Analysis of the EIAV Rev-responsive element (RRE) reveals a conserved RNA motif required for high affinity Rev binding in both HIV-1 and EIAV

A cis-acting RNA regulatory element, the Rev-responsive element (RRE), has essential roles in replication of lentiviruses, including human immunodeficiency virus (HIV-1) and equine infection anemia virus (EIAV). The RRE binds the viral trans-acting regulatory protein, Rev, to mediate nucleocytoplasmic transport of incompletely spliced mRNAs encoding viral structural genes and genomic RNA. Because of its potential as a clinical target, RRE-Rev interactions have been well studied in HIV-1; however, detailed molecular structures of Rev-RRE complexes in other lentiviruses are still lacking. In this study, we investigate the secondary structure of the EIAV RRE and interrogate regulatory protein-RNA interactions in EIAV Rev-RRE complexes. Computational prediction and detailed chemical probing and footprinting experiments were used to determine the RNA secondary structure of EIAV RRE-1, a 555 nt region that provides RRE function in vivo. Chemical probing experiments confirmed the presence of several predicted loop and stem-loop structures, which are conserved among 140 EIAV sequence variants. Footprinting experiments revealed that Rev binding induces significant structural rearrangement in two conserved domains characterized by stable stem-loop structures. Rev binding region-1 (RBR-1) corresponds to a genetically-defined Rev binding region that overlaps exon 1 of the EIAV rev gene and contains an exonic splicing enhancer (ESE). RBR-2, characterized for the first time in this study, is required for high affinity binding of EIAV Rev to the RRE. RBR-2 contains an RNA structural motif that is also found within the high affinity Rev binding site in HIV-1 (stem-loop IIB), and within or near mapped RRE regions of four additional lentiviruses. The powerful integration of computational and experimental approaches in this study has generated a validated RNA secondary structure for the EIAV RRE and provided provocative evidence that high affinity Rev binding sites of HIV-1 and EIAV share a conserved RNA structural motif. The presence of this motif in phylogenetically divergent lentiviruses suggests that it may play a role in highly conserved interactions that could be targeted in novel anti-lentiviral therapies.     

Computational modeling suggests dimerization of equine infectious anemia virus Rev is required for RNA binding

Background

The lentiviral Rev protein mediates nuclear export of intron-containing viral RNAs that encode structural proteins or serve as the viral genome. Following translation, HIV-1 Rev localizes to the nucleus and binds its cognate sequence, termed the Rev-responsive element (RRE), in incompletely spliced viral RNA. Rev subsequently multimerizes along the viral RNA and associates with the cellular Crm1 export machinery to translocate the RNA-protein complex to the cytoplasm. Equine infectious anemia virus (EIAV) Rev is functionally homologous to HIV-1 Rev, but shares very little sequence similarity and differs in domain organization. EIAV Rev also contains a bipartite RNA binding domain comprising two short arginine-rich motifs (designated ARM-1 and ARM-2) spaced 79 residues apart in the amino acid sequence. To gain insight into the topology of the bipartite RNA binding domain, a computational approach was used to model the tertiary structure of EIAV Rev.

Results

The tertiary structure of EIAV Rev was modeled using several protein structure prediction and model quality assessment servers. Two types of structures were predicted: an elongated structure with an extended central alpha helix, and a globular structure with a central bundle of helices. Assessment of models on the basis of biophysical properties indicated they were of average quality. In almost all models, ARM-1 and ARM-2 were spatially separated by >15 Å, suggesting that they do not form a single RNA binding interface on the monomer. A highly conserved canonical coiled-coil motif was identified in the central region of EIAV Rev, suggesting that an RNA binding interface could be formed through dimerization of Rev and juxtaposition of ARM-1 and ARM-2. In support of this, purified Rev protein migrated as a dimer in Blue native gels, and mutation of a residue predicted to form a key coiled-coil contact disrupted dimerization and abrogated RNA binding. In contrast, mutation of residues outside the predicted coiled-coil interface had no effect on dimerization or RNA binding.

Conclusions

Our results suggest that EIAV Rev binding to the RRE requires dimerization via a coiled-coil motif to juxtapose two RNA binding motifs, ARM-1 and ARM-2.

     

Structural Model of the Rev Regulatory Protein from Equine Infectious Anemia Virus

Rev is an essential regulatory protein in the equine infectious anemia virus (EIAV) and other lentiviruses, including HIV-1. It binds incompletely spliced viral mRNAs and shuttles them from the nucleus to the cytoplasm, a critical prerequisite for the production of viral structural proteins and genomic RNA. Despite its important role in production of infectious virus, the development of antiviral therapies directed against Rev has been hampered by the lack of an experimentally-determined structure of the full length protein. We have used a combined computational and biochemical approach to generate and evaluate a structural model of the Rev protein. The modeled EIAV Rev (ERev) structure includes a total of 6 helices, four of which form an anti-parallel four-helix bundle. The first helix contains the leucine-rich nuclear export signal (NES). An arginine-rich RNA binding motif, RRDRW, is located in a solvent-exposed loop region. An ERLE motif required for Rev activity is predicted to be buried in the core of modeled structure where it plays an essential role in stabilization of the Rev fold. This structural model is supported by existing genetic and functional data as well as by targeted mutagenesis of residues predicted to be essential for overall structural integrity. Our predicted structure should increase understanding of structure-function relationships in Rev and may provide a basis for the design of new therapies for lentiviral diseases.     

Binding sites for Rev and ASF/SF2 map to a 55-nucleotide purine-rich exonic element in equine infectious anemia virus RNA

The equine infectious anemia virus (EIAV) Rev protein (ERev) negatively regulates its own synthesis by inducing alternative splicing of its mRNA. This bicistronic mRNA contains four exons; exons 1 and 2 encode Tat, and exons 3 and 4 encode Rev. When Rev is expressed, exon 3 is skipped to produce an mRNA that contains only exons 1, 2, and 4. The interaction of ERev with its cis-acting RNA response element, the RRE, is also essential for nuclear export of intron-containing viral mRNAs that encode structural and enzymatic gene products. The primary ERev binding site and the manner in which ERev interacts with RNA or cellular proteins to exert its regulatory function have not been defined. We have performed in vitro RNA binding experiments to show that recombinant ERev binds to a 55-nucleotide, purine-rich tract proximal to the 5' splice site of exon 3. Because of its proximity to the 5' splice site and since it contains elements related to consensus exonic splicing enhancer sequences, we asked whether cellular proteins recognize the EIAV RRE. The cellular protein, ASF/SF2, a member of the serine- and arginine-rich family of splicing factors (SR proteins) bound to repeated sequences within the 55-nucleotide RRE region. Electrophoretic mobility shift and UV cross-linking experiments indicated that ERev and SR proteins bind simultaneously to the RRE. Furthermore, in vitro protein-protein interaction studies revealed an association between ERev and SR proteins. These data suggest that EIAV Rev-induced exon skipping observed in vivo may be initiated by simultaneous binding of Rev and SR proteins to the RRE that alter the subsequent assembly or catalytic activity of the spliceosomal complex.     

Exchange of the basic domain of human immunodeficiency virus type 1 Rev for a polyarginine stretch expands the RNA binding specificity, and a minimal arginine cluster is required for optimal RRE RNA binding affinity, nuclear accumulation, and trans-activation

The Rev regulatory protein of human immunodeficiency virus (HIV) facilitates the nuclear export of unspliced and partially spliced HIV RNAs. Using a Rev:MS2 phage coat protein fusion that could be targeted to bind and activate the Rev-responsive element (RRE) RNA or heterologous MS2 phage operator RNA, we analyzed the role(s) of the arginine-rich RNA binding domain in RNA binding and transactivation. The arginine-rich domain could be functionally replaced by a stretch of nine arginines. However, polyarginine substitutions expanded the RNA binding specificity of the resultant mutant Rev protein. Polyarginine insertions in place of residues 24 to 60 that excised the RNA binding and oligomerization domains of Rev preserved the activation for MS2 RNA, but not for the RRE. A nine-arginine insertion outside of the natural context of the Rev nuclear localization signal domain was incompatible with activation of either RNA target. Insertions of fewer than eight arginines impaired RRE activation. Interrupted lysine clusters and disruption of the arginine stretch with lysine or neutral residues resulted in a similar phenotype. Some of these mutants with a null phenotype for RRE activated the heterologous MS2 RNA target. Under steady-state conditions, mutants that preserved the Rev response for RRE RNA localized to the nuclei; those with poor or no Rev response accumulated mostly in the cytoplasm. Many of the cytoplasmically resident derivatives became nuclear when leptomycin B (LMB) treatment inhibited nuclear export of nuclear export signal-containing proteins. Mutants that had a null activation potential for either RNA target were particularly resistant to LMB treatment. Abbreviated nuclear residence times and differences in RRE binding affinity may have compromised their activation potential for RRE. High-affinity binding to MS2 RNA through the intact coat protein was sufficient to overcome the short nuclear residence times and to facilitate MS2 activation by some derivatives.     

Binding of equine infectious anemia virus rev to an exon splicing enhancer mediates alternative splicing and nuclear export of viral mRNAs

In addition to facilitating the nuclear export of incompletely spliced viral mRNAs, equine infectious anemia virus (EIAV) Rev regulates alternative splicing of the third exon of the tat/rev mRNA. In the presence of Rev, this exon of the bicistronic RNA is skipped in a fraction of the spliced mRNAs. In this report, the cis-acting requirements for exon 3 usage were correlated with sequences necessary for Rev binding and transport of incompletely spliced RNA. The presence of a purine-rich exon splicing enhancer (ESE) was required for exon 3 recognition, and the addition of Rev inhibited exon 3 splicing. Glutathione-S-transferase (GST)-Rev bound to probes containing the ESE, and mutation of GAA repeats to GCA within the ESE inhibited both exon 3 recognition in RNA splicing experiments and GST-Rev binding in vitro. These results suggest that Rev regulates alternative splicing by binding at or near the ESE to block SR protein-ESE interactions. A 57-nucleotide sequence containing the ESE was sufficient to mediate Rev-dependent nuclear export of incompletely spliced RNAs. Rev export activity was significantly inhibited by mutation of the ESE or by trans-complementation with SF2/ASF. These results indicate that the ESE functions as a Rev-responsive element and demonstrate that EIAV Rev mediates exon 3 exclusion through protein-RNA interactions required for efficient export of incompletely spliced viral RNAs.     

Interactions among SR proteins, an exonic splicing enhancer, and a lentivirus Rev protein regulate alternative splicing.

We examine here the roles of cellular splicing factors and virus regulatory proteins in coordinately regulating alternative splicing of the tat/rev mRNA of equine infectious anemia virus (EIAV). This bicistronic mRNA contains four exons; exons 1 and 2 encode Tat, and exons 3 and 4 encode Rev. In the absence of Rev expression, the four-exon mRNA is synthesized exclusively, but when Rev is expressed, exon 3 is skipped to produce an mRNA that contains only exons 1, 2, and 4. We identify a purine-rich exonic splicing enhancer (ESE) in exon 3 that promotes exon inclusion. Similar to other cellular ESEs that have been identified by other laboratories, the EIAV ESE interacted specifically with SR proteins, a group of serine/arginine-rich splicing factors that function in constitutive and alternative mRNA splicing. Substitution of purines with pyrimidines in the ESE resulted in a switch from exon inclusion to exon skipping in vivo and abolished binding of SR proteins in vitro. Exon skipping was also induced by expression of EIAV Rev. We show that Rev binds to exon 3 RNA in vitro, and while the precise determinants have not been mapped, Rev function in vivo and RNA binding in vitro indicate that the RNA element necessary for Rev responsiveness overlaps or is adjacent to the ESE. We suggest that EIAV Rev promotes exon skipping by interfering with SR protein interactions with RNA or with other splicing factors.     

Nuclear transport of human immunodeficiency virus type 1, visna virus, and equine infectious anemia virus Rev proteins: identification of a family of transferable nuclear export signals.

The human immunodeficiency virus type 1 Rev trans activator binds directly to unspliced viral mRNA in the nucleus and activates its transport to the cytoplasm. In additon to the sequences that confer RNA binding and nuclear localization, Rev has a carboxy-terminal region, the activation domain, whose integrity is essential for biological activity. Because it has been established that Rev constitutively exits and reenters the nucleus and that the activation domain is required for nuclear exit, it has been proposed that Rev's activation domain is a nuclear export signal (NES). Here, we used microinjection-based assays to demonstrate that the activation domain of human immunodeficiency virus type 1 Rev imparts rapid nuclear export after its transfer to heterologous substrates. NES- mediated export is specific, as it is sensitive both to inactivation by missense mutation and to selective inhibition by an excess of the wild-type, but not mutant, activation domain peptide. Examination of the Rev trans activators of two nonprimate lentiviruses, visna virus and equine infectious anemia virus, revealed that their activation domains are also potent NESs. Taken together, these data demonstrate that nuclear export can be determined by positively acting peptide motifs, namely, NESs, and suggest that Rev proteins activate viral RNA transport by providing export ribonucleoproteins with specific information that targets them to the cytoplasm.     

Two functionally distinct RNA-binding motifs in the regulatory domain of the protein kinase DAI.

The RNA-binding domain of the protein kinase DAI, the double-stranded RNA inhibitor of translation, contains two repeats of a motif that is also found in a number of other RNA-binding proteins. This motif consists of 67 amino acid residues and is predicted to contain a positively charged alpha helix at its C terminus. We have analyzed the effects of equivalent single amino acid changes in three conserved residues distributed over each copy of the motif. Mutants in the C-terminal portion of either repeat were severely defective, indicating that both copies of the motif are essential for RNA binding. Changes in the N-terminal and central parts of the motif were more debilitating if they were made in the first motif than in the second, suggesting that the first motif is the more important for RNA binding and that the second motif is structurally more flexible. When the second motif was replaced by a duplicate of the first motif, the ectopic copy retained its greater sensitivity to mutation, implying that the two motifs have distinct functions with respect to the process of RNA binding. Furthermore, the mutations have the same effect on the binding of double-stranded RNA and VA RNA, consistent with the existence of a single RNA-binding domain for both activating and inhibitory RNAs.     

The solution structure of REF2-I reveals interdomain interactions and regions involved in binding mRNA export factors and RNA

The RNA binding and export factor (REF) family of mRNA export adaptors are found in several nuclear protein complexes including the spliceosome, TREX, and exon junction complexes. They bind RNA, interact with the helicase UAP56/DDX39, and are thought to bridge the interaction between the export factor TAP/NXF1 and mRNA. REF2-I consists of three domains, with the RNA recognition motif (RRM) domain positioned in the middle. Here we dissect the interdomain interactions of REF2-I and present the solution structure of a functionally competent double domain (NM; residues 1-155). The N-terminal domain comprises a transient helix (N-helix) linked to the RRM by a flexible arm that includes an Arg-rich region. The N-helix, which is required for REF2-I function in vivo, overlaps the highly conserved REF-N motif and, together with the adjacent Arg-rich region, interacts transiently with the RRM. RNA interacts with REF2-I through arginine-rich regions in its N- and C-terminal domains, but we show that it also interacts weakly with the RRM. The mode of interaction is unusual for an RRM since it involves loops L1 and L5. NMR signal mapping and biochemical analysis with NM indicate that DDX39 and TAP interact with both the N and RRM domains of REF2-I and show that binding of these proteins and RNA will favor an open conformation for the two domains. The proximity of the RNA, TAP, and DDX39 binding sites on REF2-I suggests their binding may be mutually exclusive, which would lead to successive ligand binding events in the course of mRNA export.     

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.     

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.     

Characterization and functional implications of the RNA binding properties of nuclear factor TDP-43, a novel splicing regulator of CFTR exon 9

Variations in a polymorphic (TG)m sequence near exon 9 of the human CFTR gene have been associated with variable proportions of exon skipping and occurrence of disease. We have recently identified nuclear factor TDP-43 as a novel splicing regulator capable of binding to this element in the CFTR pre-mRNA and inhibiting recognition of the neighboring exon. In this study we report the dissection of the RNA binding properties of TDP-43 and their functional implications in relationship with the splicing process. Our results show that this protein contains two fully functional RNA recognition motif (RRM) domains with distinct RNA/DNA binding characteristics. Interestingly, TDP-43 can bind a minimum number of six UG (or TG) single-stranded dinucleotide stretches, and binding affinity increases with the number of repeats. In particular, the highly conserved Phe residues in the first RRM region play a key role in nucleic acid recognition.     

Identification of a tripartite import signal in the Ewing Sarcoma protein (EWS)

The Ewing Sarcoma (EWS) protein is a ubiquitously expressed RNA processing factor that localises predominantly to the nucleus. However, the mechanism through which EWS enters the nucleus remains unclear, with differing reports identifying three separate import signals within the EWS protein. Here we have utilized a panel of truncated EWS proteins to clarify the reported nuclear localisation signals. We describe three C-terminal domains that are important for efficient EWS nuclear localization: (1) the third RGG-motif; (2) the last 10 amino acids (known as the PY-import motif); and (3) the zinc-finger motif. Although these three domains are involved in nuclear import, they are not independently capable of driving the efficient import of a GFP-moiety. However, collectively they form a complex tripartite signal that efficiently drives GFP-import into the nucleus. This study helps clarify the EWS import signal, and the identification of the involvement of both the RGG- and zinc-finger motifs has wide reaching implications.     

Functional importance of the conserved N-terminal domain of the mitochondrial replicative DNA helicase

The mitochondrial replicative DNA helicase is an essential cellular protein that shows high similarity with the bifunctional primase-helicase of bacteriophage T7, the gene 4 protein (T7 gp4). The N-terminal primase domain of T7 gp4 comprises seven conserved sequence motifs, I, II, III, IV, V, VI, and an RNA polymerase basic domain. The putative primase domain of metazoan mitochondrial DNA helicases has diverged from T7 gp4 and in particular, the primase domain of vertebrates lacks motif I, which comprises a zinc binding domain. Interestingly, motif I is conserved in insect mtDNA helicases. Here, we evaluate the effects of overexpression in Drosophila cell culture of variants carrying mutations in conserved amino acids in the N-terminal region, including the zinc binding domain. Overexpression of alanine substitution mutants of conserved amino acids in motifs I, IV, V and VI and the RNA polymerase basic domain results in increased mtDNA copy number as is observed with overexpression of the wild type enzyme. In contrast, overexpression of three N-terminal mutants W282L, R301Q and P302L that are analogous to human autosomal dominant progressive external ophthalmoplegia mutations results in mitochondrial DNA depletion, and in the case of R301Q, a dominant negative cellular phenotype. Thus whereas our data suggest lack of a DNA primase activity in Drosophila mitochondrial DNA helicase, they show that specific N-terminal amino acid residues that map close to the central linker region likely play a physiological role in the C-terminal helicase function of the protein.     

Functional analysis of SNF, the Drosophila U1A/U2B" homolog: identification of dispensable and indispensable motifs for both snRNP assembly and function in vivo

In Drosophila, the spliceosomal protein SNF fulfills the functions of two vertebrate proteins, U1 snRNP-UlA and U2 snRNP-U2B". The structure and sequence of SNF, U1A, and U2B" are nearly identical with two RNA recognition motifs (RRM) separated by a short linker region, yet they have different RNA-binding properties: U1A binds U1 snRNA, U2B" binds U2 snRNA, and SNF binds both snRNAs. Structure/function studies on the human proteins have identified motifs in the N-terminal RRM that are critical for RNA-binding specificity but have failed to identify a function for the C-terminal RRM. Interestingly, SNF is chimeric in these motifs, suggesting a basis for its dual specificity. Here, we test the importance of these motifs by introducing site-directed mutations in the snf coding region and examining the effects of these mutations on assembly into the snRNP and on snf function in vivo. We found that an N-terminal RRM mutant protein predicted to eliminate RNA binding still assembles into snRNPs and is capable of rescuing snf's lethal phenotype only if the normally dispensable C-terminal RRM is present. We also found that the mixed motif in the "RNA-specificity" domain is necessary for SNF's dual function whereas the mixed motif in the U2A'-protein-binding region is not. Finally, we demonstrate that animals carrying a snf mutation that converts SNF from a bifunctional protein to a U1 snRNP-specific protein are viable. This unexpected result suggests that SNF's presence within the U2 snRNP is not essential for splicing.