protein which is involved in the early stages of ribosome assembly. The central 40-kDa domain of nucleolin comprises four RNA recognition motifs (RRM) which are presumed to be involved in specific interactions with pre-rRNA. In order to examine in detail the role of this central domain and the contribution of the N-terminal and C-terminal domains of nucleolin to RNA binding, we have used an Escherichia coli expression system to synthezise polypeptides corresponding to various combinations of the three domains and their subdomains. By means of an in-vitro binding assay and a synthetic RNA corresponding to a specific recognition site in pre-rRNA we have been able to demonstrate conclusively that the central 40-kDa domain is indeed responsible for the specificity of RNA recognition and that the N-terminal domain can be removed without affecting RNA binding. Most interestingly, it appears that the C-terminal 10-kDa domain, which is rich in glycine and arginine residues, is essential for efficient binding of nucleolin to RNA, but does not itself contribute to the specificity of the interaction. Circular dichroic spectroscopic probing of the RNA component shows that the C-terminal domain significantly modifies the RNA-binding properties of the central RRM core. Finally, infrared spectroscopic studies reveal that the central 40-kDa domain is structured in alpha helices and beta sheets and that the interaction with the specific pre-rRNA site induces subtle changes in the beta sheet conformation.     

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.     

Interactions between PTB RRMs Induce Slow Motions and Increase RNA Binding Affinity

Polypyrimidine tract binding protein (PTB) participates in a variety of functions in eukaryotic cells, including alternative splicing, mRNA stabilization, and internal ribosomal entry site-mediated translation initiation. Its mechanism of RNA recognition is determined in part by the novel geometry of its two C-terminal RNA recognition motifs (RRM3 and RRM4), which interact with each other to form a stable complex (PTB1:34). This complex itself is unusual among RRMs, suggesting that it performs a specific function for the protein. In order to understand the advantage it provides to PTB, the fundamental properties of PTB1:34 are examined here as a comparative study of the complex and its two constituent RRMs. Both RRM3 and RRM4 adopt folded structures that NMR data show to be similar to their structure in PRB1:34. The RNA binding properties of the domains differ dramatically. The affinity of each separate RRM for polypyrimidine tracts is far weaker than that of PTB1:34, and simply mixing the two RRMs does not create an equivalent binding platform. ¹⁵N NMR relaxation experiments show that PTB1:34 has slow, microsecond motions throughout both RRMs including the interdomain linker. This is in contrast to the individual domains, RRM3 and RRM4, where only a few backbone amides are flexible on this time scale. The slow backbone dynamics of PTB1:34, induced by packing of RRM3 and RRM4, could be essential for high-affinity binding to a flexible polypyrimidine tract RNA and also provide entropic compensation for its own formation.     

In vitro RNA selection identifies RNA ligands that specifically bind to eukaryotic translation initiation factor 4B: the role of the RNA remotif.

Translation initiation factor elF-4B is an RNA-binding protein that promotes the association of the mRNA to the 40S ribosomal subunit. One of its better characterized features is the ability to stimulate the activity of the DEAD box RNA hilicase elF-4A. In addition to an RNA recognition motif (RRM) located near its amino-terimus, elF-4B contains an RNA-binding region in its carboxy-terminal half. The elF-4A helicase stimulatory activity resides in the carboxy-terminal half of elF-4B, and the RRM has little impact on this function.To better understand the role of the elF-4B RRM, it was of interest to identify its specific RNA target sequence. To this end, it vitro RNA selection/amplifications were performed using various portions of elF-4B. These experiments were designed to test the RNA recognition specificity of the two elF-4B regions implicated in RNA binding and to assess the influence of elF-4A on the RNA-binding specificity. The RRM was shown to bind with high affinity to an RNA stem-loop structure with conserved primary sequence elements. Discrete point mutations in an in vitro-selected RNA identified residues critical for RNA binding. Neither the carboxy-terminal RNA-interaction region, nor elF-4A, influenced the structure of the high-affinity RNA ligands selected by elF-4B, and elF-4A by itself did not select any specific RNA target. Previous studies have demonstrated an interaction of elF-4B with ribosomes, and it was suggested that this association is mediated through binding to ribosomal RNA. We show that the RRM of elF-4B interacts directly with 18S rRNA and this interaction is inhibited by an excess of the elF-4B in vitro-selected RNA. ElF-4B could bind simultaneously to two different RNA molecules, supporting a model whereby elF-4B promotes ribosome binding to the 5 untranslated region of a mRNA by bridging it to 18S rRNA.     

Physical association of eukaryotic initiation factor 4G (eIF4G) with eIF4A strongly enhances binding of eIF4G to the internal ribosomal entry site of encephalomyocarditis virus and is required for internal initiation of translation

Mammalian eukaryotic initiation factor 4GI (eIF4GI) may be divided into three similarly sized regions. The central region (amino acids [aa] 613 to 1090) binds eIF3, eIF4A, and the encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES) and mediates initiation on this RNA. We identified the regions of eIF4GI that are responsible for its specific interaction with the IRES and that are required to mediate 48S complex formation on the IRES in vitro. Mutational analysis demarcated the IRES binding fragment of eIF4GI (aa 746 to 949) and indicated that it does not resemble an RNA recognition motif (RRM)-like domain. An additional amino-terminal sequence (aa 722 to 746) was required for binding eIF4A and for 48S complex formation. eIF4GI bound the EMCV IRES and beta-globin mRNA with similar affinities, but association with eIF4A increased its affinity for the EMCV IRES (but not beta-globin RNA) by 2 orders of magnitude. On the other hand, eIF4GI mutants with defects in binding eIF4A were defective in mediating 48S complex formation even if they bound the IRES normally. These data indicate that the eIF4G-eIF4A complex, rather than eIF4G alone, is required for specific high-affinity binding to the EMCV IRES and for internal ribosomal entry on this RNA.     

The structure and selectivity of the SR protein SRSF2 RRM domain with RNA

SRSF2 is a prototypical SR protein which plays important roles in the alternative splicing of pre-mRNA. It has been shown to be involved in regulatory pathways for maintaining genomic stability and play important roles in regulating key receptors in the heart. We report here the solution structure of the RNA recognition motifs (RRM) domain of free human SRSF2 (residues 9-101). Compared with other members of the SR protein family, SRSF2 structure has a longer L3 loop region. The conserved aromatic residue in the RNP2 motif is absent in SRSF2. Calorimetric titration shows that the RNA sequence 5'AGCAGAGUA3' binds SRSF2 with a K(d) of 61 ± 1 nM and a 1:1 stoichiometry. NMR and mutagenesis experiments reveal that for SFSF2, the canonical β1 and β3 interactions are themselves not sufficient for effective RNA binding; the additional loop L3 is crucial for RNA complex formation. A comparison is made between the structures of SRSF2-RNA complex with other known RNA complexes of SR proteins. We conclude that interactions involving the L3 loop, N- and C-termini of the RRM domain are collectively important for determining selectivity between the protein and RNA.     

IRES interaction with translation initiation factors: functional characterization of novel RNA contacts with eIF3, eIF4B, and eIF4GII

Translation initiation promoted by picornavirus internal ribosome entry site (IRES) elements is dependent on the association of specific IRES sequences to the initiation factor eIF4G. However the RNA determinants interacting with other components of the translational machinery are still unknown. In this study, we have identified novel RNA-protein interactions between the foot-and-mouth disease virus (FMDV) IRES and three translation initiation factors. A doublet of 116/110 kDa that crosslinked to the FMDV IRES is a component of eIF3. We show here that domain 5 holds the preferential binding site for eIF3, although this complex initiation factor can establish multiple contacts with the IRES structure. We have also identified the phylogenetically conserved hairpin of domain 5 as the RNA motif responsible for eIF4B interaction. Mutation of this stem-loop structure abrogated eIF4B, but not eIF3, binding to the IRES. Remarkably, IRES mutants severely affected in their interaction with eIF4B showed a mild reduction in IRES activity when tested in the context of a bicistronic expression vector in transfected cells. Finally, we provide evidence of the interaction of eIF4GII with FMDV IRES, the RNA determinants for this interaction being shared with its functional homolog eIF4GI. The FMDV Lb protease generated a C-terminal fragment of eIF4GII that binds to the IRES as efficiently as the intact protein. Competition experiments showed that titration of eIF4B or p110/116 interaction with the FMDV IRES required a large excess of competitor relative to eIF4G, strongly suggesting that eIF4G-IRES interaction is a limiting factor to titrate the IRES. Comparative analysis of the activity of IRES mutants affected in domains 4 and 5 regarding their pattern of RNA-protein complex formation demonstrates that while binding of eIF4B with the FMDV IRES is dispensable, interaction of eIF4G is a central feature of the activity of this element.     

X-Ray structures of the universal translation initiation factor IF2/eIF5B: conformational changes on GDP and GTP binding

X-ray structures of the universal translation initiation factor IF2/eIF5B have been determined in three states: free enzyme, inactive IF2/eIF5B.GDP, and active IF2/eIF5B.GTP. The "chalice-shaped" enzyme is a GTPase that facilitates ribosomal subunit joining and Met-tRNA(i) binding to ribosomes in all three kingdoms of life. The conserved core of IF2/eIF5B consists of an N-terminal G domain (I) plus an EF-Tu-type beta barrel (II), followed by a novel alpha/beta/alpha-sandwich (III) connected via an alpha helix to a second EF-Tu-type beta barrel (IV). Structural comparisons reveal a molecular lever, which amplifies a modest conformational change in the Switch 2 region of the G domain induced by Mg(2+)/GTP binding over a distance of 90 A from the G domain active center to domain IV. Mechanisms of GTPase function and ribosome binding are discussed.     

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.