Multiple domains of EBER 1, an Epstein-Barr virus noncoding RNA, recruit human ribosomal protein L22

EBER 1, a small noncoding viral RNA abundantly expressed in all cells transformed by Epstein-Barr virus (EBV), has been shown to associate with the human ribosomal protein L22. Here we present in vitro binding studies using purified RNAs and recombinant proteins. Electrophoretic mobility-shift assays (EMSAs) show that recombinant L22 (rL22) and maltose-binding protein (MBP)-tagged L22 protein bind EBER 1 in vitro, both forming three specific protein-dependent mobility shifts. Use of a mixture of rL22 and MBP-L22 indicates that these three shifts contain one, two, or three L22 proteins per EBER 1 molecule. EMSAs performed with EBER 1 deletion constructs and EBER 1 stem-loops inserted into a nonbinding RNA, HSUR 3, identify stem-loops I, III, and IV as L22 binding sites. The existence of multiple L22 binding sites on EBER 1 inside cells is demonstrated by in vivo UV cross-linking. Our results are discussed with respect to the function of EBER 1 in EBV-infected human B cells.     

Ribosomal protein L22 inhibits regulation of cellular activities by the Epstein-Barr virus small RNA EBER-1.

Epstein-Barr virus (EBV) is a potent mitogenic and antiapoptotic agent for B lymphocytes and is associated with several different types of human tumour. The abundantly expressed small viral RNA, EBER-1, binds to the growth inhibitory and pro-apoptotic protein kinase R (PKR) and blocks activation of the latter by double-stranded RNA. Recent evidence has suggested that expression of EBER-1 alone in EBV-negative B cells promotes a tumorigenic phenotype and that this may be related to inhibition of the pro-apoptotic effects of PKR. The ribosomal protein L22 binds to EBER-1 in virus-infected cells, but the significance of this has not previously been established. We report here that L22 and PKR compete for a common binding site on EBER-1. As a result of this competition, L22 interferes with the ability of the small RNA to inhibit the activation of PKR by dsRNA. Transient expression of EBER-1 in murine embryonic fibroblasts stimulates reporter gene expression and partially reverses the inhibitory effect of PKR. However, EBER-1 is also stimulatory when transfected into PKR knockout cells, suggesting an additional, PKR-independent, mode of action of the small RNA. Expression of L22 prevents both the PKR-dependent and -independent effects of EBER-1 in vivo. These results suggest that the association of L22 with EBER-1 in EBV-infected cells can attenuate the biological effects of the viral RNA. Such effects include both the inhibition of PKR and additional mechanism(s) by which EBER-1 stimulates gene expression.     

In vitro transcription of Drosophila ribosomal genes using Drosophila RNA polymerases

Drosophila RNA polymerases I &; II were used to transcribe a recombinant bacterial plasmid containing one copy of Drosophila ribosomal DNA. Both supercoiled and relaxed, closed circular plasmids were used. With Mg⁺² as the divalent cation, enzyme I is much more active on both forms of the plasmid; the relaxed form in particular supports almost no RNA synthesis by enzyme II. When Mn⁺² is present, differences in template efficiencies are minimal. The differences observed in the absence of Mn⁺² seem to depend only on different preferences for the physical state of the template and not on recognition of specific promotor sequences, since enzyme I shows no strand selection when transcribing these plasmids.     

In vitro selection of novel RNA ligands that bind human cytomegalovirus and block viral infection

Ribonuclease-resistant RNA molecules that bind to infectious human cytomegalovirus (HCMV) were isolated in vitro from a pool of randomized sequences after 16 cycles of selection and amplification. The two ligands (L13 and L19) characterized exhibited high HCMV-binding affinity in vitro and effectively inhibited viral infection in tissue culture. Their antiviral activity was also specific as they only reacted with two different strains of HCMV but not with the related herpes simplex virus 1 and human cells. These two ligands appeared to function as antivirals by blocking viral entry. Ultraviolet (UV) crosslinking studies suggested that L13 and L19 bind to HCMV essential glycoproteins B and H, respectively. Thus, RNA ligands that bind to different surface antigens of HCMV can be simultaneously isolated by the selection procedure. Our study demonstrates the feasibility of using these RNA ligands as a research tool to identify viral proteins required for infectivity and as an antiviral agent to block viral infection.     

Interaction of the intermediate filament protein vimentin with ribosomal subunits and ribosomal RNA in vitro

If in a low ionic strength extract of Triton X-100-resistant residual cell structures derived from Ehrlich ascites tumour (EAT) cells Mg²⁺ was chelated by EDTA, vimentin became associated with unfolded ribosomal subunits. The first molecular characterization of this association has shown that (1) vimentin binds to the RNA moiety of the ribosomes, (2) vimentin has a higher affinity for unfolded small ribosomal subunits or 18S rRNA than for unfolded large ribosomal subunits or 28S rRNA, (3) the limited degradation of vimentin by the vimentin-specific, Ca²⁺-activated proteinase, with the formation of a 48 Kd breakdown product, abolishes its affinity for rRNA, (4) the association products are rather sensitive to moderate concentrations of KCl and Mg²⁺, and (5) reductive alkylation of vimentin with pyridoxal-5-phosphate and NaBH₄ has no effect on the affinity of vimentin for rRNA. Actin and tubulin do not interact with EAT cell rRNA under the above ionic conditions.     

In vitro selection and characterization of cellulose-binding RNA aptamers using isothermal amplification

We sought to create new cellulose-binding RNA aptamers for use as modular components in the engineering of complex functional nucleic acids. We designed our in vitro selection strategy to incorporate self-sustained sequence replication (3SR), which is an isothermal nucleic acid amplification protocol that allows for the rapid amplification of RNAs with little manipulation. The best performing aptamer representative was chosen for reselection and further optimization. The aptamer exhibits robust binding of cellulose in both the powdered and paper form, but did not show any significant binding of closely related polysaccharides. The minimal cellulose-binding RNA aptamer also can be grafted onto other RNAs to permit the isolation of RNAs from complex biochemical mixtures via cellulose affinity chromatography. This was demonstrated by fusing the aptamer to a glmS ribozyme sequence, and selectively eluting ribozyme cleavage products from cellulose using glucosamine 6-phosphate to activate glmS ribozyme function.     

In vitro selection of RNA against kanamycin B.

Aminoglycosides are well-known antibiotics that function by interacting with ribosomal RNA in bacteria. In order to understand the molecular details between RNA and the drug, RNA aptamer was selected against kanamycin B. After 12 cycles of selection, RNA was cloned and sequenced. Among 9 clones, sequences of three clones were identical, suggesting the selected RNA was enriched. Among the cloned RNA molecules, the triplicated RNA was the maximum binding RNA. It showed a 180 nM affinity (KD) to the cognate aminoglycoside, as measured by a surface plasmon resonance, and a competition assay using a fluorescence anisotropy technique. The affinity of the maximum binding RNA to a similar aminoglycoside, tobramycin, was much stronger than 12 nM of KD. The binding site of the aminoglycoside in the maximum binding RNA was a stem loop located at the end of the 5' region. A stem loop structural motif, found in this study, was similar to those previously reported, even though the sequences of the RNA were totally different from the known sequences of the aminoglycoside binding site of other aptamers. The present study suggests that the aminoglycoside-binding region in RNA does not have a sequence specificity, but has a shape-specific bulged stem loop, even though it has a nanomolar affinity.     

How bacterial ribosomal protein L20 assembles with 23 S ribosomal RNA and its own messenger RNA

In bacteria, the expression of ribosomal proteins is often feedback-regulated at the translational level by the binding of the protein to its own mRNA. This is the case for L20, which binds to two distinct sites of its mRNA that both resemble its binding site on 23 S rRNA. In the present work, we report an NMR analysis of the interaction between the C-terminal domain of L20 (L20C) and both its rRNA- and mRNA-binding sites. Changes in the NMR chemical shifts of the L20C backbone nuclei were used to show that the same set of residues are modified upon addition of either the rRNA or the mRNA fragments, suggesting a mimicry at the atomic level. In addition, small angle x-ray scattering experiments, performed with the rRNA fragment, demonstrated the formation of a complex made of two RNAs and two L20C molecules. A low resolution model of this complex was then calculated using (i) the rRNA/L20C structure in the 50 S context and (ii) NMR and small angle x-ray scattering results. The formation of this complex is interesting in the context of gene regulation because it suggests that translational repression could be performed by a complex of two proteins, each interacting with the two distinct L20-binding sites within the operator.     

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.     

New insights into the interaction of ribosomal protein L1 with RNA

The RNA-binding ability of ribosomal protein L1 is of profound interest, since L1 has a dual function as a ribosomal structural protein that binds rRNA and as a translational repressor that binds its own mRNA. Here, we report the crystal structure at 2.6 A resolution of ribosomal protein L1 from the bacterium Thermus thermophilus in complex with a 38 nt fragment of L1 mRNA from Methanoccocus vannielii. The conformation of RNA-bound T.thermophilus L1 differs dramatically from that of the isolated protein. Analysis of four copies of the L1-mRNA complex in the crystal has shown that domain II of the protein does not contribute to mRNA-specific binding. A detailed comparison of the protein-RNA interactions in the L1-mRNA and L1-rRNA complexes identified amino acid residues of L1 crucial for recognition of its specific targets on the both RNAs. Incorporation of the structure of bacterial L1 into a model of the Escherichia coli ribosome revealed two additional contact regions for L1 on the 23S rRNA that were not identified in previous ribosome models.     

Requirement for C-terminal extension to the RNA binding domain for efficient RNA binding by ribosomal protein L2

Ribosomal protein L2 is a primary 23S rRNA binding protein in the large ribosomal subunit. We examined the contribution of the N- and C-terminal regions of Bacillus stearothermophilus L2 (BstL2) to the 23S rRNA binding activity. The mutant desN, in which the N-terminal 59 residues of BstL2 were deleted, bound to the 23S rRNA fragment to the same extent as wild type BstL2, but the mutation desC, in which the C-terminal 74 amino acid residues were deleted, abolished the binding activity. These observations indicated that the C-terminal region is involved in 23S rRNA binding. Subsequent deletion analysis of the C-terminal region found that the C-terminal 70 amino acids are required for efficient 23S rRNA binding by BstL2. Furthermore, the surface plasmon resonance analysis indicated that successive truncations of the C-terminal residues increased the dissociation rate constants, while they had little influence on association rate constants. The result indicated that reduced affinities of the C-terminal deletion mutants were due only to higher dissociation rate constants, suggesting that the C-terminal region primarily functions by stabilizing the protein L2-23S rRNA complex.     

The binding site for ribosomal protein L2 within 23S ribosomal RNA of Escherichia coli.

Ribosomal protein L2 from Escherichia coli binds to and protects from nuclease digestion a substantial portion of 'domain IV' of 23S rRNA. In particular, oligonucleotides derived from the sequence 1757-1935 were isolated and shown to rebind specifically to protein L2 in vitro. Other L2-protected oligonucleotides, also derived from domain IV (i.e. from residues 1955-2010) did not rebind to protein L2 in vitro nor did others derived from domain I. Given that protein L2 is widely believed to be located in the peptidyl transferase centre of the 50S ribosomal subunit, these data suggest that domain IV of 23S rRNA is also present in that active site of the ribosomal enzyme.     

Mapping the ribosomal RNA neighborhood of protein L11 by directed hydroxyl radical probing.

Ribosomal protein L11 is a highly conserved protein that has been implicated in binding of elongation factors to ribosomes and associated GTP hydrolysis. Here, we have analyzed the ribosomal RNA neighborhood of Escherichia coli L11 in 50 S subunits by directed hydroxyl radical probing from Fe(II) tethered to five engineered cysteine residues at positions 19, 84, 85, 92 and 116 via the linker 1-(p -bromoacetamidobenzyl)-EDTA. Correct assembly of the L11 derivatives was analyzed by incorporating the modified proteins into 50 S subunits isolated from an E. coli strain that lacks L11 and testing for previously characterized L11-dependent footprints in domain II of 23 S rRNA. Hydroxyl radicals were generated from Fe(II) tethered to L11 and sites of cleavage in the ribosomal RNA were detected by primer extension. Strong cleavages were detected within the previously described binding site of L11, in the 1100 region of 23 S rRNA. Moreover, Fe(II) tethered to position 19 in L11 targeted the backbone of the sarcin loop in domain VI while probing from position 92 cleaved the backbone around bases 900 and 2470 in domains II and V, respectively. Fe(II) tethered to positions 84, 85 and 92 also generated cleavages in 5 S rRNA around helix II. These data provide new information about the positions of specific features of 23 S rRNA and 5 S rRNA relative to protein L11 in the 50 S subunit and show that L11 is near highly conserved elements of the rRNA that have been implicated in binding of tRNA and elongation factors to the ribosome.     

In vitro selection of enzymatically active lipase variants from phage libraries using a mechanism-based inhibitor

The 'detergent lipase' Lipolase, from Thermomyces lanuginosa was subjected to a combinatorial protein engineering/phage display approach with the aim of identifying new enzyme variants with improved characteristics in the presence of detergents. First it was demonstrated that wild-type Lipolase could be produced in Escherichia coli retaining full activity and be displayed as an active enzyme fused to coat protein 3 on E. coli phage M13. A phagemid library designed to result in approximately two to three mutations per lipase gene was then constructed. Nine amino acids located in two regions close to the active site were targeted for randomization. Selections using a mechanism-based biotinylated inhibitor showed that phages displaying Lipolase could be specifically enriched from a population of control phages. Selections on a library phage stock in the presence of inhibitor and a commercial powder detergent resulted in a step-wise increase in the proportion of active clones. Analysis of 84 active clones revealed that they all expressed lipase activity, but with lower activities than that of a wild-type Lipolase-producing clone. In six of the seven most active clones a wild-type serine at position 83 had been replaced by threonine, a substitution known to alter the substrate chain length preference of Lipolase variants. Furthermore, the selection had enriched enzyme variants with a high degree of conservatism in one of the variegated regions, suggesting that this region is important for enzymatic activity and that the designed selection procedure was relevant. The selected variants contained primarily basic amino acid residues within the other variegated region. Taken together, the described results show that selection protocols based on enzymatic activity can be designed for this enzyme class which should be of importance for future protein engineering attempts.     

Characterization of the Escherichia coli 23S Ribosomal RNA region associated with ribosomal protein L1. Evidence for homologies with the 5''-end region of the L11 operon.

The results previously obtained upon studying the L1-23S RNA complex by the fingerprint technique have been reexamined in the light of new data on 23S RNA primary structure. The 23S RNA region that remains associated with the L1 ribosomal protein after RNase digestion of the synthetic complex lies between nucleotides 2067 and 2235 from the 5'-end of the molecule. This region contains a m7G near to the 5'-end and possesses a high degree of mutability in E. coli. Three different sequences were observed in E. coli MRE 600. All three sequences differ in two positions relative to the corresponding sequence in rrnB cistron from E. coli K12. Striking homology is observed between the 23S RNA region associated with protein L1 and the 5'-part of L11 operon. This observation supports the model of feedback regulation of r-proteins synthesis proposed by Yates et al. (PNAS, 77, 1837) and strongly suggests that the region of 23S RNA located between positions 2155 and 2202 is essential for the binding of protein L1.     

Studies on the RNA and protein binding sites of the E. coli ribosomal protein L10.

We have used modification of specific amino acid residues in the E. coli ribosomal protein L10 as a tool to study its interactions with another ribosomal protein, L7/L12, as well as with ribosomal core particles and with 23S RNA. The ribosome and RNA binding capability of L10 was found to be inhibited by modification of one more of its arginine residues. This treatment does not affect the ability of L10 to bind four molecules of L7/L12 in a L7/L12-L10 complex. Our results support the view that L10's role in promoting the L7/L12-ribosome association is due primarily to its ability to bind to both 23S RNA and L7/L12 simultaneously.