mRNA Binding Sites Research Articles

Identification of a Binding Site for ASF/SF2 on an RNA Fragment Derived from the Hepatitis delta Virus Genome

The hepatitis delta virus (HDV) is a small (∼1700 nucleotides) RNA pathogen which encodes only one open reading frame. Consequently, HDV is dependent on host proteins to replicate its RNA genome. Recently, we reported that ASF/SF2 binds directly and specifically to an HDV-derived RNA fragment which has RNA polymerase II promoter activity. Here, we localized the binding site of ASF/SF2 on the HDV RNA fragment by performing binding experiments using purified recombinant ASF/SF2 combined with deletion analysis and site-directed mutagenesis. In addition, we investigated the requirement of ASF/SF2 for HDV RNA replication using RNAi-mediated knock-down of ASF/SF2 in 293 cells replicating HDV RNA. Overall, our results indicate that ASF/SF2 binds to a purine-rich region distant from both the previously published initiation site of HDV mRNA transcription and binding site of RNAP II, and suggest that this protein is not involved in HDV replication in the cellular system used.

HomoTarget: A new algorithm for prediction of microRNA targets in Homo sapiens

MiRNAs play an essential role in the networks of gene regulation by inhibiting the translation of target mRNAs. Several computational approaches have been proposed for the prediction of miRNA target-genes. Reports reveal a large fraction of under-predicted or falsely predicted target genes. Thus, there is an imperative need to develop a computational method by which the target mRNAs of existing miRNAs can be correctly identified. In this study, combined pattern recognition neural network (PRNN) and principle component analysis (PCA) architecture has been proposed in order to model the complicated relationship between miRNAs and their target mRNAs in humans. The results of several types of intelligent classifiers and our proposed model were compared, showing that our algorithm outperformed them with higher sensitivity and specificity. Using the recent release of the mirBase database to find potential targets of miRNAs, this model incorporated twelve structural, thermodynamic and positional features of miRNA:mRNA binding sites to select target candidates.

A complex network of small non‐coding RNAs regulate motility in Escherichia coli

Small Hfq-dependent non-coding regulatory RNAs (sRNAs) that alter mRNA stability and expression by pairing with target mRNAs have increasingly been shown to be important in influencing the behaviour of bacteria. In Escherichia coli, flhD and flhC, which encode the master regulator of flagellar synthesis, are co-transcribed from a promoter that is regulated by multiple transcription factors that respond to different environmental cues. Here, we show that the 5' untranslated region (5' UTR) of the flhDC mRNA also serves as a hub to integrate additional environmental cues into the decision to make flagella. Four sRNAs, ArcZ, OmrA, OmrB and OxyS, negatively regulated and one sRNA, McaS, positively regulated motility and flhDC expression by base-pairing with the 5' UTR of this mRNA. Another sRNA, MicA, positively regulated motility independent of regulation of flhDC. Furthermore, we demonstrate that the regulation of motility by the ArcB/A two component system is in part due to its regulation of ArcZ. flhDC is the first mRNA that has been shown to be both positively and negatively regulated by direct pairing to sRNAs. Moreover, both positive regulation by McaS and negative regulation by ArcZ require the same binding site in the flhDC mRNA.

MiR-429 regulation of osmotic stress transcription factor 1 (OSTF1) in tilapia during osmotic stress

The Nile tilapia represents an excellent model for osmoregulation study. Osmotic stress transcription factor 1 (OSTF1) identified in tilapia gill epithelium is a critical element of osmosensory signal transduction by means of transcriptional regulation. Thus, tight regulation of OSTF1 level is necessary for tilapia osmotic adaptation. microRNAs (miRNAs), have emerged as a crucial regulator of gene expression at post-transcriptional level. We reasoned that OSTF1 expression could be regulated by miRNAs. By bioinformatics analysis, we identified a putative miR-429 binding site in the OSTF1 mRNA. Interestingly, miR-429 is down-regulated in tilapia upon osmotic stress, consistent with OSTF1 protein up-regulation. miR-429 directly regulates OSTF1 expression by targeting its 3′-UTR, and inhibition of miR-429 substantially increases OSTF1 level in vivo. Moreover, miR-429 loss of function could influence the regulation of plasma osmolality and ion concentration responding to osmotic stress. Taken together, miR-429 is an endogenous regulator of OSTF1 expression, which participates in a regulatory circuit that allows rapid gene program transitions in response to osmotic stress.

Analysis of MDM2 and MDM4 Single Nucleotide Polymorphisms, mRNA Splicing and Protein Expression in Retinoblastoma

Retinoblastoma is a childhood cancer of the developing retina that begins in utero and is diagnosed in the first years of life. Biallelic RB1 gene inactivation is the initiating genetic lesion in retinoblastoma. The p53 gene is intact in human retinoblastoma but the pathway is believed to be suppressed by increased expression of MDM4 (MDMX) and MDM2. Here we quantify the expression of MDM4 and MDM2 mRNA and protein in human fetal retinae, primary retinoblastomas, retinoblastoma cell lines and several independent orthotopic retinoblastoma xenografts. We found that MDM4 is the major p53 antagonist expressed in retinoblastoma and in the developing human retina. We also discovered that MDM4 protein steady state levels are much higher in retinoblastoma than in human fetal retinae. This increase would not have been predicted based on the mRNA levels. We explored several possible post-transcriptional mechanisms that may contribute to the elevated levels of MDM4 protein. A proportion of MDM4 transcripts are alternatively spliced to produce protein products that are reported to be more stable and oncogenic. We also discovered that a microRNA predicted to target MDM4 (miR191) was downregulated in retinoblastoma relative to human fetal retinae and a subset of samples had somatic mutations that eliminated the miR-191 binding site in the MDM4 mRNA. Taken together, these data suggest that post-transcriptional mechanisms may contribute to stabilization of the MDM4 protein in retinoblastoma.

Glycyl-tRNA synthetase specifically binds to the poliovirus IRES to activate translation initiation

Adaptation to the host cell environment to efficiently take-over the host cell's machinery is crucial in particular for small RNA viruses like picornaviruses that come with only small RNA genomes and replicate exclusively in the cytosol. Their Internal Ribosome Entry Site (IRES) elements are specific RNA structures that facilitate the 5′ end-independent internal initiation of translation both under normal conditions and when the cap-dependent host protein synthesis is shut-down in infected cells. A longstanding issue is which host factors play a major role in this internal initiation. Here, we show that the functionally most important domain V of the poliovirus IRES uses tRNAGly anticodon stem–loop mimicry to recruit glycyl-tRNA synthetase (GARS) to the apical part of domain V, adjacent to the binding site of the key initiation factor eIF4G. The binding of GARS promotes the accommodation of the initiation region of the IRES in the mRNA binding site of the ribosome, thereby greatly enhancing the activity of the IRES at the step of the 48S initiation complex formation. Moonlighting functions of GARS that may be additionally needed for other events of the virus–host cell interaction are discussed.

MiR‐365: a mechanosensitive microRNA stimulates chondrocyte differentiation through targeting histone deacetylase 4

Mechanical stress plays an essential role in tissue development and remodeling. In this study, we determined the role of microRNA in chondrocyte mechanotransduction. Using microarray, we identified miR-365 as a mechanoresponsive microRNA in parallel to mechanical induction of Indian hedgehog (Ihh) in primary chicken chondrocytes cultured in 3-dimensional collagen scaffoldings under cyclic loading (1 Hz, 5% elongation). Interestingly, expression of miR-365 is elevated in the prehypertrophic zone of the growth plate, coinciding with the Ihh expression region in vivo. MiR-365 significantly stimulates chondrocyte proliferation and differentiation. MiR-365 increases expression of Ihh and the hypertrophic marker type X collagen, whereas anti-miR-365 inhibits the expression of these genes. We identified histone deacetylase 4 (HDAC4), an inhibitor of chondrocyte hypertrophy, as a target of miR-365. MiR-365 inhibits both endogenous HDAC4 protein levels as well as the activity of a reporter gene bearing the 3'-untranslated region of HDAC4 mRNA. Conversely, inhibition of endogenous miR-365 relieves the repression of HDAC4. Mutation of the miR-365 binding site in HDAC4 mRNA abolishes miR-365-mediated repression of the reporter gene activity. Overexpression of HDAC4 reverses miR-365 stimulation of chondrocyte differentiation markers including Ihh, Col X, and Runx2. Moreover, inhibition of miR-365 abolishes mechanical stimulation of chondrocyte differentiation. Taken together, miR-365 is the first identified mechanically responsive microRNA that regulates chondrocyte differentiation via directly targeting HDAC4.

Differentiated prostatic antigen expression in LNCaP cells following treatment with bispecific antisense oligonucleotides directed against BCL-2 and EGFR

Antisense oligonucleotides (oligos) have been administered against in vivo and in vitro prostate cancer models employing LNCaP and PC-3 cell lines. While most oligos consist of a single mRNA binding site targeting a single gene product or those with sequence homology, our lab has developed bispecific oligos directed toward two unrelated proteins. In LNCaP cells, we initially identified bispecifics that increased the expression of prostate-specific membrane antigen (PSMA) while not affecting secreted prostate-specific antigen (PSA). We postulated that surface antigen expression is increased by bispecifics able to form double-stranded regions, acting as interferon (IFN-γ) inducers. In other systems, when induced, IFN-γ promotes cell surface antigen expression, including HLA and receptors for tumor necrosis factor. To test this hypothesis, we measured the effect of oligo treatment on both IFN-γ induction and the expression of another secreted product of differentiated prostate cells, prostatic acid phosphatase (PAP). This study initially evaluated the inhibition of in vitro propagating LNCaP cells employing mono- and bispecific oligos directed against bcl-2 (the second bispecific binding site was against the epidermal growth factor receptor). Employing RT-PCR, the expression of non-targeted proteins encoded by mRNA for PSMA, PSA, PAP, and IFN-γ was subsequently valuated. When LNCaP prostate tumor cells were incubated with oligos and compared to lipofectin-containing controls significant growth inhibition resulted. Employing RT-PCR, the levels of mRNA encoding PSMA were unexpectedly found to be elevated following treatment with the bispecific oligos but not with a monospecific directed solely against bcl-2. No differences were detected in mRNA levels encoding PSA following treatment with either oligo type. IFN-γ was significantly induced only by bispecific oligos, and PAP expression was similar to PSA. These data support the hypothesis that double strand-forming bispecific oligos induce IFN-γ that enhances cell surface PSMA expression. Expression of tumor-associated surface antigens could increase their recognition and targeting by immunologic defense mechanisms and increase the effectiveness of tumor vaccines.

GHRH antagonist causes DNA damage leading to p21 mediated cell cycle arrest and apoptosis in human colon cancer cells

We investigated the mechanisms of inhibitory effect of growth hormone-releasing hormone (GHRH) antagonist JMR-132 on the growth of HT29, HCT-116 and HCT-15 human colon cancer cells in vitro and in vivo. High-affinity binding sites for GHRH and mRNA for GHRH and splice variant-1 (SV1) of the GHRH receptor were found in all three cell lines tested. Proliferation of HT-29, HCT-116 and HCT-15 cells was significantly inhibited in vitro by JMR-132. Time course studies revealed that the treatment of human HCT-116 colon cancer cells with 10μM GHRH antagonist JMR-132 causes a significant DNA damage as shown by an increase in olive tail moment (OTM) and loss of inner mitochondrial membrane potential (∆Ψm). Western blotting demonstrated a time-dependent increase in protein levels of phospho-p53 (Ser46), Bax, cleaved caspase-9, -3, cleavage of poly(ADP-ribose)polymerase (PARP) and a decrease in Bcl-2 levels. An augmentation in cell cycle checkpoint protein p21Waf1/Cip1 was accompanied by a cell cycle arrest in S-phase. DNA fragmentation visualized by the comet assay and the number of apoptotic cells increased time dependently as determined by flow cytometric annexinV and PI staining assays. In vivo, JMR-132 decreased the volume of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic mice up to 75% (p

CAMP-Mediated Stimulation of Tyrosine Hydroxylase mRNA Translation Is Mediated by Polypyrimidine-Rich Sequences within Its 3′-Untranslated Region and Poly(C)-Binding Protein 2

Tyrosine hydroxylase (TH) plays a critical role in maintaining the appropriate concentrations of catecholamine neurotransmitters in brain and periphery, particularly during long-term stress, long-term drug treatment, or neurodegenerative diseases. Its expression is controlled by both transcriptional and post-transcriptional mechanisms. In a previous report, we showed that treatment of rat midbrain slice explant cultures or mouse MN9D cells with cAMP analog or forskolin leads to induction of TH protein without concomitant induction of TH mRNA. We further showed that cAMP activates mechanisms that regulate TH mRNA translation via cis-acting sequences within its 3'-untranslated region (UTR). In the present report, we extend these studies to show that MN9D cytoplasmic proteins bind to the same TH mRNA 3'-UTR domain that is required for the cAMP response. RNase T1 mapping demonstrates binding of proteins to a 27-nucleotide polypyrimidine-rich sequence within this domain. A specific mutation within the polypyrimidine-rich sequence inhibits protein binding and cAMP-mediated translational activation. UV-cross-linking studies identify a approximately 44-kDa protein as a major TH mRNA 3'-UTR binding factor, and cAMP induces the 40- to 42-kDa poly(C)-binding protein-2 (PCBP2) in MN9D cells. We show that PCBP2 binds to the TH mRNA 3'-UTR domain that participates in the cAMP response. Overexpression of PCBP2 induces TH protein without concomitant induction of TH mRNA. These results support a model in which cAMP induces PCBP2, leading to increased interaction with its cognate polypyrimidine binding site in the TH mRNA 3'-UTR. This increased interaction presumably plays a role in the activation of TH mRNA translation by cAMP in dopaminergic neurons.

Sequence-specific bacterial growth inhibition by peptide nucleic acid targeted to the mRNA binding site of 16S rRNA

Peptide nucleic acid (PNA) targeted to the functional domains of 23S rRNA can inhibit translation and cell growth. However, effective inhibition of translation and cell growth using 16S rRNA-targeted PNA has still not been achieved. Here, we report that PNA targeted to the functional site of 16S rRNA could inhibit both gene expression in vitro and bacterial growth in pure culture with sequence specificity. We used 10-mer PNAs conjugated with a cell-penetrating peptide, which targeted the mRNA binding site at the 3' end of 16S rRNA. Using 0.6 microM of the peptide-PNAs, cell-free ss-galactosidase production decreased by 50%, whereas peptide-PNAs with one or two mismatches to the target sequence showed much weaker inhibition effects. To determine the growth inhibition and bactericidal effects of the peptide-PNA conjugate, we performed OD measurement and viable cell counting. We observed dose- and sequence-dependent inhibition of cell growth and bactericidal effects. These growth inhibitory effects are observed both in the Gram-negative bacterium of Escherichia coli and the Gram-positive bacteria Bacillus subtilis and Corynebacterium efficiens, although inhibitory concentrations were different for each bacterial species. These results present possibilities for 16S rRNA sequence-based specific bacterial growth inhibition using a peptide-PNA conjugate.

Treatment of prostate and breast tumors employing mono- and bi-specific antisense oligonucleotides targeting apoptosis inhibitory proteins clusterin and bcl-2

Antisense oligonucleotides (oligos) have demonstrated their efficacy in inhibiting the growth of prostate and breast tumor cells. Previous studies employed first generation, phosphorothioated, cDNA oligos synthesized complimentary to mRNA encoding transforming growth factor-alpha (TGF-alpha), epidermal growth factor receptor (EGFR), the anti-apoptosis protein bcl-2, and the androgen receptor (AR). In an effort to construct oligos with greater than one mRNA binding site, bi-specifics have been developed which target combinations of the above proteins, and these have been shown at least as effective as the mono-specific oligos from which their sequences were derived. While all bi-specifics have inhibitory effects, which can be enhanced by the combined administration of an additional chemotherapeutic agent, those bi-specifics which target bcl-2 and EGFR were reported to be the most effective. The experiments presented here are an effort to evaluate a new group of bi-specifics whose targets include the chaperone protein clusterin, whose expression is up regulated in many tumors and activity is known to inhibit apoptosis. Of particular interest were those bi-specifics constructed to target both clusterin and bcl-2 (also an apoptosis inhibitory protein). Cell lines targeted included both prostate LNCaP and PC-3, as well as the breast derived MCF-7. In order to identify agents which enhance oligo activity, but contribute less toxicity, oligos were tested both alone and in combination with either the immune inhibitor Rapamycin, or the chemotherapeutic (and more toxic) Taxol. Results indicate that bi-specifics targeting clusterin are statistically effective, and are similarly enhanced by Rapamycin, or Taxol. When bi-specifics including clusterin as a target, were tested against LNCaP and MCF-7 cells, the level of activity was intermediate between that of the mono-specific compounds tested separately. In experiments which compared both, bi-specifics which included a target for clusterin had inhibitory activity similar to the previously described bi-specifics directed towards bcl-2 and EGFR.

Multigene targeting of signal transduction pathways for the treatment of breast and prostate tumors

Previous studies have demonstrated that monospecific antisense oligonucleotides (oligos) directed against mRNA encoding proteins associated with tumor growth, death, and survival are efficacious against breast and prostate tumors. Targeted proteins, associated with different signal transduction pathways, have included transforming growth factor-alpha [TGF-alpha (MR(1))], its binding site the epidermal growth factor receptor [EGFR (MR(2))] sharing sequence homology to the breast cancer prognostic marker Her-2/neu, an apoptosis inhibiting protein [bcl-2 (MR(4))], and the androgen receptor [AR (MR(5))]. In attempts to enhance antisense therapy, recent reports describe how two of the binding sites mentioned above can be sequentially placed within a single complementary (bispecific) strand and administered either in the presence or absence of additional therapeutic agents. When tested against breast and prostate tumor cell lines specific differences were noted: MCF-7 breast cancer cells were more receptive to the inhibitory effects of monospecific oligos, whereas PC-3 and LNCaP prostate cells were particularly responsive to bispecifics. In an effort to identify agents which enhance the activity of oligos and which possess less toxicity than traditionally employed chemotherapeutics, Rapamycin, an immunosuppressive agent known to regulate tumor growth and signal transduction mediated by the mTOR receptor, is compared to paclitaxel in combination therapy employing monospecific or bispecific oligos. Bispecifics were constructed recognizing the binding sites for TGF-alpha and EGFR mRNA [TGF-alpha/EGFR (MR(12)) and EGFR/TGF-alpha (MR(21))]; another pair recognized binding sites for EGFR and bcl-2 [EGFR/bcl-2 (MR(24)) and bcl-2/EGFR (MR(42))]; while a third pair employed only against the LNCaP prostate cell line recognized bcl-2 and the androgen receptor [bcl-2/AR (MR4(45)) and AR/bcl-2 (MR(54))]. Oligo pairs differ in their 5'-3' linear binding site orientations, and were tested in vitro against MCF-7 breast and PC-3 and LNCaP prostate tumor cell lines. Following cell attachment, incubations were done for 2 days with the agents followed by 2 days in their absence. Five experiments evaluated the effect of monospecific or bispecific antisense oligos in combination with an LD(50) dosage of either Rapamycin or paclitaxel and led to the conclusion that although these agents act via different mechanisms, they are comparable in effectiveness.

The G-quartet containing FMRP binding site in FMR1 mRNA is a potent exonic splicing enhancer

The fragile X mental retardation protein (FMRP) is a RNA-binding protein proposed to post-transcriptionally regulate the expression of genes important for neuronal development and synaptic plasticity. We previously demonstrated that FMRP binds to its own FMR1 mRNA via a guanine-quartet (G-quartet) RNA motif. However, the functional effect of this binding on FMR1 expression was not established. In this work, we characterized the FMRP binding site (FBS) within the FMR1 mRNA by a site directed mutagenesis approach and we investigated its importance for FMR1 expression. We show that the FBS in the FMR1 mRNA adopts two alternative G-quartet structures to which FMRP can equally bind. While FMRP binding to mRNAs is generally proposed to induce translational regulation, we found that mutations in the FMR1 mRNA suppressing binding to FMRP do not affect its translation in cellular models. We show instead that the FBS is a potent exonic splicing enhancer in a minigene system. Furthermore, FMR1 alternative splicing is affected by the intracellular level of FMRP. These data suggest that the G-quartet motif present in the FMR1 mRNA can act as a control element of its alternative splicing in a negative autoregulatory loop.

Neuropeptide FF2 receptor distribution in the human brain: An immunohistochemical study

Human neuropeptide FF2 (hFF2) receptor has been postulated to mediate central autonomic regulation by virtue of its ability to bind with high affinity to many amidated neuropeptides. In the present immunohistochemical study, we identified hFF2 positive neurons in the forebrain and medulla oblongata of individuals, who died suddenly of mechanical trauma or hypothermia. Morphologically, these neurons demonstrated features identified with both projection neurons and interneurons. In the forebrain, the highest density of hFF2 expressing neurons was observed in the anterior amygdaloid area and dorsomedial hypothalamic nucleus, especially in its caudal part. A lesser density of hFF2 neurons was identified in the ventromedial hypothalamic nucleus, lateral and posterior hypothalamic areas whereas few cells were visualized in the paraventricular hypothalamic nucleus, perifornical nucleus, horizontal limb of the diagonal band, ventral division of the bed nucleus of the stria terminalis, nucleus basalis of Meynert and ventral tegmental area. In the medulla, significant numbers of hFF2 neurons were observed in the dorsal motor nucleus of vagus and to a lesser extent in the area of catecholaminergic cell groups, A1/C1. These data provide first immunohistochemical evidence of hFF2 localization in the human brain, which is consistent with that reported for tissue distribution of FF2 mRNA and FF2 binding sites within the brain of a variety of mammalian species. The distribution of hFF2 may help in identifying the role of amidated neuropeptides in the human brain within the context of central autonomic and neuroendocrine regulation.

Interactions of elongation factor EF‐P with the Escherichia coli ribosome

EF-P (eubacterial elongation factor P) is a highly conserved protein essential for protein synthesis. We report that EF-P protects 16S rRNA near the G526 streptomycin and the S12 and mRNA binding sites (30S T-site). EF-P also protects domain V of the 23S rRNA proximal to the A-site (50S T-site) and more strongly the A-site of 70S ribosomes. We suggest that EF-P: (a) may play a role in translational fidelity and (b) prevents entry of fMet-tRNA into the A-site enabling it to bind to the 50S P-site. We also report that EF-P promotes a ribosome-dependent accommodation of fMet-tRNA into the 70S P-site.

Molecular switch in tandem winged-helix motifs of elongation factor SelB

Elongation factor SelB is responsible for cotranslational incorporation of the 21(st) amino acid selenocysteine (Sec) into proteins. UGA stop codon is recoded as a Sec codon in presence of a downstream mRNA hairpin. Prokaryotic SelB has EF-Tu-like N-terminal domains and a C-terminal extension containing four tandem winged-helix motifs (WH1-4). The C-terminal extension recognizes the mRNA hairpin. Crystal structures of the Escherichia coli WH3/4 domains and WH1-4 domains from Moorella thermoacetica each bound to mRNA were determined. These structures provide insights into how the molecular switch that may allow communication between tRNA and mRNA binding sites is formed and illustrate how RNA acts as an activator of the switch. The structures show that tandem WH motifs not only provide an excellent scaffold for RNA binding but can also have an active role in the function of protein-RNA complexes.

Protein S3 in the human 80S ribosome adjoins mRNA 3′ of the A-site codon

The protein environment of mRNA 3' of the A-site codon (the decoding site) in the human 80S ribosome was studied using a set of oligoribonucleotide derivatives bearing a UUU triplet at the 5'-end and a perfluoroarylazide group at one of the nucleotide residues at the 3'-end of this triplet. Analogues of mRNA were phased into the ribosome using binding at the tRNAPhe P-site, which recognizes the UUU codon. Mild UV irradiation of ribosome complexes with tRNAPhe and mRNA analogues resulted in the predominant crosslinking of the analogues with the 40S subunit components, mainly with proteins and, to a lesser extent, with rRNA. Among the 40S subunit ribosomal proteins, the S3 protein was the main target for modification in all cases. In addition, minor crosslinking with the S2 protein was observed. The crosslinking with the S3 and S2 proteins occurred both in triple complexes and in the absence of tRNA. Within triple complexes, crosslinking with S15 protein was also found, its efficiency considerably falling when the modified nucleotide was moved from positions +5 to +12 relative to the first codon nucleotide in the P-site. In some cases, crosslinking with the S30 protein was observed, it was most efficient for the derivative containing a photoreactive group at the +7 adenosine residue. The results indicate that the S3 protein in the human ribosome plays a key role in the formation of the mRNA binding site 3' of the codon in the decoding site.

Combination chemotherapy employing bispecific antisense oligonucleotides having binding sites directed against an autocrine regulated growth pathway and bcl-2 for the treatment of prostate tumors

In previous studies we demonstrated that antisense oligonucleotides (oligos) against transforming growth factor-alpha (TGF-alpha [MR1]), its binding site the epidermal growth factor receptor (EGFR [MR2]), and the anti-apoptosis protein bcl-2 (MR4) are efficacious against prostate tumors. In recent reports we also describe how two of these mRNA directed binding sites can be synthesized sequentially within a single linear complementary strand and administered either in the presence or absence of additional therapeutic agents. In these continuing experiments "bispecific" oligo pairs were further evaluated in the presence or absence of Cytoxan, Taxol, or DES. One oligo pair recognized the binding sites for TGF-alpha and EGFR mRNA (TGF-alpha/EGFR [MR12] and EGFR/TGF-alpha [MR21]); another pair recognized binding sites for EGFR and bcl-2 (EGFR/bcl-2 [MR24] and bcl-2/EGFR [MR42]). Oligo pairs differ in their linear 5' to 3' binding site orientations, and were tested in vitro against PC-3 and LNCaP prostate tumor cell lines. Following cell attachment, incubations were for 2 days with the agents followed by 2 days in their absence. When tested against PC-3 cells and combined with LD50 Cytoxan, MR2, MR4, MR24, MR42 significantly inhibited 47.3, 45.7, 68.3, and 64.9%; with LD50 Taxol MR2, MR4, MR24, MR42 significantly inhibited 49.8, 45.8, 64.1, and 59.2%; and with LD50 DES MR2, MR4, MR24, MR42 significantly inhibited 66.6, 67.6, 64.3, and 67.2% respectively. Each agent significantly increased the inhibition produced by either oligo alone.LNCaP cells were also incubated with mono- and bispecific oligos in either the presence or absence of chemotherapeutics. MR2, MR4, MR24, MR42 produced significant inhibitions of 57.4, 58.4, 69.4, and 68.6% with LD50 Cytoxan; 70.4, 70.1, 73.6, and 74.0% with LD50 Taxol; and 49.8, 50.1, 59.6, and 53.9%, respectively with LD50 DES.A complete PC-3 experiment compared MR1, MR2, MR4, MR12, MR21, MR24 and MR42, in the presence of LD50 Cytoxan. Each oligo combined with Cytoxan significantly inhibited: MR1 by 51.0, MR2 by 55.0, MR4 by 58.0; MR12 by 56.0; MR21 by 61.1, MR24 by 65.5 and MR42 by 66.0%. Bispecifics directed against two different pathways, MR24, and MR42 were the most effective.A complete LNCaP experiment compared the same series of oligos also in the presence of LD50 Cytoxan. Each oligo combined with Cytoxan significantly inhibited: MR1 by 49.0, MR2 by 50.0, MR4 by 53.0; MR12 by 52.0; MR21 by 58.6, MR24 by 53.9 and MR42 by 58.0%.

Structural Insight into a Molecular Switch in Tandem Winged-helix Motifs from Elongation Factor SelB

Elongation factor SelB is responsible for co-translational incorporation of selenocysteine (Sec) into proteins. The UGA stop codon is recoded as a Sec codon in the presence of a downstream mRNA hairpin. In prokaryotes, in addition to the EF-Tu-like N-terminal domains, a C-terminal extension containing four tandem winged-helix motifs (WH1-4) recognizes the mRNA hairpin. The 2.3–Å resolution crystal structure of the Escherichia coli WH3/4 domains bound to mRNA with mutagenesis data reveal that the two WH motifs use the same structural elements to bind RNA. The structure together with the 2.6–Å resolution structure of the WH1-4 domains from Moorella thermoacetica bound to RNA revealed that a salt bridge connecting WH2 to WH3 modules is disrupted upon mRNA binding. The results provide a structural basis for the molecular switch that may allow communication between tRNA and mRNA binding sites and illustrate how RNA acts as an activator of the switch. The structures show that tandem WH motifs not only provide an excellent scaffold for RNA binding but can also have an active role in the function of protein–RNA complexes.