The organization of RNA contacts by PTB for regulation of FAS splicing.

Post-transcriptional steps of gene expression are regulated by RNA binding proteins. Major progress has been made in characterizing RNA-protein interactions, from high resolution structures to transcriptome-wide profiling. Due to the inherent technical challenges, less attention has been paid to the way in which proteins with multiple RNA binding domains engage with target RNAs. We have investigated how the four RNA recognition motif (RRM) domains of Polypyrimidine tract binding (PTB) protein, a major splicing regulator, interact with FAS pre-mRNA under conditions in which PTB represses FAS exon 6 splicing. A combination of tethered hydroxyl radical probing, targeted inactivation of individual RRMs and single molecule analyses revealed an unequal division of labour between the four RRMs of PTB. RNA binding by RRM4 is the most important for function despite the low intrinsic binding specificity and the complete lack of effect of disrupting individual RRM4 contact points on the RNA. The ordered RRM3-4 di-domain packing provides an extended binding surface for RNA interacting at RRM4, via basic residues in the preceding linker. Our results illustrate how multiple alternative low-specificity binding configurations of RRM4 are consistent with repressor function as long as the overall ribonucleoprotein architecture provided by appropriate di-domain packing is maintained.

The helicase-primase complex as a target for effective herpesvirus antivirals.

Herpes simplex virus and varicella-zoster virus have been treated for more that half a century using nucleoside analogues. However, there is still an unmet clinical need for improved herpes antivirals. The successful compounds, acyclovir; penciclovir and their orally bioavailable prodrugs valaciclovir and famciclovir, ultimately block virus replication by inhibiting virus-specific DNA-polymerase. The helicase-primase (HP) complex offers a distinctly different target for specific inhibition of virus DNA synthesis. This review describes the synthetic programmes that have already led to two HP-inhibitors (HPI) that have commenced clinical trials in man. One of these (known as AIC 316) continues in clinical development to date. The specificity of HPI is reflected by the ability to select drug-resistant mutants. The role of HP-antiviral resistance will be considered and how the study of cross--resistance among mutants already shows subtle differences between compounds in this respect. The impact of resistance on the drug development in the clinic will also be considered. Finally, herpesvirus latency remains as the most important barrier to a therapeutic cure. Whether or not helicase primase inhibitors alone or in combination with nucleoside analogues can impact on this elusive goal remains to be seen.

Defining the roles and interactions of PTB.

PTB (polypyrimidine tract-binding protein) is an abundant and widely expressed RNA-binding protein with four RRM (RNA recognition motif) domains. PTB is involved in numerous post-transcriptional steps in gene expression in both the nucleus and cytoplasm, but has been best characterized as a regulatory repressor of some ASEs (alternative splicing events), and as an activator of translation driven by IRESs (internal ribosome entry segments). We have used a variety of approaches to characterize the activities of PTB and its molecular interactions with RNA substrates and protein partners. Using splice-sensitive microarrays we found that PTB acts not only as a splicing repressor but also as an activator, and that these two activities are determined by the location at which PTB binds relative to target exons. We have identified minimal splicing repressor and activator domains, and have determined high resolution structures of the second RRM domain of PTB binding to peptide motifs from the co-repressor protein Raver1. Using single-molecule techniques we have determined the stoichiometry of PTB binding to a regulated splicing substrate in whole nuclear extracts. Finally, we have used tethered hydroxyl radical probing to determine the locations on viral IRESs at which each of the four RRM domains bind. We are now combining tethered probing with single molecule analyses to gain a detailed understanding of how PTB interacts with pre-mRNA substrates to effect either repression or activation of splicing.

Detection and seroprevalence of morbillivirus and other paramyxoviruses in geriatric cats with and without evidence of azotemic chronic kidney disease.

BACKGROUND: Feline morbillivirus (FeMV) is associated with the presence of tubulo-interstitial nephritis (TIN) in cats, however the seroprevalence of FeMV in the UK and the association between the presence of FeMV and renal azotemia is unknown HYPOTHESIS/OBJECTIVES: To identify whether paramyxoviruses are present in urine samples of geriatric cats and to develop an assay to assess FeMV seroprevalence. To investigate the relationship between both urinary paramyxovirus (including FeMV) excretion and FeMV seroprevalence and azotemic chronic kidney disease (CKD). ANIMALS: Seventy-nine cats (40 for FeMV detection; 72 for seroprevalence). METHODS: Retrospective cross-sectional, case control study. Viral RNA was extracted from urine for RT-PCR. PCR products were sequenced for virus identification and comparison. The FeMV N protein gene was cloned and partially purified for use as an antigen to screen cat sera for anti-FeMV antibodies by Western Blot. RESULTS: Feline morbillivirus RNA from five distinct morbilliviruses were identified. Detection was not significantly different between azotemic CKD (1/16) and nonazotemic groups (4/24; P = .36). Three distinct, non-FeMV paramyxoviruses were present in the nonazotemic group but their absence from the azotemic group was not statistically significant (P = .15). 6/14 (43%) azotemic cats and 40/55 (73%) nonazotemic cats were seropositive (P = .06). CONCLUSIONS AND CLINICAL IMPORTANCE: Feline morbillivirus was detected in cats in the UK for the First time. However, there was no association between virus prevalence or seropositivity and azotemic CKD. These data do not support the hypothesis that FeMV infection is associated with the development of azotemic CKD in cats in the UK.

An AU-rich stem-loop structure is a critical feature of the perinuclear localization signal of c-myc mRNA.

In eukaryotic cells, several mRNAs including those of c-myc and c-fos are localized to the perinuclear cytoplasm and associated with the cytoskeleton. The localization element of c-myc mRNA is present within its 3'UTR (3'-untranslated region) but the precise nature of this signal has remained unidentified. Chemical/enzymatic cleavage with RNases (ribonucleases) and lead have identified single-stranded and double-stranded regions in RNA transcripts of nucleotides 194-280 of the c-myc 3'UTR. Combined with computer predicted structure these results indicate that this region folds so that part of it forms a stem-loop structure. A mutation, that has been previously shown to prevent localization, leads to a different secondary RNA structure in this region as indicated by altered cleavage patterns. Competitive gel-retardation assays, using labelled transcripts corresponding to nucleotides 205-280 of c-myc 3'UTR, and fibroblast extracts revealed that the stem-loop region was sufficient for RNA-protein complex formation. In situ hybridization studies in cells transfected with reporter constructs, in which all or parts of the region corresponding to this stem-loop were linked to beta-globin, indicated that this region was sufficient for localization and that deletion of the nucleotides corresponding to the proposed upper-stem or terminal loop prevented localization. Our hypothesis is that an AU-rich stem-loop structure within nt 222-267 in the c-myc 3'UTR forms the perinuclear localization signal. Bioinformatic analysis suggests that this signal shares features with 3'UTRs of other localized mRNAs and that these features may represent a conserved form of signal in mRNA localization mechanisms.

Annexin A2 binds to the localization signal in the 3' untranslated region of c-myc mRNA.

Messenger RNA trafficking, which provides a mechanism for local protein synthesis, is dependent on cis-acting sequences in the 3' untranslated regions (3'UTRs) of the mRNAs concerned acting together with trans-acting proteins. The C-MYC transcription factor is a proto-oncogene product involved in cell proliferation, differentiation and apoptosis. Localization of c-myc mRNA to the perinuclear cytoplasm and its association with the cytoskeleton is determined by a signal in the 3'UTR. Here we show the specific binding of a trans-acting factor to the perinuclear localization element in the 3'UTR of c-myc mRNA and identify this protein as annexin A2. Gel retardation and UV cross-linking experiments showed that proteins in fibroblast extracts formed complexes with the region of c-myc 3'UTR implicated in localization; a protein of approximately 36 kDa exhibited specific, Ca(2+)-dependent binding. Binding was reduced by introduction of a mutation that abrogates localization. Using RNA-affinity columns followed by gel electrophoresis and mass spectrometry this protein was identified as annexin A2. The RNA-protein complex formed by cell extracts was further retarded by anti-(annexin A2). Purified annexin A2 bound to the same region of the c-myc 3'UTR but binding was reduced by introduction of a mutation, as with cell extracts. It is proposed that binding of annexin A2 to the localization signal in the c-myc mRNA leads to association with the cytoskeleton and perinuclear localization. The data indicate a novel functional role for the RNA-binding properties of annexin A2 in perinuclear localization of mRNA and the association with the cytoskeleton.

Baseline sensitivity of HSV-1 and HSV-2 clinical isolates and defined acyclovir-resistant strains to the helicase-primase inhibitor pritelivir.

Fifty-nine US isolates of HSV-1 and HSV-2 obtained between 1998 and 2004 were tested for sensitivity to the helicase-primase inhibitor, pritelivir (AIC316, BAY 57-1293) by plaque-reduction assay. All isolates, which were collected prior to any clinical use of primase-helicase inhibitors, were sensitive and showed mean EC50 values of 0.026 and 0.029µM for HSV-1 and HSV-2, respectively. Furthermore, several laboratory-selected acyclovir-resistant HSV mutants were also sensitive to pritelivir. These data provide a baseline for HSV sensitivity to pritelivir in general population before it is introduced and broadly used to treat HSV infection. The data also validate pritelivir as an appropriate therapy for nucleoside-resistant HSV infections.

Mesoionic heterocyclic compounds as candidate messenger RNA cap analogue inhibitors of the influenza virus RNA polymerase cap-binding activity.

BACKGROUND: An unusual feature of influenza viral -messenger RNA (mRNA) synthesis is its dependence upon host cell mRNAs as a source of capped RNA primers. A crucial activity of the influenza polymerase is to steal these primers by binding and cleaving the caps from host mRNAs. The recent structural analysis of the cap-binding fragment of the influenza virus PB2 protein has highlighted the importance of the mesoionic properties of the N7-methylguanine (N(7m)G) component of the mRNA cap in this interaction. METHODS: A series of mesoionic heterocycles with 5,6-fused ring systems analogous to the N(7m)G component of mRNA cap structures were synthesized and examined for the ability to inhibit the cap-binding activity of the influenza virus RNA polymerase complex using a bead-based in vitro cap-binding assay. RESULTS: None of the compounds tested were able to significantly inhibit binding and subsequent endonucleolytic cleavage of a synthetic radiolabelled capped mRNA substrate by recombinant influenza virus polymerase in vitro. CONCLUSIONS: Compounds analogous to the mesoionic N(7m)G component of mRNA cap structures comprise a large class of potential inhibitors of the influenza virus polymerase. Although this preliminary assessment of a small group of related analogues was unsuccessful, further screening of this class of compounds is warranted.

Elongation factor 1alpha binds to the region of the metallothionein-1 mRNA implicated in perinuclear localization--importance of an internal stem-loop.

In eukaryotic cells, mRNA localization can provide local protein synthesis. Metallothionein-1 (MT-1) mRNA is associated with the perinuclear cytoskeleton, and this is essential for subsequent nuclear import of the protein. The present study defines the cis-acting localization signal and a trans-acting binding protein. Gel retardation and UV cross-linking assays using MT-1 3'UTR transcripts and CHO cell extracts revealed formation of a complex containing a approximately 50-kDa protein. Only localization-positive mutant transcripts competed for binding of this protein. Using an RNA affinity technique, Western blotting, mass spectrometry, and a supershift assay, the protein was identified as Elongation factor 1alpha (eEF1alpha). Mutation and deletion analysis showed that two regions, nucleotides 21-36 and 66-76, were required for both binding and localization. RNA-folding prediction combined with chemical and enzymatic probing experiments suggest that these regions are in juxtaposition within a stem/internal loop structure. Mutations that are predicted to alter this structure abrogate protein binding. Our hypothesis is that the cis-acting signal in MT-1 3'UTR is formed by this stem/internal loop, that it binds eEF1alpha, and that eEF1alpha-cytoskeleton interactions play a role in perinuclear mRNA localization.

Zipcodes and postage stamps: mRNA localisation signals and their trans-acting binding proteins.

Messenger RNA (mRNA) localisation is a widespread mechanism within eukaryotic cells that provides local synthesis of proteins close to where they function. In general, this mRNA targeting involves the cytoskeleton and signals within the 3' untranslated region (3'UTR) of the transcript. In this paper, the authors review what is known of the nature of the localisation signals and the proteins that interact with them in animal cells. Specific examples are selected to illustrate the emerging pattern of how these signals are formed by the mRNA and the key RNA-binding proteins. The signals are usually restricted to relatively short regions of the 3'UTR, but their precise nature varies, with both sequence and structure playing key roles. Repeat motifs and functional redundancy also appear as common features of these signals. The trans-acting factors involved in localisation include proteins having other roles in nuclear events, proteins that shuttle between the nucleus and the cytoplasm and translational factors. In addition, there is evidence of homology among these proteins and the mechanisms of localisation across eukaryotic species.

Can resistant starch and/or aspirin prevent the development of colonic neoplasia? The Concerted Action Polyp Prevention (CAPP) 1 Study.

Loss of function of the adenomatous polyposis coli (APC) tumour suppressor gene through truncating mutations or other means is an early event in most colo-rectal cancer (CRC). The APC gene encodes a large multifunctional protein that plays key roles in several cellular processes, including the wnt signalling pathway where an intact APC protein is essential for down regulation of beta-catenin. The APC protein also plays a role in regulation of cell proliferation, differentiation, apoptosis, cell-cell adhesion, cell migration and chromosomal stability during mitosis. Acquisition of a non-functional APC gene can occur by inheritance (in the disease familial adenomatous polyposis (FAP)) or by a sporadic event in a somatic cell. Whilst there is strong epidemiological evidence that variation in diet is a major determinant of variation in CRC incidence, conventional adenoma recurrence trials in sporadic cases of the disease have been relatively unsuccessful in identifying potentially protective food components. Since the genetic basis of CRC in FAP and in sporadic CRC is similar, intervention trials in FAP gene carriers provide an attractive strategy for investigation of potential chemo-preventive agents, since smaller numbers of subjects and shorter time frames are needed. The Concerted Action Polyp Prevention (CAPP) 1 Study is using a 2 x 2 factorial design to test the efficacy of resistant starch (30 g raw potato starch-Hylon VII (1:1, w/w)/d) and aspirin (600 mg/d) in suppressing colo-rectal adenoma formation in young subjects with FAP. Biopsies of macroscopically-normal rectal mucosa are also being collected for assay of putative biomarkers of CRC risk.