Analysis of the interaction with the hepatitis C virus mRNA reveals an alternative mode of RNA recognition by the human La protein

Human La protein is an essential factor in the biology of both coding and non-coding RNAs. In the nucleus, La binds primarily to 3' oligoU containing RNAs, while in the cytoplasm La interacts with an array of different mRNAs lacking a 3' UUU(OH) trailer. An example of the latter is the binding of La to the IRES domain IV of the hepatitis C virus (HCV) RNA, which is associated with viral translation stimulation. By systematic biophysical investigations, we have found that La binds to domain IV using an RNA recognition that is quite distinct from its mode of binding to RNAs with a 3' UUU(OH) trailer: although the La motif and first RNA recognition motif (RRM1) are sufficient for high-affinity binding to 3' oligoU, recognition of HCV domain IV requires the La motif and RRM1 to work in concert with the atypical RRM2 which has not previously been shown to have a significant role in RNA binding. This new mode of binding does not appear sequence specific, but recognizes structural features of the RNA, in particular a double-stranded stem flanked by single-stranded extensions. These findings pave the way for a better understanding of the role of La in viral translation initiation.     

Cellular proteins from human monocytes bind to dengue 4 virus minus-strand 3' untranslated region RNA

The synthesis of plus and minus RNA strands of several RNA viruses requires as a first step the interaction of some viral regulatory sequences with cellular and viral proteins. The dengue 4 virus genome, a single-stranded, positive-polarity RNA, is flanked by two untranslated regions (UTR) located in the 5' and 3' ends. The 3'UTR in the minus-strand RNA [3'UTR (-)] has been thought to function as a promoter for the synthesis of plus-strand RNA. To study the initial interaction between this 3'UTR and cellular and viral proteins, mobility shift assays were performed, and four ribonucleoprotein complexes (I through IV) were formed when uninfected and infected U937 cells (human monocyte cell line) interacted with the 3'UTR (-) of dengue 4 virus. Cross-linking assays with RNAs containing the complete 3'UTR (-) (nucleotides [nt] 101 to 1) or a partial sequence from nt 101 to 45 and nt 44 to 1 resulted in specific binding of some cellular proteins. Supermobility shift and immunoprecipitation assays demonstrated that the La protein forms part of these complexes. To determine the region in the 3' UTR that interacted with the La protein, two deletion mutants were generated. The mutant (del-96), with a deletion of nt 96 to 101, was unable to interact with the La protein, suggesting that La interacted with the 5' portion of the 3'UTR (-). Complex I, which was the main ribonucleoprotein complex formed with the 3'UTR (-) and which had the fastest electrophoretic migration, contained proteins such as calreticulin and protein disulfide isomerase, which constitute important components of the endoplasmic reticulum.     

The sequence of the stem and flanking sequences at the 3' end of histone mRNA are critical determinants for the binding of the stem-loop binding protein.

Complexes of different electrophoretic mobility containing the stem-loop binding protein, a 45 kDa protein, bound to the stem-loop at the 3' end of histone mRNA, are present in both nuclear and cytoplasmic extracts from mammalian cells. We have determined the effect of changes in the loop, in the stem and in the flanking sequences on the affinity of the SLBP for the 3' end of histone mRNA. The sequence of the stem is particularly critical for SLBP binding. Specific sequences both 5' and 3' of the stem-loop are also required for high-affinity binding. Expanding the four base loop by one or two uridines reduced but did not abolish SLBP binding. RNA footprinting experiments show that the flanking sequences on both sides of the stem-loop are critical for efficient binding, but that cleavages in the loop do not abolish binding. Thus all three regions of the RNA sequence contribute to SLBP binding, suggesting that the 26 nt at the 3' end of histone mRNA forms a defined tertiary structure recognized by the SLBP.     

Autoantigens interact with cis-acting elements of rubella virus RNA.

Rubella virus (RV) infections in adult women can be associated with acute and chronic arthritic symptoms. In many autoimmune individuals, antibodies are found targeting endogenous proteins, called autoantigens, contained in ribonucleoprotein complexes (RNPs). In order to understand the molecular mechanisms involved in the RV-associated pathology, we investigated the nature of cellular factors binding RV RNA and whether such RNPs were recognized by antibodies in infected individuals. Previously, we noted that cellular proteins associated with the RV 5'(+) stem-loop (SL) RNA are recognized by serum with Ro reactivity. To better understand the nature of the autoantigens binding RV cis-acting elements, serum samples from individuals with various autoimmune diseases were tested for their ability to immunoprecipitate RNPs containing labeled RV RNAs. A subset of serum samples recognizing autoantigen La, or Ro and La, immunoprecipitated both the RV 5'(+)SL and 3'(+)SL RNA-protein complexes. Autoantigens binding the RV 5'(+)SL and 3'(+)SL RNAs differed in molecular mass, specificities for respective RNA binding substrates, and sensitivity to alkaline phosphatase treatment. The La autoantigen was found to interact with the RV 5'(+)SL RNA as determined by immunological techniques and binding reactions with mixtures containing recombinant La protein. To test whether there is a correlation between La binding to an RV RNA element and the appearance of an anti-La response, we measured anti-La titers in RV-infected individuals. Significant anti-La activity was detected in approximately one-third of RV-infected individuals 2 years postinfection.     

DNA-protein interactions at the telomeric repeats of Schizosaccharomyces pombe.

Gel retardation assays using a probe containing the repeat region of a Schizosaccharomyces pombe chromosomal telomere identified four specific DNA- protein complexes in S. pombe total protein extracts (I, I', IIa and IIb). The proteins responsible for these complexes bound to the telomeric repeat region irrespective of whether or not the repeats were in close proximity to the end of a DNA molecule, and none of them bound strongly to single-stranded DNA. The protein responsible for complex I (TeRF I) was separated from the activity responsible for complexes IIa and IIb (TeRF II) using heparin-Sepharose chromatography. Both factors were efficiently cross-competed by an oligonucleotide containing the 18 bp sequence 5'-GGTTACAGGTTACAGGTT-3', which corresponds to two complete telomeric repeat units. Mutation of the T residues at positions 4 and 11 in the oligonucleotide dramatically reduced binding by TeRF II, but had no affect on binding by TeRF I. The protein responsible for complex I' did not bind strongly to either the wild-type or mutant oligonucleotide.     

La antigen recognizes and binds to the 3''-oligouridylate tail of a small RNA.

The La antigen is a cellular protein which interacts with many RNA species that are products of RNA polymerase III, including the adenovirus virus-associated (VA) RNAs. We demonstrate that the efficiency of antigen binding in vitro is determined by the number of U residues at the RNA 3' terminus. Forms of VA RNAI with more than two terminal U residues are fully bound, forms with two U residues are partially bound, and forms with fewer than two U residues are not bound at all. The antigen can be covalently linked to VA RNA by UV irradiation, and the site of cross-linking is shown to contain the 3' terminus of the RNA. We conclude that the antigen recognizes the U-rich 3' tail of VA RNA, and presumably that of other polymerase III products, and that it binds at or close to this site.     

Sequence-specific binding of the influenza virus RNA polymerase to sequences located at the 5' ends of the viral RNAs.

The enzymatic activity of recombinant influenza virus RNA polymerase is strictly dependent on the addition of a template RNA containing 5' and 3' viral sequences. Here we report the analysis of the binding specificity and physical characterization of the complex by using gel shift, modification interference, and density gradient techniques. The 13S complex binds specifically to short synthetic RNAs that mimic the partially double stranded panhandle structures found at the termini of both viral RNA and cRNA. The polymerase will also bind independently to the single-stranded 5' or 3' ends of viral RNA. It binds most strongly to specific sequences within the 5' end but is unable to bind these sequences in the context of a completely double stranded structure. Modification interference analysis identified the short sequence motifs at the 5' ends of the viral RNA and cRNA templates that are critical for binding.     

Substrate recognition and catalysis by the exoribonuclease RNase R

RNase R is a processive, 3' to 5' hydrolytic exoribonuclease that together with polynucleotide phosphorylase plays an important role in the degradation of structured RNAs. However, RNase R differs from other exoribonucleases in that it can by itself degrade RNAs with extensive secondary structure provided that a single-stranded 3' overhang is present. Using a variety of specifically designed substrates, we show here that a 3' overhang of at least 7 nucleotides is required for tight binding and activity, whereas optimum binding and activity are achieved when the overhang is 10 or more nucleotides in length. In contrast, duplex RNAs with no overhang or with a 4-nucleotide overhang bind extremely poorly to RNase R and are inactive as substrates. A duplex RNA with a 10-nucleotide 5' overhang also is not a substrate. Interestingly, this molecule is bound only weakly, indicating that RNase R does not simply recognize single-stranded RNA, but the RNA must thread into the enzyme with 3' to 5' polarity. We also show that ribose moieties are required for recognition of the substrate as a whole since RNase R is unable to bind or degrade single-stranded DNA. However, RNA molecules with deoxyribose or dideoxyribose residues at their 3' termini can be bound and degraded. Based on these data and a homology model of RNase R, derived from the structure of the closely related enzyme, RNase II, we present a model for how RNase R interacts with its substrates and degrades RNA.     

BHK cell proteins that bind to the 3' stem-loop structure of the West Nile virus genome RNA.

The first 83 3' nucleotides of the genome RNA of the flavivirus West Nile encephalitis virus (WNV) form a stable stem-loop (SL) structure which is followed in the genome by a smaller SL. These 3' structures are highly conserved among divergent flaviviruses, suggesting that they may function as cis-acting signals for RNA replication and as such might specifically bind to cellular or viral proteins. Cellular proteins from uninfected and WNV-infected BHK-21 S100 cytoplasmic extracts formed three distinct complexes with the WNV plus-strand 3' SL [(+)3'SL] RNA in a gel mobility shift assay. Subsequent competitor gel shift analyses showed that two of these RNA-protein complexes, complexes 1 and 2, contained cell proteins that specifically bound to the WNV (+)3'SL RNA. UV-induced cross-linking and Northwestern blotting analyses detected WNV (+)3'SL RNA-binding proteins of 56, 84, and 105 kDa. When the S100 cytoplasmic extracts were partially purified by ion-exchange chromatography, a complex that comigrated with complex 1 was detected in fraction 19, while a complex that comigrated with complex 2 was detected in fraction 17. UV-induced cross-linking experiments indicated that an 84-kDa cell protein in fraction 17 and a 105-kDa protein in fraction 19 bound specifically to the WNV (+)3'SL RNA. In addition to binding to the (+)3'SL RNA, the 105-kDa protein bound to the SL structure located at the 3' end of the WNV minus-strand RNA. Initial mapping studies indicated that the 84- and 105-kDa proteins bind to different regions of the (+)3'SL RNA. The 3'-terminal SL RNA of another flavivirus, dengue virus type 3, specifically competed with the WNV (+)3'SL RNA in gel shift assays, suggesting that the host proteins identified in this study are flavivirus specific.     

R150A mutant of F TraI relaxase domain: reduced affinity and specificity for single-stranded DNA and altered fluorescence anisotropy of a bound labeled oligonucleotide

F factor TraI is a helicase and a single-stranded DNA nuclease ("relaxase") essential for conjugative DNA transfer. A TraI domain containing relaxase activity, TraI36, was generated previously. Substituting Ala for Arg150 (R150A) of TraI36 reduces in vitro relaxase activity. The mutant has reduced affinity, relative to wild type, for a 3'-TAMRA-labeled 22-base single-stranded oligonucleotide. While both R150A and wild-type TraI36 bind oligonucleotide, only wild type increases steady-state fluorescence anisotropy of the labeled 22-base oligonucleotide upon binding. In contrast, binding by either protein increases steady-state anisotropy of a 3'-TAMRA-labeled 17-base oligonucleotide. Time-resolved intensity data for both oligonucleotides, bound and unbound, require three lifetimes for adequate fits, at least one more than the fluorophore alone. The preexponential amplitude for the longest lifetime increases upon binding. Time-resolved anisotropy data for both oligonucleotides, bound and unbound, require two rotational correlation times for adequate fits. The longer correlation time increases upon protein binding. Correlation times for the protein-bound 17-base oligonucleotide are similar for both proteins, with the longer correlation time in the range of molecular tumbling of the protein-DNA complex. In contrast, protein binding causes less dramatic increases in correlation times for the 22-base oligonucleotide relative to the 17-base oligonucleotide. Binding studies indicate that R150 contributes to recognition of bases immediately 3' to the DNA cleavage site, consistent with the apparent proximity of R150 and the 3' oligonucleotide end. Models in which the R150A substitution alters single-stranded DNA flexibility at the oligonucleotide 3' end or affects fluorophore-DNA or fluorophore-protein interactions are discussed.     

Evidence for a substrate-binding subsite in ribonuclease T1. Crystal structure of the complex with two guanosines, and model building of the complex with the substrate guanylyl-3',5'-guanosine

The enzyme ribonuclease T1 cleaves single-stranded RNA at the 3'-side of guanosine. The structure of the complex with two guanosines has been analyzed at 1.8-A resolution and refined to a crystallographic R value of 14.0%. One guanosine occupies the guanosine recognition site as observed in previously analyzed complexes of ribonuclease T1 with guanosine phosphates. The other is bound to a base-unspecific subsite marking the binding locus of the nucleoside 3'-proximal to guanosine in a cleavable RNA chain. The positions of the guanosine bound to the recognition site and of the guanine base at the subsite were used to guide model building of the substrate guanylyl-3',5'-guanosine bound to the active site of ribonuclease T1. After energy minimization and a 7-ps stochastic dynamics simulation, a plausible model of the enzyme-substrate complex was obtained which may serve as a reference point in consideration of the mechanisms of RNA hydrolysis by ribonuclease T1.     

Characterization of multimeric complexes formed by the human PTB1 protein on RNA

Polypyrimidine tract binding protein (PTB or hnRNP I) has several known functions in eukaryotic cells, including exon exclusion during alternative splicing events, mRNA stabilization, and regulation of viral translation and replication. PTB contains four RNA Binding Domains (RBDs, or RRMs), all of which can potentially bind RNA, but their roles in the various biological functions of PTB are not clear. We investigate the properties of the complexes formed by human PTB1 on two target RNAs: the rat GABAA receptor gamma2 subunit pre-mRNA and the Hepatitis C Virus 3' NonTranslated RNA. The GABA RNA contains four polypyrimidine tracts in the intron and exon, while the HCV NTR contains a 75-nt U-rich tract and a highly structured 3'-terminus. Electrophoretic mobility shift assays show that PTB1 protein first binds to both RNAs with nanomolar affinities, but subsequent protein addition leads to formation of higher-order complexes. Stoichiometry experiments show that the ultimate complexes contain up to eight PTB1 proteins per RNA strand. Protein constructs containing two tandem RBDs also bind the two RNAs, but with different affinities and stoichiometries. Nuclease protection assays show that PTB1 protects the polypyrimidine tracts in the GABA RNA, as does a construct consisting of RBD3 and RBD4; however, a construct containing RBD1 and RBD2 enhances cleavage of bound RNA. The binding mechanisms of PTB1 are unique to the full-length protein; these modes appear to include direct association with the RNA as well as weaker intermolecular protein associations.     

Three different cellular proteins bind to complementary sites on the 5'-end-positive and 3'-end-negative strands of mouse hepatitis virus RNA.

The termini of viral genomic RNA and its complementary strand are important in the initiation of viral RNA replication, which probably involves both viral and cellular proteins. To detect the possible cellular proteins involved in the replication of mouse hepatitis virus RNA, we performed RNA-protein binding studies with RNAs representing both the 5' and 3' ends of the viral genomic RNA and the 3' end of the negative-strand complementary RNA. Gel-retardation assays showed that both the 5'-end-positive- and 3'-end-negative-strand RNA formed an RNA-protein complex with cellular proteins from the uninfected cells. UV cross-linking experiments further identified a 55-kDa protein bound to the 5' end of the positive-strand viral genomic RNA and two proteins 35 and 38 kDa in size bound to the 3' end of the negative-strand cRNA. The results of the competition assay confirmed the specificity of this RNA-protein binding. No proteins were found to bind to the 3' end of the viral genomic RNA under the same conditions. The binding site of the 55-kDa protein was mapped within the 56-nucleotide region from nucleotides 56 to 112 from the 5' end of the positive-strand RNA, and the 35- and 38-kDa proteins bound to the complementary region on the negative-strand RNA. The 38-kDa protein was detected only in DBT cells but was not detected in HeLa or COS cells, while the 35-kDa protein was found in all three cell types. The juxtaposition of the different cellular proteins on the complementary sites near the ends of the positive- and negative-strand RNAs suggests that these proteins may interact with each other and play a role in mouse hepatitis virus RNA replication.     

Mechanical studies of single ribosome/mRNA complexes

Methodology was developed for specifically anchoring Escherichia coli 70S ribosomes onto a chemically modified, cysteine-reactive glass surface. Immobilized ribosomes maintain the capability of binding a polyuridylic acid (poly(U)) template, enabling investigation of mechanical properties of individual ribosome-poly(U) complexes using laser tweezers. Streptavidin-coated polystyrene microspheres bound specifically to the biotinylated 3' end of long (up to 10,000 bases) poly(U) strands. A novel optical method was built to control the position of the laser trap along the microscope optical axis at 2 nm resolution, facilitating measurement of the force-extension relationship for poly(U). Some immobilized ribosome-poly(U) complexes supported 100 pN of force applied at the 3' end of the mRNA. Binding of N-acetylated Phe-tRNA(Phe), an analog of the initiator fMet-tRNA(Met), enhanced the population of complexes that could withstand high forces. The persistence length of poly(U) RNA homopolymer, modeled as a worm-like chain, was found to be 0.79 +/- 0.05 nm and the backbone elasticity was 900 +/- 140 pN, similar to values for single-stranded DNA.     

The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3).

To assess the RNA helicase activity of hepatitis C virus (HCV) nonstructural protein 3 (NS3), a polypeptide encompassing amino acids 1175 to 1657, which cover only the putative helicase domain, was expressed in Escherichia coli by a pET expression vector. The protein was purified to near homogeneity and assayed for RNA helicase activity in vitro with double-stranded RNA substrates prepared from a multiple cloning sequence and an HCV 5' nontranslated region (5'-NTR) or 3'-NTR. The enzyme acted successfully on substrates containing both 5' and 3' single-stranded regions (standard) or on substrates containing only the 3' single-stranded regions (3'/3') but failed to act on substrates containing only the 5' single-stranded regions (5'/5') or on substrates lacking the single-stranded regions (blunt). These results thus suggest 3' to 5' directionality for HCV RNA helicase activity. However, a 5'/5' substrate derived from the HCV 5'-NTR was also partially unwound by the enzyme, possibly because of unique properties inherent in the 5' single-stranded regions. Gel mobility shift analyses demonstrated that the HCV NS3 helicase could bind to either 5'- or 3'-tailed substrates but not to substrates lacking a single-stranded region, indicating that the polarity of the RNA strand to which the helicase bound was a more important enzymatic activity determinant. In addition to double-stranded RNA substrates, HCV NS3 helicase activity could displace both RNA and DNA oligonucleotides on a DNA template, suggesting that HCV NS3 too was disposed to DNA helicase activity. This study also demonstrated that RNA helicase activity was dramatically inhibited by the single-stranded polynucleotides. Taken altogether, our results indicate that the HCV NS3 helicase is unique among the RNA helicases characterized so far.