Drosophila melanogaster. We found that Importin-α2 was expressed in the nervous system where it was required for normal active zone density at the NMJ and axonal commissure formation in the central nervous system. Other aspects of synaptic morphology at the NMJ and the localization of other synaptic markers appeared normal in importin-α2 mutants. Importin-α2 also functioned in development of the body wall musculature. Mutants in importin-α2 exhibited errors in muscle patterning and organization that could be alleviated by restoring muscle expression of Importin-α2. Thus, Importin-α2 is needed for some processes in the development of both the nervous system and the larval musculature.Nuclear import is required for communication between the cytoplasm and the nucleus and to enact lasting changes in gene transcription following stimuli. Binding to an Importin-α molecule in the cytoplasm is often required to mediate nuclear entry of a signaling protein. As multiple isoforms of Importin-α exist, some may be responsible for the entry of distinct cargoes rather than general nuclear import. Indeed, in neuronal systems, Importin-α isoforms can mediate very specific processes such as axonal tiling and communication of an injury signal. To study nuclear import during development, we examined the expression and function of Importin-α2 in  To ensure a proper long-term response to stimuli, cells must have communication between the cytoplasm and the nucleus. Signals arising from diverse pathways must be imported into the nucleus where they can affect gene transcription. This process is largely accomplished through the function of importin proteins The importin family comprises two major classes of protein: Importin-α and Importin-β Drosophila genome importin-α1 is involved in wing patterning importin-α2 may be involved in cell proliferation and cell cycle progression importin-α3 is involved in cell fate decisions importin-α3 fail to import a synaptic dSmad2 signal into the nucleus and show defects in proper axonal tiling of photoreceptors importin-α1 and importin-α2 mutants survive through to adulthood importin-α1 and importin-α2.There are three evolutionary clades of Importin-α homologues and each is singly represented in the importin-α2 is required in the muscle for normal patterning and organization of the larval body-wall musculature.We recently identified a role for Importin-α2 outside the germline. Importin-α2 contributes to postsynaptic development of the neuromuscular junction (NMJ) by permitting the import of a Fz2-derived signal, the C-terminal peptide of the Fz2 receptor, into muscle nuclei Drosophila strains were raised at 25°C on cornmeal-molasses food. The isogenic stock y, w; FRT42D was used as the wild-type control. The following alleles and transgenic strains were used: D3imp-α2, D14imp-α2Df(3L) α1S1, referred to here as DfImp-α1Larvae were grown on standard grape juice agar plates at low density at 25°C. Wandering third-instar larvae were dissected and processed for immunohistochemistry as described Larvae were imaged using an LSM 510 Meta laser scanning confocal microscope  and either a 63× 1.4 NA or 40× 1.0 NA objective. Images were processed in separate channels using the LSM software or Adobe Photoshop CS2 .Scoring for bouton counts, commissural defects and muscle patterning errors were performed using a Nikon  E800 fluorescent microscope. For boutons, NMJs on muscles 6 and 7 of segments A2 and A3 on both the right and left sides were analyzed; bouton number was normalized to muscle surface area. Commissures were scored as defective if they had discontinuous axons at the midline of the ventral nerve cord. Muscle patterning was analyzed in segments A2 through A7. Genotypes were processed in the same tube and imaged with identical settings. Bruchpilot (Brp) and GluRIIC puncta were manually counted at MN4b synapses on muscle 4 from confocal z-stacks and terminal area was calculated using the threshold function in Metamorph for the HRP channel as previously Fluorescent intensity was measured in ImageJ as previously n) is described either in the figure legend or in the Statistical analysis was conducted using Prism 5 software  and significance (relative to wild-type unless otherwise noted) was calculated using a one-way analysis of variance (ANOVA) with a Dunnett post-hoc test to a control sample. When only two samples were tested, an unpaired student's t-test was used. Values are given as mean ± SEM; sample size . In aggregate, although no deviations from the normal pattern were observed in 96 wild-type segments examined, a quarter of the 228 mutant hemisegments contained defects and every importin-α2 mutant larva had at least 1 defective hemisegment (Importin-α2 is present in muscle nuclei and is required for normal postsynaptic development by mediating a synapse-to-nucleus Fz2 signal e fibers . Most fre fibers . Additioe fibers , arrow oe fibers . We alsoe fibers . Unlike isegment . Expressisegment . The proimportin-α2 in Drosophila nerve and muscle. Rather than preventing all nuclear import and causing cell lethality, importin-α2 mutants display specific defects in active zone development, central axon projections, and muscle patterning. These defects arise in the context of larvae whose overall patterning and development appear to be normal.We have identified an array of consequences of the loss of Drosophila Importin-α2 in the nervous system based on transcript expression Previous work suggested roles for importin-α2 phenotypes described here, can entail regulation of the synapse. In particular, studies of Aplysia and hippocampal neurons indicated an essential role of an Importin-α in long-term synaptic plasticity Drosophila NMJ indicated a role in controlling the density of release sites and their accompanying postsynaptic receptor clusters. While there were no obvious phenotypes involving synaptic morphology and bouton number at the neuromuscular junction (Neuronal functions of the Importin-α family have been reported recently in several systems junction , active junction . The addimportin-α2 also showed defects in the organization of the central nervous system. Specifically, the axon tracts that cross the midline of the CNS and express the cell adhesion molecule Fasciclin III In addition to these peripheral defects, mutants in Drosophila larval body walls importin-α2 mutants. The importance of signaling pathways in establishing the normal pattern has been demonstrated previously by mutations in the toll/dorsal pathway and in dystroglycan and POMT1. The phenotypes of these mutants and others entail muscle duplications, absent muscles, branched muscles and errors in muscle insertion importin-α2 mutants and arose from the loss of Importin-α2 expression in the muscle (dystroglycan and POMT1 that affect larval muscle integrity also display patterning defects akin to and at similar frequencies to those of the importin-α2 mutants The stereotypical patterning and unique identification of each muscle in e muscle . While tDrosophila Importin-α2 is required in multiple cell types for distinct aspects of development but is not required for general development or cell viability may reflect a high degree of cargo specificity for this Importin-α importin-α2. The identification of those phenotypes is a critical step towards identifying the mechanisms and cargo molecules through which the importins contribute to cellular processes.In current models of nuclear import, Importin-α binds cargo molecules destined for nuclear entry Figure S1Active Zone Density is Normal in importin1 Mutants. A C Representative confocal images of larval NMJs from muscle 4 stained with antibodies against Bruchpilot magenta, GluRIIC green and HRP blue in wildtype control animals y,w FRT42D . Each Brp punctum is closely apposed by a corresponding GluRIIC punctum. D F Representative confocal images of importin1 mutants w Df3L 1S1  stained as above. No changes in active zone apposition are apparent. Scale bar 10 m. G J Histograms of NMJ area G, Brp density H, GluRIIC density I and the ratio of Brp to GluRIIC J at wildtype and importin1 mutants NMJs at muscle 4. No significant changes in these parameters are evident. n 12 NMJs for each genotype error bars represent SEM.TIFClick here for additional data file.


Classical nuclear localization signal (NLS) dependent nuclear import is carried out by a heterodimer of importin α and importin β. NLS cargo is recognized by importin α, which is bound by importin β. Importin β mediates translocation of the complex through the central channel of the nuclear pore, and upon reaching the nucleus, RanGTP binding to importin β triggers disassembly of the complex. To date, six importin α family members, encoded by separate genes, have been described in humans.We sequenced and characterized a seventh member of the importin α family of transport factors, karyopherin α 7 (KPNA7), which is most closely related to KPNA2. The domain of KPNA7 that binds Importin β (IBB) is divergent, and shows stronger binding to importin β than the IBB domains from of other importin α family members. With regard to NLS recognition, KPNA7 binds to the retinoblastoma (RB) NLS to a similar degree as KPNA2, but it fails to bind the SV40-NLS and the human nucleoplasmin (NPM) NLS. KPNA7 shows a predominantly nuclear distribution under steady state conditions, which contrasts with KPNA2 which is primarily cytoplasmic.KPNA7 is a novel importin α family member in humans that belongs to the importin α2 subfamily. KPNA7 shows different subcellular localization and NLS binding characteristics compared to other members of the importin α family. These properties suggest that KPNA7 could be specialized for interactions with select NLS-containing proteins, potentially impacting developmental regulation. Eukaryotic cells are defined by the separation of DNA from the rest of the cell by the nuclear envelope, a double bilayer made selectively permeable by Nuclear Pore Complexes (NPC) . TranspoD. melanogaster homologue of β catenin  as an importin α. Using RT-PCR, we amplified a product of the expected size from RNA prepared from the prostate cancer cell line LNCaP. We sequenced the cDNA [Genbank:EU126604], and found that it matched that of the predicted gene, leading us to conclude that the gene is transcribed in human cells. This gene is the seventh member of the importin α family of transport receptors, therefore we named it karyopherin alpha 7 (KPNA7). The complete human importin α family (KPNA1-7) is shown in Table There are six known members of the human importin α family. We used BLAST to query the human genome with the importin α family member KPNA2, and retrieved a sequence predicted to encode a seventh member of the importin α family. Because the sequence of the putative importin α was generated by gene prediction, we set out to experimentally validate the expression and function of the predicted importin α gene [GenBank:Drosophila melanogaster importin α proteins were included in the phylogram , xenopus  and zebra fish . In all animals, importin α proteins are classified into 3 subfamilies, α1, α2, and α3 . While vgram Fig  to help The alignment of KPNA7 with KPNA's 1-6 Fig , as the in vitro translated, 35S-Methionine labeled KPNA proteins . Deletion of the KPNA7 IBB resulted in complete loss of KPNA7 binding to importin β  that might influence the outcome of these binding assays, we used recombinant proteins to formally test whether the N-terminus of KPNA7 functions as an IBB. GST fusions of the IBB domains from KPNA2 and KPNA7 were immobilized on glutathione beads, recombinant importin β was added, and bound fractions analyzed by immunoblotting. Importin β bound to the IBB domains of both KPNA2 and KPNA7; the interactions were Ran-sensitive because they were reduced significantly by including RanQ69L (preloaded with GTP) in the binding reaction Fig. , a reactThe competitive autoinhibition of NLS binding by the IBB may be altered by changes in the IBB affinity for importin α. Thus we decided to test whether the KPNA7ΔIBB would show enhanced binding to the SV40-NLS compared to full length KPNA7. Unexpectedly, neither full length nor IBB deletion forms of KPNA7 display significant levels of binding to the SV40 NLS Fig . The dif1PJM, [Crystal structures have been solved for several importin α proteins, yeast SRP1 , mouse a1PJM, ). The RB1PJM, . The res1PJM,  and 16/11PJM,  are perf35S Methionine-labeled KPNA proteins , Tejomurtula et al. reported the sequence of bovine KPNA7 and showed that it binds bovine nucleoplasmin [After we deposited the human KPNA7 sequence to GenBank . Double-label immunofluorescence microscopy was performed on HeLa cells with the KPNA7 polyclonal antibody and a KPNA2 monoclonal antibody. This revealed that KPNA7 is predominantly nuclear under conditions where KPNA2 is largely cytoplasmic Fig . By immuHere we describe the characterization of a new member of the human importin α family, KPNA7. Sequence analysis revealed that KPNA7 is most similar to KPNA2 (54%), and that it is the most divergent member of the importin α family. Sequence divergence in KPNA7 occurs throughout the protein, including the IBB domain. Our binding data indictates that the IBB of KPNA7 binds importin β stronger than other KPNA proteins. The NLS binding surface is highly conserved in all of the importin α family members, but we observed clear differences among the different family members for binding the RB NLS, SV40 NLS, and the NPM NLS. KPNA7 bound to the RB NLS with equivalent affinity to KNPA2, but failed to bind both the SV40 NLS and the NPM NLS. In addition, by immunofluorescence microscopy KPNA7 localizes to the nucleus, a distribution that contrasts with the predominantly cytoplasmic localization of KPNA2. Our data emphasizes that despite a high degree of structural conservation, there are important differences in the NLS binding specificity and nucleocytoplasmic trafficking of importin -a family members.Based on the conservation of the residues which make up the RB NLS and SV40 NLS binding surfaces of KPNA2, we reached the conclusion that the NLS binding surface is almost perfectly conserved in all seven members of the importin α family. It was surprising, therefore to observe KPNA-specific binding to both the RB NLS and SV40 NLS. Differences in the IBBs of importin α family members could have potentially altered the affinity for NLS binding through autoinhibition , but delKPNA7 is the most divergent of the seven proteins in the human importin α family, and the IBB domain is more divergent than the ARM repeats, which maintain identity in the amino acids necessary for interaction with both the RB NLS and the SV40 NLS. The lack of conservation in the KPNA7 IBB suggests that it does not have the same selective pressures as KPNAs 1 through 6. There are two known functions for the IBB, binding to importin β, and autoinhibition through binding to importin α. Importin β binding could be affected by the amino acid changes in the KPNA7 N-terminus, but our data show very strong binding to importin β. This increased binding to importin β may represent an enhanced affinity of the IBB for importin β, but it could also be due to decreased autoinhibitory binding of the IBB to importin α.A striking feature of KPNA7 is its localization to the nucleus under steady state conditions, while KPNA2 is predominantly cytoplasmic. This nuclear localization suggests that nucleocytoplasmic shuttling of KPNA7 differs from that of the other members of the importin α family. There are three possibilities for the altered subcellular distribution; 1) enhanced nuclear import by importin β; 2) decreased nuclear export by CAS; and 3) inefficient release of KPNA7 from target NLS, resulting in nuclear retention. The most likely of these situations, given our evidence that KPNA7 binds importin β with much higher affinity than other a family members is that this enhanced binding results in increased nuclear import of KPNA7.The recent study on bovine KPNA7 showed that it is expressed preferentially in the embryo, suggesting a possible role in early in development . Given tWe identified and characterized KPNA7, a highly divergent member of the importin a family of nuclear import receptors. The importin α family in vertebrates comprises seven proteins, KPNA1 through KPNA7. Sequence analysis and molecular modeling revealed the residues that form the NLS binding surface are nearly perfectly conserved throughout the importin a family. Nonetheless, there are striking differences in NLS recognition amongst the different KPNA proteins. Tertiary structure in KPNA proteins could play a major role in creating binding surfaces that can discriminate between different NLS's.http://www.mbio.ncsu.edu/BioEdit/BioEdit.html. The Protdist neighbor function was used to create phylogenetic trees, and Dendroscope http://www-ab.informatik.uni-tuebingen.de/software/dendroscope was used to visualize the phylogenetic trees.RNA from the prostate cancer cell line LnCaP was used as the template to amplify the open reading frame of KPNA7. Multiple alignments were made using the ClustalW alignment tool in Bioedit The parent vector used for generating the KPNA proteins was pCMVTNT (Promega). T7 or HA tags were cloned as annealed oligonucleotides with a 5' Kozak sequence into pCMVTNT using Xho1 and EcoR1 sites. KPNAs 1 through 6 were purchased from Origene, PCR amplified and cloned into pCMVTNT-T7 using EcoR1 and Sma1 sites  or Kpn1 and Sma1 sites (KPNA4). Importin β was cloned using Mlu1 and Xba1 sites.in vitro with the TNT Coupled Reticulocyte Lysate System  using T7 polymerase and 35S-methionine . 10 μg of each GST fusion protein was bound to glutathione beads, and incubated with 250,000 CPM of translated protein for 4 hours at 4°C. The beads were washed with buffer  and eluted in 2x SDS sample buffer. Samples were resolved on 10% acrylamide gels, fixed in methanol/acetic acid, incubated in Autofluor , dried and exposed to film at -80°C. Importin β binding to GST-IBB domains was performed in a similar manner except that His-tagged, recombinant importin β  and Ran  were used. Binding was assessed by semi-quantitative immunoblotting using IRDye conjugated secondary antibody and the Odyssey infrared imaging system .GST fusions of the indicated NLS constructs were expressed and purified by standard methods. His-tagged RanQ69L protein was expressed in the E. coli strain BL21 by induction for 16 hrs at 18 C, and purified on Talon beads. Recombinant Ran protein was loaded with GTP (100 uM) in the presence of EDTA (10 mM) for 20 min at 30 C. Bound nucleotide was stabilized by the addition of excess MgCl2 (20 mM), and unincorporated nucleotide was removed by chromatography on a PD-10 gel filtration column. The pCMVTNT-T7 KPNA constructs were translated A peptide corresponding to a unique sequence (DQDYEFIDYEC) in the C-terminus of KPNA7 was synthesized, coupled to Keyhole Limpet hemocyanin, and injected into rabbits . Antibody was purified from serum using immobilized peptide.2. For transfection experiments, cells were plated on glass coverslips for 24 hours and transfected with KNPA plasmid using Transfectin (Biorad) according to the manufacturer's protocol. Cells were grown for an additional 24 hours and processed for immunofluorescence microscopy. Coverslips were washed with PBS, fixed with 4% formaldehyde for 20 minutes, permeabilized with 0.5% TritonX-100 for 5 minutes, blocked (2% BSA and 2% NCS in PBS), and incubated with primary antibody. Primary Antibodies used were 16B12 anti-HA antibody , Rch1 antibody , and anti-KPNA7. Secondary antibodies used were Donkey anti-Mouse FITC  and Donkey anti-Rabbit Cy3 .HeLa cells were grown in DMEM containing 5% Newborn Calf Serum (NCS), 5% Fetal Bovine Serum, 1 mM Sodium Pyruvate at 37°C in 5% COJBK carried out the sequence analysis, molecular modeling, immunofluorescence microscopy, and drafted the manuscript. AMT performed KPNA7 plasmid construction, antibody production and immunoblotting. AS performed the binding assays. DG performed PCR amplification for the KPNA7 transcript. BMP designed the study and edited the manuscript. All authors read and approved the final manuscript.


Incorporation of these probes using improved RNA ligation strategies promises to change the landscape of structural biology of supramacromolecules probed by biophysical tools such as nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography and Raman spectroscopy. Finally, some of the structural and dynamic problems that can be addressed using these technological advances are outlined.The ability to adopt complex three-dimensional (3D) structures that can rapidly interconvert between multiple functional states (folding and dynamics) is vital for the proper functioning of RNAs. Consequently, RNA structure and dynamics necessarily determine their biological function. In the post-genomic era, it is clear that RNAs comprise a larger proportion (>50%) of the transcribed genome compared to proteins (≤2%). Yet the determination of the 3D structures of RNAs lags considerably behind those of proteins and to date there are even fewer investigations of dynamics in RNAs compared to proteins. Site specific incorporation of various structural and dynamic probes into nucleic acids would likely transform RNA structural biology. Therefore, various methods for introducing probes for structural, functional, and biotechnological applications are critically assessed here. These probes include stable isotopes such as  Increasingly RNA molecules have taken center stage as key informational, structural, catalytic, and gene-regulatory molecules . ContrarThe calculation of 3D structures of RNA molecules by NMR and X-ray crystallography have, therefore, lagged substantially behind those of proteins. For NMR, this lag is partly because of extensive signal overlap and the rapid signal decay that occurs with increasing molecular weight. For X-ray crystallography, RNA structures have been difficult to solve partly because of RNA flexibility and the difficulty of finding suitable constructs that are able to diffract to high-resolution, as well as finding suitable heavy atom derivatives for phase determination. Recently, a number of emerging technologies make the prospects of tackling large supramacromolecular RNA complexes both probable and doable by various biophysical methods such as NMR, fluorescent resonance energy transfer (FRET), Raman, and X-ray crystallography. Several excellent reviews describe the technical details of NMR RNA structure calculation , advanceNMR spectroscopic and X-ray crystallographic analyses require large milligram quantities of pure and functionally active materials. Chemical or biochemical methods allow synthesis of large quantities of RNA. While the chemical synthetic method affords site-specific incorporation of both standard and non-standard nucleobases into RNA, the labeled precursors are usually expensive and difficult to synthesize. Although chemical synthesis of RNA has improved considerably over the past decade, this method is highly inefficient beyond 50 nucleotides (nt) . In thisin vitro transcription with various DNA dependent polymerases such as SP6, T3, or T7 is a widely used enzymatic method . In addition, the energy of transition for deuterium is very different from that of hydrogen such that the deuterium resonances are absent in a proton NMR spectral region. Of the two types of deuterium labeling, random fractional deuteration, wherein organisms are grown on partially deuterated carbon sources in a partial D2O solvent mixture, is not as effective for RNA NMR as it is for protein NMR. In contrast, specific deuteration, where 2H is placed in a specific location of a macromolecule, results in homogenous labeled molecules without uniform reduction in signal intensity  by the action of enolase and pyruvate kinase. 3-phosphoglycerate mutase converts the relatively inexpensive D-(−)-3-phosphoglyceric acid (3PGA) to D-(+)-2-phosphoglyceric acid (2PGA), enolase forms PEP from 2PGA, and then pyruvate kinase catalyses the conversion of ADP to ATP -d-glucose and conducting the reaction in D2O without G6P isomerase, 84% of the starting glucose is converted to the four NTPs with the ribose fully deuterated, a label called d6-NTP. By repeating this reaction in water and in the presence of G6P isomerase, the H1′ and H2′ ribose positions are selectively protonated in a background of deuterated ribose. This label is called d4-NTP. By carrying out the reaction in H2O with G6P isomerase using completely deuterated and uniformly 13C labeled -d-glucose, d4-13C-ribose NTPs are easily prepared [2O using completely deuterated and uniformly 13C labeled glucose shown above, d5-13C-ribose NTPs are easily prepared.Using this general strategy, the Williamson group has produced several isotopic labeling patterns , 81–82. prepared –71. By s4-13C-ribose NTPs are valuable for preparing RNA uniformly 13C-labeled in the ribose ring with H3′, H4′, H5′/H5″ protons specifically replaced with deuterons [4 labeling pattern retains most of the useful structural information: the H1′-H2′ scalar coupling that reports on ribose pucker; base to H1′ NOE that provides the glycosidic torsion; and exchangeable protons that provide base pairing information [4, d4-13C, d6, and combinations of d4 and d6) are important for NMR because their incorporation into an RNA molecule is valuable for recording NMR experiments that are easy to interpret and analyze. For example, the nuclear Overhauser spectroscopy (NOESY) experiment of such a sample has very interpretable base to ribose proton region that is difficult to assign otherwise [5-13C-ribose pattern have only the 2′-position protonated, and all the other ribose protons are specifically replaced with deuterons as described in detail next.All five isotopic labeled patterns are valuable for NMR spectroscopic analysis of RNA structure and dynamics. The deuterons . This laormation . These stherwise , 75. RNA5-13C-NTPs can be useful for relaxation analysis [13C-labeled and the pyrimidine bases were 2H-labeld at the C5 position. This labeling strategy represents a significant improvement over partial (50%) deuteration within a fully ~100% 13C-enriched background because the latter selects ~25% signals with concomitant compromise in sensitivity. With close to 100% 2H label at C1′, C3′, C4′, and C5′ carbon sites, the authors probed the backbone dynamics of most of the deuterated sites in HIV-2 TAR RNA [The research group of Kay, in collaboration with the Williamson group, showed that danalysis . Here, t TAR RNA .19F) nucleus can be incorporated into RNA bases [19F can provide a wealth of geometrical and dynamical information about RNA molecules. 19F is not endogenously expressed in biological molecules making it a valuable probe, it has 100% natural abundance and a gyromagnetic ratio comparable to that of a proton (γF~0.94γH) which means it possesses an intrinsic sensitivity almost as high as that of a proton. Importantly, 19F also offers chemical shift dispersion (~900 ppm) ~50 times larger than that obtainable from protons. Recently, the research groups of Henning and Williamson proposed a facile T7 RNA polymerase based incorporation of 5-Fluoro Cytidine, 5-Fluoro Uracil and 2-Fluoro Adenine into a 30 nt HIV-2 TAR RNA construct [As another excellent example of the versatility of this enzymatic approach, Fluorine-19  and further development of other labels will greatly facilitate structure determination of large RNAs especially using ligation strategies (See below).13C and 15N  currently lists at $750 for 0.1 g from Cambridge Isotope Laboratories. Incorporating the commercially available bases into the enzymatic labeling scheme would, therefore, be a costly method. Second, 7 out of the 18 enzymes required for synthesis are not commercially available and must be produced from over-expressing E.coli strains. Of these seven, only ribokinase and APRT have robust activities of 350–700 U . The other five are only moderately over-expressed with activities of 28–40 U, making the enzyme preparation labor intensive [Two limitations of this enzymatic approach must be overcome to make this method widely adopted by the biophysics community. First, the price for bases with various labels is high. Uracil uniformly labeled with ntensive , 76. By X-ray crystallography is the other powerful approach for 3D structure determination of macromolecules. A substantial barrier for RNA structure determination by X-ray crystallography is heavy atom derivatization for phase determination , 83. TheIn the past RNA crystallization tags have been used to address this problem. One example is the “soak and pray” method, wherein heavy atoms are soaked into the crystal to help with anomalous scattering –86. OtheSp diasteromer to synthesize RNA [One method, multiple anomalous dispersion (MAD), aims to solve the phase problem by using intrinsic or exogenous anomalous scatterers. Incorporation of Selenium, an attractive element for derivatization of biomolecules, into proteins led to an explosive growth of new protein crystal structures. In contrast to proteins, derivatizing nucleic acids for X-ray crystallography has lagged behind. Recently the groups of Egli, Huang, and Micura have developed a selenium derivatization strategy for nucleic acids . Two posize RNA . The funGiven the current limitations of chemical modifications of RNA for phase determinations, the research groups of Batey and Kieft proposed a direct soaking method. GU wobble pair motifs are intentionally engineered into seven variants of the SRP RNA to provide heavy atom binding sites with high occupancy and low B factors . They deSolving high-resolution structures of RNA molecules by X-ray crystallography and NMR spectroscopy is central to obtaining insights into fundamental biological processes. As discussed earlier, major hurdles for NMR structural analyses are crowding and signal decay, and for X-ray crystallographic analyses these are preparation of suitable heavy atom derivatives that are essential for calculation of crystallographic phases and generation of an electron density map. While improved methods for chemical synthesis of RNA exist, generating larger RNAs that are site-specifically modified with probes to help overcome some of these obstacles is more difficult. A very promising approach is joining RNA pieces together, which can be accomplished in at least four different ways: by chemical ligation, by DNA splinted DNA T4 ligation, by T4 RNA ligation, or by deoxyribozyme mediated ligation. In this section, the salient features of each method, as well as their advantages and limitations are presented.Chemical coupling with cyanogen bromide or a water soluble carbodiimide allows synthesis of both 3′-5′- and 2′-5′-phosphodiesters. A terminal monophosphate is required, which is donated by either the 5′- or 3′-substrate, while another substrate supplies the nucleophilic oxygen such as a 3′-OH. The segments to be joined are then hybridized to a complementary template. While the yields for coupling DNA duplexes can be as high as 96%, the efficiency for RNA oligomers is much lower at 4–16% –96. Unfoin vitro transcribed donor RNA can be initiated with GMP or a 5′-XpG-3′ dinucleotide, or the transcribed RNA can be dephosphorylated and then rephosphorylated. For the in vitro transcribed RNAs, abortive initiation products are significant, and therefore they must be purified away from the full length donor RNA to prevent competition for the cDNA template. T4 DNA ligase will only ligate full (N) length acceptors. But a significant amount of 3′-end heterogeneity caused by N+1 products can inhibit ligation efficiency by sequestering donor substrates into unproductive hybrid complexes with the cDNA template [One of the most widely used methods for ligating RNA molecules employs T4 DNA ligase . DNA ligtemplate . The mai4 and R5 in 1 and Y2 in An alternative method for RNA ligation, used extensively prior to 1992 –102. RNA1 X2 X3 R3 R4 in Stark and colleagues did exactly that and the following modifications to improve the ligation efficiency to near completion in 30 minutes . First, The question still remains: how well do these DNA splinted ligation schemes work in the context of structured RNA elements? Unfortunately very few studies have documented the influence of structure on DNA splinted ligation efficiencies using T4 RNA ligase. Preliminary studies from our own group suggest that even large (~500 nt), structured RNAs can be ligated to obtain yields of ~33% .in vitro selection experiments. Unlike the T4 protein ligases, the two promising ligases require a 2′, 3′-diol for the acceptor fragment and a 5′-triphosphate functional group for the donor RNA fragment . The Zn2+-dependent ligase is less efficient at ligation than 9DB1 (40–50%). Nonetheless it ligates under practical in vitro conditions that reduce the potential for RNA degradation. At 23°C, pH 7.5 and 1 mM Zn2+, this ligase has a t1/2 of 30 minutes. Together, both ligases provide a useful alternative to protein-mediated RNA ligation. In the future, efforts to evolve such DNAzymes to work under more physiological conditions to ligate 3′-5′-bonds of any two RNA sequences will be extremely valuable.Another alternative for the synthesis of native 3′-5′-RNA linkage is to use deoxyribozymes –105. Silfragment . The twoIn the post-genomic structural biology era, large size matters: most molecular machines are mega Dalton machines, making traditional NMR techniques inadequate for the task. For these large systems, cryo-electron microscopic reconstructions can provide images of the overall shape but without the requisite molecular details. X-ray crystallography is ideally suited for providing these atomic details, yet is limited to well structured and conformationally homogeneous systems. NMR spectroscopy is ideally positioned to tackle such systems. As discussed above, the problems of overlap and size that plague traditional approaches can be effectively circumvented using these emerging technologies. In particular, modular multi-component biological systems are particularly suited for a divide-and-conquer approach that can take advantage of these technologies. By divide-and-conquer we mean working on multi-component systems that can be broken down into folded “domains” and then probing the labeled components singly or in various combinations with unlabeled components. In this last section, two biological problems that are amenable to this approach are described.Retroviruses are a family of RNA viruses that cause a number of diseases such as AIDS, neurological disorders, tumors, and leukemias . The strPylaiella littoralis into a highly active form by combining an unstructured 22 nt substrate, a 36 nt domain 5 (D5) hairpin, and a 493 nt domains 1 through 3 (D123) [32P labeled probes [A large riboncucleoprotein complex called the spliceosome  mediates3 (D123) –112. Thi3 (D123) . We have3 (D123) , 112. Pad probes , 112–113d probes .Using a divide-and-conquer approach, we have shown that NMR experiments can provide a detailed view of the binding interface between the catalytically important D5 and the large 100 kDa D123 fragment. The regions of D5 that undergo the greatest chemical shift changes correspond to those areas predicted by footprinting experiments . Using NIn conclusion, using very simple NMR experiments on multidomain structures, wherein one component is preferentially labeled with NMR-active nuclei, one can probe in atomic detail the binding interface of numerous molecular machines , 114. In


A method is described that allows an accurate mapping of 3' ends of RNAs. In this method a labeled DNA probe, containing the presumed 3' end of the RNA under analysis is allowed to anneals to the RNA itself. Mung-bean nuclease is then used to digest single strands of both RNA and DNA. Electrophoretic fractionation of "protected" undigested, labeled DNA is than performed using a sequence reaction of a known DNA as length marker. This procedure was applied to the analysis of both a polyA RNA (Interleukin 10 mRNA) and non polyA RNAs (sea urchin 18S and 26S rRNAs). This method might be potentially relevant for the evaluation of the role of posttrascriptional control of IL-10 in the pathogenesis of the immune and inflammatory mediated diseases associated to ageing. This might allow to develop new strategies to approach to the diagnosis and therapy of age related diseases. The denatured RNAs were then used to anneal, at 65°C for 5 minutes, with PCR amplified  labelled DNA fragments, pre-incubated at 95°C for 3 minutes, which we know to contain the 3' ends of correspondent RNAs  [Aliquots (10 μg each) of total RNA extracted as indicated elsewhere  from botC000001]  side by In lanes 2, 4 and 6, bands of 127 nucleotides (nt), 120 nt and 110 nt, corresponding to the IL-10, 18S and 26S RNA digestions, respectively, indicates the length of the "protected" regions, permitting an accurate mapping of respective 3' ends by comparison with the sequence reaction. In lanes 1, 3 and 5, IL-10, 18S and 26S undigested probes, respectively, were fractionated.P. lividus unfertilized eggs. If a "long run" of the electrophoresis shown in Figure The accuracy and sensibility of our system is also demonstrated by the presence of a faint band in lane 4, which represents the 3' end of a 21S rRNA, precursor of the mature 18S rRNA. The presence of low amounts of this precursor was previously demonstrated [Taking into account the well known role of IL-10 in longevity and in age-related diseases -7, we harRNA: ribosomal RNA; mRNA: messenger RNA; IL10: interleukin 10; dNTP: deoxyribonucleotides; nt: nucleotides.The authors declare that they have no competing interests.DB carried out both the molecular and the electrophoretic procedures and helped to draft the manuscript. GS participated to preparation of the materials used in the experiments and participated to all the experiments described in the text. GLP participated to all the experiments described in the text. GIF participated to all the experiments described in the text. RB senior author; conceived of the study, and participated in its design and coordination and wrote the manuscript.


Caliciviridae failed to lead to recovery of MNV. This was due to our observation that vaccinia virus infection had a negative effect on MNV replication. In contrast, FWPV infection had no deleterious effect and allowed the recovery of infectious MNV from cells previously transfected with MNV cDNA constructs. These studies also indicated that the nature of the 3′-terminal nucleotide is critical for efficient virus recovery and that inclusion of a hepatitis delta virus ribozyme at the 3′ end can increase the efficiency with which virus is recovered. This system now allows the recovery of genetically defined noroviruses and will facilitate the analysis of the effects of genetic variation on norovirus pathogenesis.Despite the significant disease burden caused by human norovirus infection, an efficient tissue-culture system for these viruses remains elusive. Murine norovirus (MNV) is an ideal surrogate for the study of norovirus biology, as the virus replicates efficiently in tissue culture and a low-cost animal model is readily available. In this report, a reverse-genetics system for MNV is described, using a fowlpox virus (FWPV) recombinant expressing T7 RNA polymerase to recover genetically defined MNV in tissue culture for the first time. These studies demonstrated that approaches that have proved successful for other members of the family  Caliciviridae are responsible for many diseases in both man and animals. Those that infect humans, namely members of the genera Norovirus and Sapovirus, are a major cause of acute gastroenteritis and are thought to be responsible for >85 % of all non-bacterial gastroenteritis outbreaks in Europe between 1995 and 2000 , porcine enteric calicivirus . Both NS7 and VP2 were purified under denaturing conditions (8 M urea) on nickel agarose and used for immunization of New Zealand White rabbits. All immunizations and test bleeds were carried out by Eurogentec.MNV 1 strain CW1, the murine macrophage line RAW 264.7 and antisera to the MNV VP1 protein were kindly supplied by Herbert Virgin IV . MVA-T7 (All cells were maintained in Dulbecco's modified Eagle's medium (Gibco) containing 10 % fetal calf serum. The Huh 7.5 cell line was kindly provided by Charlie Rice . The BSR-T7 cell line was obtained from Karl-Klaus Conzelmann .FS), containing a frame shift in the RNA-dependent RNA polymerase [NS7 in Fig. 1(a)XhoI, followed by mung-bean nuclease digestion and religation. A transfer vector containing the AfeI–SacII fragment of the MNV-1 genome (pSL301 : MNV AfeI–SacII) was generated by cloning the fragment into pSL301 (Invitrogen). To repair the frame-shift mutation in pT7 : MNV-GFS, the AfeI–SacII fragment from the transfer vector pSL301 : MNV AfeI–SacII was inserted into pT7 : MNV-GFS to generate pT7 : MNV-GFS/R.A full-length cDNA clone of MNV 1 CW1 matching the passage 3 consensus sequence (BglII restriction site was introduced at position 3959 in the MNV genome by the introduction of a single-nucleotide change (C to A) at position 3959 to generate pT7 : MNV-G/BglII. The restriction site was introduced by PCR amplification of the region using the primers IGIC44 and 4450R , digestion with AfeI and KpnI and subsequent insertion into the pSL301 : MNV AfeI–SacII transfer vector (see above). The mutated fragment was subcloned into pT7 : MNV-G via the AfeI and SacII sites. Insertion of the desired mutation, which did not affect the encoded polypeptide sequence, was confirmed by sequencing.A NheI site was PCR-amplified from pRZ . The ligated product was subsequently digested with SacII and EcoRV and inserted into pT7 : MNV-G that had been digested with SacII and SnaBI. The resultant plasmid, pT7 : MNV-G 3′Rz, contained the MNV genomic RNA flanked by a truncated T7 RNA polymerase promoter at the 5′ end and a hepatitis delta virus ribozyme at the 3′ end. The RNA produced from this construct contained no additional non-viral nucleotides. A similar construct that lacked the 3′ ribozyme, but which contained a correct 3′-terminal nucleotide, referred to as pT7 : MNV-G 3′Rp, was generated by ligation of the 3′ SacII–NheI fragment from pT7 : MNV-G 3′Rz with SacII- and SnaBI-digested pT7 : MNV. The resulting construct, pT7 : MNV-G 3′Rp, was identical to pT7 : MNV-G 3′Rz, but lacked the 3′ hepatitis delta virus ribozyme.To insert a hepatitis delta virus ribozyme into pT7 : MNV-G at the 3′ end of the genome and to repair the 3′-terminal nucleotide, a derivative of the ribozyme containing a 5′ NotI and SwaI and inserted into pTriEx1.1 (Novagen) between the NotI and MscI sites. The construct was then sequenced fully to confirm that it matched the reported CW1 passage 3 consensus sequence.The MNV 1 subgenomic RNA cDNA expression construct pT7 : MNV-SG was generated by RT-PCR amplification of RNA purified from MNV 1 CW1-infected cells, using primers IGIC21 and IGIC22 . The amplified product was digested with 50 in RAW 264.7 cells, using microscopic visualization for the appearance of cytopathic effect. In some cases, plaque morphology was also examined by plaque assay (50 on RAW 264.7 cells.Cells were infected with poxviruses expressing T7 RNA polymerase at an m.o.i. (based on the virus titre in chick embryo fibroblasts) of 0.5–1.0 p.f.u. per cell. Each cDNA expression construct or VPg-linked viral RNA, purified as described previously (−1), RNase A (10 units ml−1) and RNase T1 (400 units ml−1) at 37 °C for 2 h. Encapsidated RNA was partially purified by centrifugation through a 30 % sucrose cushion for 1 h at 55 000 r.p.m. using a Beckman SW55 Ti rotor. The viral pellet was DNase- and RNase-treated prior to extraction of the viral RNA, which used the GenElute system (Sigma). The viral RNA was detected by RT-PCR amplification using the primers 7155F and 7400R , resulting in the amplification of a 258 bp product.To remove unencapsidated MNV RNA or excess cDNA, clarified lysates from transfected cells were treated with DNase I . Seven overlapping PCR products were amplified, covering the entire genome, and the sequence was determined by using a series of primers  and that VACV infection can have a detrimental effect on MNV replication . To examine the requirement for the viral capsid proteins VP1 and VP2, cells were co-transfected with either empty vector or a cDNA construct containing the MNV subgenomic RNA (pT7 : MNV-SG). Western blot analysis demonstrated that high levels of NS7 were detected in cells transfected with either VPg-linked viral RNA or pT7 : MNV-G , but was detected readily when cells were transfected with an identical construct in which the frame shift had been repaired . Infectious virus was reproducibly produced only from cells that had been transfected with purified viral RNA or transfected with cDNA constructs for both the full-length genomic and subgenomic RNAs  at position 3959 resulted in the introduction of a BglII site . The recovered viruses, referred to as CW1-R and CW1-Bgl, were subsequently amplified in tissue culture, and RT-PCR analysis of the region encoding NS7 was performed by using primers 3734F and 4450R. BglII digestion of the 746 bp amplified products demonstrated the presence of a single BglII site in the amplicons from the parental and recombinant MNV, CW1 and CW1-R  was subsequently compared with that of the parental MNV CW1 by examining both plaque size and growth characteristics  cell line, due to its high efficiency of transfection and our observation that transfection of purified viral RNA resulted in the production of >10h Table 2, yieldedIn this report, we describe the generation of the first norovirus reverse-genetics system, using an FWPV recombinant to deliver T7 RNA polymerase. Previously described reverse-genetics systems for FCV  have relAlso worth noting is the fact that the T7 RNA polymerase in the MVA-T7 used in this study is under the control of a late (p11) poxvirus promoter .Our inability to recover MNV by transfection of s on FCV , PEC (ChDuring our study, we also examined the ability of the BSR-T7 cell line, a BHK cell line derivative that expresses T7 RNA polymerase constitutively, to allow the recovery of MNV in the absence of FWPV infection Table 2, similarGiven the previously reported role of interferon in controlling calicivirus replication , failed Our results would also suggest that the sequence of the 3′-terminal nucleotide of the MNV genome is critical for efficient recovery, as constructs containing an incorrect 3′-terminal nucleotide required the co-expression of the MNV subgenomic RNA for recovery (Fig. 3aSeroprevalence studies have highlighted that, as well as functioning as a model for the human noroviruses, MNV is a significant pathogen in its own right, with 22 % of mouse colonies in the USA and Canada found to be seropositive (


Saccharomyces cerevisiae) has been engineered for xylose utilization to enable production of fuel ethanol from lignocellulose raw material. One unresolved challenge is that S. cerevisiae lacks a dedicated transport system for pentose sugars, which means that xylose is transported by non-specific Hxt transporters with comparatively low transport rate and affinity for xylose.Baker's yeast (Candida intermedia, Pichia stipitis and Arabidopsis thaliana, respectively, were expressed in isogenic strains of S. cerevisiae and the transport kinetics and utilization of xylose was evaluated. Expression of the Gxf1 and Sut1 transporters led to significantly increased affinity and transport rates of xylose. In batch cultivation at 4 g/L xylose concentration, improved transport kinetics led to a corresponding increase in xylose utilization, whereas no correlation could be demonstrated at xylose concentrations greater than 15 g/L. The relative contribution of native sugar transporters to the overall xylose transport capacity was also estimated during growth on glucose and xylose.In this study, we compared three heterologous xylose transporters that have recently been shown to improve xylose uptake under different experimental conditions. The transporters Gxf1, Sut1 and At5g59250 from S. cerevisiae transporters increased significantly during cultivation in xylose and at low glucose concentration.Kinetic characterization and aerobic batch cultivation of strains expressing the Gxf1, Sut1 and At5g59250 transporters showed a direct relationship between transport kinetics and xylose growth. The Gxf1 transporter had the highest transport capacity and the highest xylose growth rate, followed by the Sut1 transporter. The range in which transport controlled the growth rate was determined to between 0 and 15 g/L xylose. The role of catabolite repression in regulation of native transporters was also confirmed by the observation that xylose transport by native  Saccharomyces cerevisiae) is used for industrial production of fuel ethanol from starch (corn) and sucrose (sugar cane). Lignocellulose biomass derived from forest and agricultural byproducts has been investigated as an alternative substrate, which does not compete with food and feed production  labeled xylose . The reactions were initiated by mixing 20 μL of buffered cell solution with 20 μL of radioactive substrate, giving final substrate concentrations between 3 and 150 mM. The reaction was quenched after 20 seconds by addition of 3.5 mL ice-cold 100 mM sorbitol solution. The resulting solution was immediately filtered through a 2.5 cm GF/C filter  and washed with 2 × 50 mL ice-cold water. Washed filters were transferred to scintillation vials containing 5 mL scintillation cocktail  and the corresponding radioactivity was measured in a liquid scintillation counter . The baseline radioactivity was obtained in parallel measurements by adding the radioactive substrate to the cell solution after the quenching solution had been added. All measurements were performed in biological duplicate for each strain and condition. Matlab software  was used for non-linear regression analysis of kinetic data.Initial xylose uptake rates were determined using a previously described method  with min620 of 0.2. Cells were grown at 30°C in an orbital shaker at 200 rpm. The growth rate was followed by measuring the optical density (OD) at 620 nm. All measurements were performed in biological duplicate for each investigated strain and each condition.Yeast strains were inoculated in 25 mL xylose medium  in 250 mL baffled shake flasks at a starting ODS. cerevisiae strain TMB 3662  xylose uptake. The measured xylose transport rate was plotted against substrate concentration and fitted to a single-component Michaelis-Menten equation by non-linear regression , all strains still displayed the same growth rate, but it was reduced to 0.04 h-1. At low xylose concentration (4 g/L), the specific growth rate was further reduced to 0.002 to 0.008 h-1. However, at this concentration, strains expressing the heterologous transporters grew significantly faster than the control strain . At this concentration, the strains expressing heterologous transporters and the control strain all displayed the same specific growth rate (μ), 0.16 h-S. cerevisiae strains expressing the Gxf1 and Sut1 transporters showed increased xylose transport performance compared with the control strain expressing only native transporters  increased when cells were grown at 0.5 g/L glucose (28 × 10-7 min-1 × mgDW-1) and 60 g/L xylose (13 × 10-7 min-1 × mgDW-1) compared with 20 g/L glucose (10 × 10-7 min-1 × mgDW-1). Cultivation at the low glucose concentration (0.5 g/L) yielded the highest xylose transport performance, which was comparable with cells expressing the Gxf1 transporter during cultivation on 20 g/L glucose   Figure . Express) Figure . The oventration . In agrentration . The indP. stipitis cDNA library in a S. cerevisiae HXT null strain and subsequent reconstitution of growth on glucose [P. stipitis [C. intermedia, and subsequent screening of a C. intermedia cDNA library in an S. cerevisiae HXT null strain identified the GXF1 transporter gene [S. cerevisiae but also the highest transport capacity.Among the transporters investigated in this work, the Gxf1 transporter clearly improved the xylose transport capacity and growth rate more than the Sut1 and At5g59250 transporters (Figure  glucose . By contstipitis . The higter gene . Thus, tS. cerevisiae strains under identical conditions. A direct relationship was seen between transport kinetics of the individual transporters and xylose utilization under transport limiting growth conditions. The relative xylose transport performance of native S. cerevisiae transporters was also found to be significantly increased during cultivation at low glucose concentrations, which points towards the importance of designing an appropriate cofermentation strategy for industrial ethanol production from lignocellulose biomass.In the current study, the transporters Gxf1, Sut1 and At5g59250 were compared in isogenic The authors declare that they have no competing interests.DR participated in the design of the study, performed the experimental work and wrote the manuscript. BHH participated in the design of the study and commented on the manuscript. PR participated in the design of the study and commented on the manuscript. All the authors have read and approved the final manuscript.


X-ray crystallographic and complementary functional studies have contributed significantly to the current understanding of gene transcription. Here, recent structure–function studies on various aspects of the elongation phase of transcription are summarized. in vitro transcription experiments led to a mechanistic understanding of various additional features of the EC, including its inhibition by the fungal toxin α-amanitin, the tunability of the active site by the elongation factor TFIIS, the recognition of DNA lesions and the use of RNA as a template.RNA polymerase II (Pol II) is the eukaryotic enzyme that is responsible for transcribing all protein-coding genes into messenger RNA (mRNA). The mRNA-transcription cycle can be divided into three stages: initiation, elongation and termination. During elongation, Pol II moves along a DNA template and synthesizes a complementary RNA chain in a processive manner. X-ray structural analysis has proved to be a potent tool for elucidating the mechanism of Pol II elongation. Crystallographic snapshots of different functional states of the Pol II elongation complex (EC) have elucidated mechanistic details of nucleotide addition and Pol II translocation. Further structural studies in combination with  Initial crystals  including the two additional subunits Rpb4 and Rpb7 were subsequently obtained but displayed a high solvent content of 80% and only diffracted to around 4 Å resolution. This resulted in a backbone model of the complete enzyme  and a second mobile metal ion  . The Brownian ratchet model of translocation assumes that Brownian motion gives rise to oscillation of the EC between pre-translocation and post-translocation states, establishing the translocation equilibrium. Substrate NTPs can only bind in the post-translocation state and would act like the pawl of a ratchet. X-ray structural evidence for the existence of the translocation equilibrium was recently obtained with an EC labelled with 5-bromouracil in the template strand . In this putative intermediate the DNA–RNA hybrid adopts a post-translocation state , whereas the state of the downstream DNA is intermediary between pre-translocation and post-translocation. The template base entering the active centre (the templating base) was found in a new ‘pre-templating’ position above the central bridge helix . Two Pol II elements, the trigger loop and the bridge helix, were observed in new conformations, suggesting their involvement in facilitating translocation. α-Amanitin apparently traps the trigger loop and bridge helix in these conformations with direct and indirect contacts, thereby inhibiting nucleotide incorporation and translocation. An independent study also revealed the direct contact between the trigger loop and α-amanitin and additionally showed a role of the trigger loop in substrate selection and fidelity , or intrastrand cross-links induced by the anticancer drug cisplatin  was combined with expertise in preparing functional crystallization-grade ECs of the complete 12-subunit Pol II (our group). Bulky DNA lesions were introduced into the DNA-template strand at several different positions around the polymerase active site and the resulting Pol II ECs were studied structurally Fig. 6 and in Rc; Tornaletti et al., 1997et al., 2004et al., 2007et al., 2007et al., 2004Pol II stalls when a CPD in the DNA-template strand reaches the enzyme active site after nucleotide incorporation opposite both CPD thymines . Now only UMP can be incorporated opposite the 5′-thymine ] forms 1,2-d(GpG) DNA intrastrand cross-links (cisplatin lesions) that stall Pol II and trigger transcription-coupled DNA repair . The cisplatin lesion can be stably accommodated in a Pol II EC at position +2/+3 of the template strand, but translocation to position +1/+2 is disfavoured . Modelling suggested that a conformational change of the bridge helix would be required to accommodate the lesion in the active site. Secondly, a G·A mismatch base pair would be formed at position −1 in contrast to a stabilizing T·A base pair in the case of the CPD lesion.The anticancer drug cisplatin [In conclusion, the mechanism of recognition by transcribing Pol II is different for the two dinucleotide lesions. At a cisplatin lesion, Pol II stalls because the lesion cannot be delivered to the active site, whereas it stalls at a CPD lesion after delivery to the active site and specific UMP misincorporation opposite the 5′-thymine. Bypass of the CPD lesion is only possible by artificially replacing the resulting T·U mismatch by a T·A match. Remarkably, bypass of the cisplatin lesion is also possible, but only by artificially providing a starting transcript that extends at least up to the 3′-guanine. In this case, bypass is even possible in presence of a G·A mismatch with the 3′-guanine of the cisplatin dimer.5.et al., 2007in vitro enzyme assays revealed that the RNA-dependent RNA polymerase (RdRP) activity resides in the site used during transcription, but is slower and less processive than the DNA-dependent activity. The RdRP activity of Pol II provides a missing link in molecular evolution, because it suggests that Pol II evolved from an ancient replicase that duplicated RNA genomes. There is compelling evidence that the ancient RdRP activity of Pol II is still relevant for the replication of the RNA genome of the hepatitis δ virus (HDV) and it may also be used in certain cellular processes as many organisms lack dedicated single-subunit RdRPs.Although Pol II generally uses DNA as a template, there is also evidence that Pol II can use RNA templates. Recent structures have shown that an RNA template–product duplex can bind to the site normally occupied by the DNA–RNA hybrid and provided the structural basis for the phenomenon of RNA-dependent RNA synthesis by Pol II (Lehmann 6.in vitro transcription experiments allowed exploration of the basic mechanisms of transcription elongation, including the nucleotide-addition cycle, and additional features such as the mechanism of TFIIS function, DNA-damage recognition and RNA-templated RNA synthesis. Further aspects of transcription elongation still await further characterization using the available system. Further investigations could focus on the regulation by additional protein factors or RNA molecules, transcriptional mutagenesis and fidelity and the effect of other kinds of DNA lesions, e.g. oxidative lesions, to name a few.Combining X-ray crystallographic analysis of Pol II ECs with


Elevated levels of FMR1 mRNA in blood have been implicated in RNA toxicity associated with a number of clinical conditions. Due to the extensive inter-sample variation in the time lapse between the blood collection and RNA extraction in clinical practice, the resulting variation in mRNA quality significantly confounds mRNA analysis by real-time PCR.Here, we developed an improved method to normalize for mRNA degradation in a sample set with large variation in rRNA quality, without sample omission. Initially, RNA samples were artificially degraded, and analyzed using capillary electrophoresis and real-time PCR standard curve method, with the aim of defining the best predictors of total RNA and mRNA degradation.FMR1, DNMT1, GUS, B2M and GAPDH) occurred during the early to moderate stages of degradation; (ii) chromatographic features for the 18S, 28S and the inter-peak region were the most reliable predictors of total RNA degradation, however their use for target gene normalization was inferior to internal control genes, of which GUS was the most appropriate. Using GUS for normalization, we examined in the whole blood the relationship between the FMR1 mRNA and CGG expansion in a non-coding portion of this gene, in a sample set (n = 30) with the large variation in rRNA quality. By combining FMR1 3' and 5' mRNA analyses the confounding impact of mRNA degradation on the correlation between FMR1 expression and CGG size was minimized, and the biological significance increased from p = 0.046 for the 5' FMR1 assay, to p = 0.018 for the combined FMR1 3' and 5' mRNA analysis.We found that: (i) the 28S:18S ratio and RNA quality indicator (RQI) were good predictors of severe total RNA degradation, however, the greatest changes in the quantity of different mRNAs (FMR1 mRNA toxicity in blood, it may also have more general implications for gene expression studies in fresh and archival tissues.Our observations demonstrate that, through the use of an appropriate internal control and the direct analysis of multiple sites of target mRNA, samples that do not conform to the conventional rRNA criteria can still be utilized to obtain biologically/clinically relevant data. Although, this strategy clearly has application for improved assessment of  FMR1 gene [FMR1 mRNA which is believed to be pathogenic in these conditions, has been supported in animal models where it leads to increased cell death in Drosophila [FMR1 mRNA levels in carriers of small CGG expansions, apart from being essential in the mechanisms involved in RNA toxicity, may also have a diagnostic and prognostic significance.The mutations in an X linked MR1 gene -4. FragiMR1 gene . It is pMR1 gene -8. In feMR1 gene . A toxicosophila  and miceosophila . Althougosophila , and psyosophila  manifestFMR1 expression is primarily regulated by the state of methylation of its promoter. DNMT1 is an enzyme pivotal in this process [FMR1 related disorders. Thus, expression of both genes, FMR1 and DNMT1, is of interest in fresh and archival samples from individuals with diseases characterized by abnormal expansions within the CGG region. Unfortunately, archival samples and samples extracted from blood have variable total RNA and mRNA degradation, which we have found to be a confounding factor in the gene expression analysis, unless a method was established to normalize for FMR1 and DNMT1 mRNA degradation. process -16. ThisFMR1 and DNMT1 have utilized GUS and HPRT as internal control genes [Previous real-time PCR studies normalized gene expression against total cell number, total RNA or mRNA of internal control gene/s, reviewed in . The maiol genes ,21,22. Hol genes .18S, 28S and 5S, and/or by examining the 28S:18S ratio and the RNA Qiality Indicator (RQI) values. However, these approaches have their limitations which we addressed in the study. We also evaluated in depth the use of internal control genes and the capillary electrophoresis features for the normalization of mRNA degradation in partially degraded and intact RNA from patient lymphoblasts and blood. We then defined the best predictor of target gene mRNA degradation, utilizing it for normalization in the whole blood RNA with a high variability of rRNA quality. In this sample set we examined the relationship between the FMR1 mRNA and the CGG expansion in a non-coding portion of this gene, and found that by incorporating the 3' and 5' mRNA analysis data from patients with small to intermediate CGG expansions, we have significantly improved upon the current approach used to examine the FMR1 mRNA toxicity, demonstrating the clinical relevance of our strategy.In this study the Experion system (Bio-rad) was used to assess the quality of rRNA by subjective/visual interpretation of the chromatographs for absence of peaks other than those of FMR1 alleles – 6 to 40 repeats); grey zone carriers (41 to 55 repeats), premutation carriers (55 to 200 repeats) and full-mutation carriers (>200 repeats) [6 cell/ml in RPMI medium /10% Foetal bovine serum (CSL) at 37°C, 5% CO2 overnight. Cell pellets were resuspended in RNA lysis buffer, , and stored at -80°C until RNA extraction.Fourteen EBV transformed lymphoblast cell lines from healthy controls (repeats) -26 were Total RNA was extracted and purified using the RNeasy extraction kit . RNA concentrations were measured in triplicate using a NanoDrop ND-1000 Spectrophotometer, with purity being determined by the A260/A280 ratio using the expected values between 1.8 and 2. Each RNA sample was then diluted to 2 ng/ul. Sixty ul of each sample was left at room temperature for 0, 18, 24 and 96 hours. At each time point an aliquot of 15 ul was collected from each sample, 5 ul of which were used for capillary electrophoresis (Bio-rad) and 2 ul for cDNA synthesis.FMR1 expression and CGG expansion was approved by the La Trobe University Human Ethics Committee, the Southern Tasmanian Health and Medical Human Research Ethics Committees, and by the Institutional Review Board of the University of California at Davis. The methods used for CGG repeat sizing and FMR1 mRNA assessments, as well as the results of correlation between these two measures in a larger sample, which included the present participants, were reported in the earlier study [FMR1ex3.4, FMR1ex13.14 and GUS.The original study that investigated the relationship between er study . An aliqer study . After mTotal RNA quality was assessed using the RNA HighSens Kit as per manufacturer's instructions (Bio-rad). Objective and subjective analysis of each RNA profile was performed. Objective assessment involved automatic collection of a number of  parameters which included percentage area under the lower marker (LM), small fragment region (SR), 5S, fast region (FR), 18S, Inter region (IR), 28S, post region (PR), the 28S:18S ratio and RQI Figure . SubjectFMR1ex3.4, FMR1ex13.14, DNMT1, GAPDH, B2M, and GUS, using the relative standard curve method. The target gene and the internal control gene dynamic linear ranges were performed on a series of doubling dilutions of RNA (160-0.5 ng/ul) of a selected peripheral bood mononuclear cell sample. Previously published sequences were used for RT-PCR primers and TaqMan probe for: FMR1exon3/4 and GUS [FMR1exon13/exon14 [DNMT1 [FMR1exon3/4, FMR1exon13/14 and DNMT1 primers and probes were used at concentrations of 18 uM and 2 uM, respectively. GAPDH and B2M primer/probe mixes were obtained from PrimerDesign (PerfectProbe ge-PP-12-hu kit) and used at concentration of 2 uM. Each sample was assayed in duplicate 10 μl PCR reactions, consisting of 5.8 mM MgCl2, 1 μl Buffer A (Applied Biosystems), 3.35 μl RNase-free water, 1.2 mM dNTPs, 0.01 units/μl of AmpliTaq Gold, 0.5 μl of TaqMan probe and 0.5 μl forward and 0.5 μl reverse primers, and 1 μl of the reverse transcription (cDNA) reaction. The annealing temperature for thermal cycling protocol was 60°C for 40 cycles. Samples were quantified in arbitrary units (au) in relation to the standard curves performed on each plate.Reverse transcription was performed one reaction per sample using the Multiscribe Reverse Transcription System, 50 units/ul (Applied Biosystems). The 7900HT Fast Real Time PCR (Applied Biosystems) was used to quantify 3/exon14 , and DNM4 [DNMT1 . FMR1exoFMR1 and DNMT1 mRNA to 18S, 28S, GAPDH and GUS, expressed as a function of the degradation time, was assessed by fitting the values to a simple linear regression. The sign test was then used to examine the null hypothesis that the median slope was zero against the two-sided alternative of it not being equal to 0. The relationship between the qPCR results for FMR1ex3.4 and FMR1ex13.14 assays with the CGG expansion size was assessed using a significance test in a linear regression. The analysis was conducted using the publicly available R statistical computing package, version 1.191 .Generalized estimating equations (GEE) were used for the objective assessment of the chromatographs and mRNA integrity. Normalization of FMR1 and DNMT1 mRNA quantities were examined using the relative standard curve method, in RNA samples 496, 497, 488, 584 and 585 artificially degraded at room temperature for 0, 18, 24 and 96 hours from 5 lymphoblast cell lines (Table FMR1 (5' and 3') and DNMT1 . This was also reflected in the 28S:18S ratios and RQI values, where there was no change between 0 and 24 hours, but a large decrease between 24 and 96 hours. In contrast, FMR1ex13.14 mRNA level decreased by more than half within the first 18 hours and remained at this level for the following time points. A similar trend was observed for FMR1ex3.4 mRNA, which decreased between 18 and 24 hours of incubation, and remained at the same level through to 96 hours. In contrast, there were no major changes in DNMT1 mRNA quantities for sample 496 throughout the time course.Sample 496 Figure ; Panel I28S peak at 0 hours  could be used as predictors of the total RNA degradation as reflected by the degradation time. The second aim was to objectively delineate whether FMR1 and DNMT1 mRNA degradation correlated with the degradation time, and if not, which features could be used as predictors of the target gene mRNA integrity.Objective assessment of total RNA and mRNA degradation was performed in RNA samples from 14 cell lines , best represented by the small fragmentation region % area. In contrast, B2M mRNA could only be used to predict early total RNA degradation (p = 0.093). Furthermore, it was not associated with any other chromatographic and gene expression parameters examined. For the target transcripts, DNMT1 and FMR1ex3/4, mRNA degradation was closely associated with moderate to severe degradation of total RNA (p < 0.001), and was best predicted by the small fragmentation region % area, and by GUS and GAPDH mRNA, all of which had similar degradation kinetics. In contrast, FMR1ex13.14 mRNA degradation appeared to be closely associated with early, moderate and severe total RNA degradation (p < 0.001), and was best predicted by % areas of 18S, 28S and the inter-peak region (p < 0.001). This analysis demonstrated that the degradation of mRNA was different between most internal controls and target genes examined, and that the degradation kinetics of specific mRNAs were not necessarily the same as those for total RNA and rRNA.FMR1 and DNMT1 mRNA degradation as determined by the objective assessment of chromatographs and mRNA integrity. 18S % area, 28S % area, GUS mRNA or GAPDH mRNA quantity were used for normalization, and expressed as a function of the degradation time. The normalization method that provided the most constant (least significant) values throughout the time course was considered as the most optimal of the predictors tested for the target gene mRNA degradation  and 28S (p = 0.066) % area were poor normalization features compared to GAPDH (p = 0.388) and GUS (p = 0.774). Similarly, for FMR1ex3.4, both 18S (p = 0.11) and 28S (p = 0.11) % area normalization provided less constant values than GAPDH (p = 0.51) and GUS (p = 0.254). For FMR1ex13.14, 18S (p = 0.11) and 28S (p = 0.11) % area as well as GAPDH (p = 0.11) normalization provided less constant values than GUS (p = 0.34). Thus, it appeared that normalization of the target genes to 18S and 28S chromatographic features was overall inferior to the use of internal control genes, GAPDH and GUS. Although 18S and 28S % areas could be still be used as normalization markers for FMR1ex3.4 and FMR1ex13.14, the most optimal of the predictors tested for both DNMT1 and FMR1 was GUS.The target gene expression in the RNA samples from the cell lines were normalized to the best predictors of FMR1 expression was significantly elevated in carriers of CGG expansion, compared with normal controls of a similar age, and that the expression was proportional to the size of CGG expansions within the grey zone and lower premutation range [We have previously demonstrated using freshly extracted RNA, that on range . SubsequFMR1ex3.4/GUS mRNA levels assessed here closely corresponded to the levels of the earlier study in freshly extracted RNA samples[FMR1ex3.4/GUS and FMR1ex13.14/GUS assays indicating that most samples had intact FMR1 mRNA    Figure  indicati) Figure .FMR1ex3.4/GUS and FMR1ex13.14/GUS assays , did not follow the common pattern of correlation between the s Figure , and thee Figure . Two of e Figure . Furthers Figure  and 4C. s Figure  and 4D. FMR1 mRNA in a sample set showing large variability in rRNA quality, and demonstrate it's clinical/biological relevance. Previous studies have normalized FMR1 expression to GUS [GUS as an internal control for our sample set, where we have observed a large variation in rRNA degradation, particularly as there are no previous studies that examined the rates of mRNA degradation for both FMR1 and GUS. Since different mRNA species degrade depending on their length and secondary structure [GUS was a suitable control for FMR1 mRNA degradation, and if not, which of the capillary electrophoresis and real-time PCR parameters would provide a better normalization method. Because of the potential relevance of DNMT1 to FMR1 gene regulation [DNMT1 as as target gene in some of the studies.The specific aim of this study was to establish an optimal method to normalize for the degradation of target gene n to GUS ,21. Howetructure ,23,29, wgulation -33, we hWe initially demonstrated using the Experion system that for the artificially degraded RNA samples, both the 28S:18S ratio and the RQI were most useful as predictors of severe RNA degradation, whereas the greatest changes in stability of different transcripts examined occurred during early to moderate stages of RNA degradation. Thus, the 28S:18S ratio and the RQI were not suitable predictors of mRNA stability, at least in our settings. Since, the RQI is closely related to a more widely used RNA Integrity Number (RIN) from an analogous Agilent system , our findings suggest that RIN may be also inappropriate as a normalization tool in our settings. Furthermore, the subjective assessment of general chromatographic features did not provide a useful estimation of mRNA degradation, as real-time PCR could still be used to obtain biologically relevant mRNA data in samples with chromatographs indicating severe rRNA degradation. In another approach, we determined that of the 10 selected chromatographic features, 18S, 28S and the inter-peak region % areas were the most reliable predictors of total RNA degradation when examined as a function of the degradation time. However, the normalization of the target genes to 18S and 28S chromatographic features was found to be inferior to the use of the internal control genes.Primarily these observations indicate that the degradation kinetics of rRNA may be heavily size dependent, as the small 5S rRNA subunit, 160 nucleotides, was found to be a good predictor of only early to moderate RNA degradation, whereas the 18S and 28S % areas, 1770 and 3770 nucleotides respectively, could be used to predict RNA degradation at early, moderate and late stages of degradation. For mRNA quantitation, by qPCR, however, it has been previously suggested that the length of the amplicon, rather than that of the whole mRNA molecule, may be a more important indicator of degradation kinetics, particularly because fragmentation of a long mRNA may only result in a loss of the molecule for qPCR detection if the RNA break occurs within the product sequence .FMR1ex3.4 (5') and FMR1ex13.14 (3') qPCR data throughout the RNA degradation time course. Since most FMR1 transcripts contain the 195-bp exon 14 [FMR1 transcripts of a similar size and abundance, which is experimentally reflected by the highly significant correlation between the two assays for high quality total RNA samples from the peripheral blood of patients with small to intermediate size expansions  with small to intermediate size expansions in RNA samples of high total RNA quality.Click here for file


Ex7 and CAREx8). We found low-level expression of the CAREx8 isoform in well-differentiated human airway epithelia. Surprisingly, in contrast to CAREx7, CAREx8 localizes to the apical membrane of epithelia where it augments adenovirus infection. Interestingly, despite sharing a similar class of PDZ-binding domain with CAREx7, CAREx8 differentially interacts with PICK1, PSD-95, and MAGI-1b. MAGI-1b appears to stoichiometrically regulate the degradation of CAREx8 providing a potential mechanism for the apical localization of CAREx8 in airway epithelial. In summary, apical localization of CAREx8 may be responsible for initiation of respiratory adenoviral infections and this localization appears to be regulated by interactions with PDZ-domain containing proteins.Adenovirus is an important respiratory pathogen. Adenovirus fiber from most serotypes co-opts the Coxsackie-Adenovirus Receptor (CAR) to bind and enter cells. However, CAR is a cell adhesion molecule localized on the basolateral membrane of polarized epithelia. Separation from the lumen of the airways by tight junctions renders airway epithelia resistant to inhaled adenovirus infection. Although a role for CAR in viral spread and egress has been established, the mechanism of initial respiratory infection remains controversial. CAR exists in several protein isoforms including two transmembrane isoforms that differ only at the carboxy-terminus (CAR The Coxsackievirus and Adenovirus Receptor (CAR) plays a vital role in cell adhesion and viral infection Alternative splicing plays an important role in eukaryotes. During pre-mRNA splicing, the spliceosome cleaves intron sequences, and joins exons together, forming an mRNA. Regulation of the spliceosome can result in alternative splicing of mRNA, determining which exons are present or absent in template mRNA. Alternative splicing not only regulates protein expression, but also allows multiple proteins to be expressed from the same gene resulting in significant proteomic diversity CAR is encoded by a highly conserved, alternatively spliced gene with five described transcripts. Three alternative transcripts encode CAR, lacking the transmembrane domain, yielding a soluble extracellular domain  et al as a 7 exon protein Human CAR was first described by Bergelson th exon form occurs within the 7th exon. Thus, these two isoforms contain identical extracellular and transmembrane domains, which predicts identical adenovirus binding and serotype preference. The majority of the cytoplasmic domain is identical except for the last 26 (CAREx7) or 13 (CAREx8) amino acids. Although comprised of distinct sequences, the last 4 amino acids of both isoforms encode class I PSD95/DlgA/ZO-1 (PDZ) binding domain sequences (-X-(S/T)-X-Ф, where X  =  any amino acid and Ф  =  any hydrophobic amino acid). Interacting PDZ-domain-containing proteins for human CAREx7 include MAGI-1b, PICK1, PSD-95, MUPP-1, LNX1, and ZO-1 Ex7 and CAREx8 interact with LNX2 and this interaction appears to be modulated by both the PDZ binding domain of each isoform as well as an upstream sequence common to both isoforms The splicing event to create the 8Ex8 has never been described. We used a computational approach to identify human exon 8 encoding CAR, and investigated the functional significance of this isoform in primary human airway epithelia.In contrast with the murine isoform, the protein for human CARwww.genome.ucsc.edu). Similar sequences were identified for human, chimpanzee, dog and rat (http://www.bork.embl-heidelberg.de/polyPhen/), which assigns scores, derived from likelihood matrices, as “benign”, “possibly damaging”, “probably damaging”, or “unknown”. The change P343A was designated as “benign.” Although an intuitive difference between these two amino acids exists, based on these analyses, additional studies to determine whether protein function was affected were not pursued. Primers were designed for RT-PCR to evaluate the presence of hCAREx8 in several human cell lines including HeLa, 293, A549, Caco-2, and primary human airway epithelia. Transcripts for both hCAREx7 and hCAREx8 were detected in all cell lines and primary cells examined (data not shown). A panel of human RNA was subsequently screened for the full-length hCAREx7  or lung tissue (in vivo), RNA was extracted from human airway epithelia greater than two weeks of age or total lung tissue, respectively, and subjected to quantitative RT-PCR XΦ) at the C-terminus , these and the upstream sequences are distinct. Thus we hypothesized that PDZ-interactions may be responsible for altered localization in human airway epithelia. We have previously shown that hCAREx7 interacts with PICK1 and PSD-95 via a PDZ binding domain specific interaction such that co-localization by immunocytochemistry reveals hCAREx7 is able to pull these proteins out of the cytoplasm and co-localize at the junctions between cells. We further hypothesized that hCAREx8 may interact with some hCAREx7 partners. COS-7 cells were co-transfected with hCAREx8 and PSD-95-GFP and immunocytochemistry was performed to determine localization. hCAREx8 localized to the junctions as observed in Ex8, PSD-95-GFP localizes diffusely within the cytoplasm Ex8, the localization of PSD-95-GFP was altered to co-localize with hCAREx8 at the junctions of cells (Ex8 (hCAREx8-PDZ) was similar to full length hCAREx8 . These data suggest that a transient interaction between MAGI-1b and hCAREx8 results in the disappearance of hCAREx8.hCARI-1b-GFP . To investaining  was presstaining . Most ofunctions . The preng cells .This wasng cells . hCAREx8Ex8, we hypothesized that there would be a stoichiometric relationship between these proteins. COS-7 cells were electroporated with a dose response of hCAREx8 (0-30 µg plasmid DNA) in the presence of constant (15 µg plasmid) MAGI-1b-GFP or GFP. hCAREx8 expression was evaluated by immunocytochemistry (red fluorescence) using fluorescence microscopy and Western blot. Fluorescence intensity was calculated as average pixel count per field of view . Moreover, no effect was observed on GFP or MAGI-1b-GFP fluorescence upon increasing hCAREx8 (data not shown).Since some MAGI-1b-GFP expressing cells also expressed some, albeit mislocalized, hCARw n = 6, . Increasrescence . In contpression , and revEx8 in the presence of MAGI-1b-GFP, CHO-K1 cells were transfected with hCAREx8 or MAGI-1b-GFP, or co-transfected with hCAREx8, at a constant dose, with MAGI-1b-GFP at increasing doses. All transfections were balanced with GFP plasmid to maintain equal amounts of DNA. Transfected cells were transduced with Ad-β-gal 48h later. β-galactosidase expression was determined 24 hr post-transduction . To determine the requirement of the hCAREx8 PDZ binding domain, a stop codon was added to the cDNA of hCAREx8 by site-directed mutagenesis resulting in a protein missing the last 4 amino acids (-ITVV). Similar to wild-type hCAREx8  or the hCAREx8 was swapped with hCAREx7 . hCAREx7, hCAREx8, hCAREx7/8, or hCAREx8/7 were each co-transfected with PICK1-GFP. As previously shown, co-transfection of COS-7 cells with hCAREx7 and PICK1-GFP results in accumulation of hCAREx7 at the junctions of cells  inhibitors, a finding with significant implications for oncolytic adenoviral cancer therapy RNA splicing is an important event that significantly increases proteomic diversity and may impart cell specificity. Despite the importance of hCAR in viral infection, cell adhesion, and development, the genetic and splicing regulation of the gene for hCAR, Several transcripts have been described for hCAR. However, the importance of each splice variant is unclear. Secreted splice variants that interact with the extracellular domain of the transmembrane form of hCAR could clearly alter homophilic transmembrane hCAR interactions and hence modulate junctional remodeling. These variants could also play a physiological role modulating interactions between hCAR and its ligand, AMICA1/JAML, found on transmigrating lymphocytes and dendritic cells Ex7 isoform, only one interaction has been described for the alternative, less prevalent mouse CAREx8 isoform. Both mouse CAREx7 and CAREx8 interact with Ligand-of-Numb Protein-X2 (LNX2), an intracellular scaffolding protein that may play a role in Notch signaling Considering the importance of junctional organization, interactions, and signaling, alternative transmembrane forms could play equally important roles in junctional remodeling and responses to ligation with soluble isoforms or ligands on other cell types. Two transmembrane isoforms are known for CAR. Although several interactions have been discovered for both the mouse and human CAREx7 results in junctional localization of MAGI-1b Ex8 is unique to other MAGI-1b interactions since it results in the loss of hCAREx8. Epithelia express MAGI-1b where it localizes to the basolateral junctions. hCAREx7 also localizes to the basolateral junctional adhesion complex. The data presented herein is consistent with MAGI-1b interacting with hCAREx7 at the junctions between epithelial cells. It is also consistent with the unexpected apical localization of hCAREx8. If hCAREx8 were to go to the basolateral junctions, it would be predicted to interact with MAGI-1b and be degraded. Thus the only place it could exist in the cell is in an apical compartment devoid of MAGI-1b. Alternatively, there may be a specific mechanism for the apical localization of hCAREx8 that remains to be discovered. Apical localization is consistent with elevated adenovirus infection after expression of hCAREx8. Thus this isoform of hCAR would be able to interact with adenovirus on the luminal air-exposed surface and mediate the initial infection of an epithelium. It is notable that the increase of infection is similar to the extracellular domain of hCAR conjugated to a glycophosphatidyl-inositol (GPI) tail, which explicitly is apically localized in airway epithelia In vitro, the C-terminus of hCAR is not required for adenovirus infection in vivo. Furthermore, it is interesting to speculate that mutations altering the splicing or transcript abundance of the hCAREx8 isoform may be responsible for viral susceptibility.Our previous work revealed that interaction with hCAREx7, CFTR traffics to the apical membrane of airway epithelia where it behaves as a chloride channel. Interactions between the PDZ-binding domain of CFTR and the PDZ domain of Na+/H+ exchanger-3 regulatory factors 1 and 2 (NHERF1 and NHERF2) act to stabilize CFTR at the cell surface et al have recently shown that the delicate balance of interactions regulating cell surface maintenance and lysosomal degradation upon cycling is due to the relative affinity of the PDZ interactions Ex7) appears to be dominant in directing the localization of the PDZ-domain containing proteins while MAGI-1b is dominant in the interaction with hCAREx8 resulting in the loss of this protein. To our knowledge, all descriptions of PDZ-directed degradation have been during mature protein cycling. In contrast, we have observed immediate loss of hCAREx8 implying regulation during early protein synthesis or quality control stages.PDZ-based regulation has been described for other membrane proteins Ex7 may be a gatekeeper for neutrophil transmigration but hCAREx8 may either tether neutrophils to the apical surface or sense how many neutrophils are present. Alternatively, these two isoforms may play a role in dendritic cell surveillance and maintaining the seal around dendritic cell filopodia.The question remains why differential compartmentalization of these hCAR isoforms would exist. The signals transduced by these isoforms, despite having the same extracellular ligands, may differ. Differential localization of these two isoforms could result in the cell being able to discern whether the signal is from the apical or basolateral compartment and mount the appropriate response. For example, both isoforms would recognize AMICA1/JAML present on the surface of neutrophils and dendritic cells. hCAREx8 alters the resulting interaction such that PICK1 does not interact, PSD-95 does interact to localize at cellular junctions, and MAGI-1b results in degradation of the hCAREx8 protein. In contrast, the 22 amino acids from hCAREx7 allow a stable interaction between these three proteins and the ITVV PDZ-sequence from hCAREx8 resulting in co-localization at the junctions of cells. The exact mechanism requires further investigation and will lead to a greater understanding of this important class of protein interaction domains.These data also provide support for the importance of sequences upstream of the PDZ binding domain in dictating target PDZ domains and subsequent activity. Both the GSIV (Ex7) and ITVV (Ex8) sequences bind all three targets investigated, MAGI-1b, PICK1 and PSD95. However, the presence of the upstream 9 unique amino acids from hCAREx8 localizes to the apical surface where it may play a key role in the initiation of adenoviral, and potentially coxsackievirus, pulmonary infection. We propose a model for the regulation of this localization based upon isoform-specific PDZ binding domain interactions with MAGI-1b. Although PDZ based interactions are known to be key regulators of membrane microdomain structure, stability and signaling, we have shown that this interaction may also regulate protein expression. Furthermore, PDZ based interactions are influenced by several factors, including PDZ binding domain and upstream sequence context. Affinity of interaction between the PDZ domain and PDZ binding domain may be higher or lower depending on surrounding sequences resulting in the specificity of interaction. Surprisingly, either interacting partner can be dominant in dictating the result of the interaction . Further elucidation of these mechanisms may provide a novel target for either down regulation of the adenovirus receptor to limit viral infection or alternatively up regulation for the purpose of adenoviral-based therapies.In summary, human airway epithelia express several isoforms of hCAR. Importantly, human CARThis study was conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by the Institutional Review Board of the University of Iowa (IRB ID No. 9507432). Primary human airway epithelia were isolated from discarded and deidentified trachea and bronchi of donor lungs. This study used discarded lung tissue, thus the IRB deemed consent was not needed.Ex7 5490 and Rabbit anti-hCAREx8 5678 were produced in rabbits immunized with peptides of 13 c-terminal amino acids (CVMIPAQSKDGSIV and FKYPYKTDGITVVC respectively). COS-7 cells were from ATCC , and maintained under standard culture conditions . CHO-K1 cells were from BD Biosciences  and maintained under standard culture conditions . Ad serotype 5 containing the β-galactosidase , eGFP, RFP , or hCAR gene have previously been described FLAG M2 antibody (Ab) was purchased from Sigma , mouse anti-CD55 (DAF) was from BD Pharmingen , mouse anti-Ezrin was from Santa Cruz , mouse anti-acetylated α-tubulin was from Sigma , Alexa-488 and -568 conjugated goat anti-mouse or anti-rabbit Abs, mouse and rabbit anti-GFP were from Molecular Probes . RmcB Ab  was produced by the University of Iowa Hybridoma Core. Rabbit anti-hCAR 1605 was produced in rabbits immunized with a GST fusion to the intracellular c-terminus (aa 261–365) as previously described www.genome.ucsc.edu) to determine the human sequence for exon 8. Comparison of hCAR P343 to mCAR A343 using Conseq software identified this amino acid as an exposed or buried residue respectively with a non-structural role. Conservation could not be determined due to insufficient data. The score assigned by Conseq was validated using PolyPhen (http://www.bork.embl-heidelberg.de/polyPhen/).We performed a BLAT search with the mouse sequence . The fragment was digested with EcoRI and BamHI and cloned into pcDNA3.1(-) or an Ad5-CMV shuttle plasmid. DNA sequencing and Western blot confirmed the expected protein. hCAREx8 was modified with two FLAG tags (DYKDDDDK) added between amino acid 22 and 23, similar to previously described modifications in hCAREx7. Site-directed mutagenesis was performed to generate hCAREx7/8 according to manufacturer's standard protocol  with the following primer: GSIV-ITVV, 5′-CCAGCACAGAGCAAGGATATCACTGTAGTATAGGGATCCGAGCTC.hCAREx8/7 according to manufacturer's standard protocol  with the following primer: ITVV-GSIV, 5′-CCTTACAAGACTGATGGAGGTTCAATTGTATAAGGATCAAGGGTGSite-directed mutagenesis was performed to generate CAREx8-PDZ according to manufacturer's standard protocol  with the following primer: ITVV-*TVV, 5′-CCTTACAAGACTGATGGATAAACAGTTGTATAAGGATCAAGGGTGG.Site-directed mutagenesis was performed to generate hCAREx7 or hCAREx8 from the basolateral surface as previously described Primary human airway epithelia were isolated from trachea and bronchi of donor lungs and seeded onto collagen coated, semi-permeable membranes  and grown at the air-liquid interface as previously described Ex7, hCAREx8 or eGFP using the Amaxa Nucleofector I  according to manufacturer's standard protocol for primary mammalian epithelial cells . Approximately 3×105 cells were seeded onto collagen coated, semi-permeable membranes as described above. Epithelia were infected with Ad-β-gal (MOI 10 pfu/cell) for 1 hr at 37°C, washed twice and lysed 48 hr post infection. β-galactosidase expression per mg protein was determined as previously described Primary airway epithelial cells seeded on plastic were trypsinized, washed and cells electroporated with 2.5 µg of plasmids encoding hCARChinese hamster ovary cells were plated in 24 well dishes and transfected using Lipofectamine 2000  following the manufacturer's protocol. Twenty-four hours after transfection cells were infected with Ad serotype 5 containing the β-galactosidase gene  (MOI 100) for 1 hr at 37°C. 48 hours later cells were lysed and β-galactosidase expression per mg protein was determined as previously described 2, 10mM K2HPO4, 10mM KH2PO4, 25mM HEPES, 2mM EGTA, 5mM MgCl2, 2mM ATP and glutathione) and put in an electroporation cuvette  for 30 minutes on ice. After electroporation, cells were seeded onto 10cm dishes for immunoprecipitation (IP) and collagen coated glass chamber slides for immunofluorescence studies 2 days later.COS-7 cells were electroporated by standard methodologies. Briefly, 10 million cells were mixed with 20 µg of plasmid DNA for single transfection, 15 µg of each DNA for double transfections, or 10 µg of each for triple transfections, in 400 µl of cytomix . Cells were incubated with primary Ab, washed extensively and incubated with goat anti-mouse Alexa-568 secondary Ab. After washing, slides were coverslipped with Vectashield mounting media . Images were acquired with a BioRad MRC-1024 Laser Scanning Confocal Microscope  mounted on a Nikon E600 microscope  using a 60X oil immersion lens. Fluorescence imaging was performed on an Olympus IX71 X-Cite 120 fluorescence microscope  followed by quantitation using Image J.CXADR were designed with Primer Express software (Applied Biosystems). The sequences for hCAREx7 were: hCAREx7-F, 5′-TGCCAGAAGCTACATCGGCAGTAA; hCAREx7-R, 5′-ATAGACCCATCCTTGCTCTGTGCT; hCAREx7 probe, 5′-AAGTCGAATGGGTGCGATTCCTGTGA ; PCR product 141bp. The sequences for the hCAREx8 were: hCAREx8-F, 5′-AGGGAAGATGTGCCACCTCCAAA; hCAREx8-R, 5′-CAACTGTAATTCCATCAGTCTTGTAAG; hCAREx8 probe, 5′-ACTGCCAGAAGCTACATCGGCAGTAA ; 165bp. Samples were run using TaqMan Fast Universal PCR Master Mix (Applied Biosystems) on a 7500 Fast Real-Time PCR System (Applied Biosystems). Transcript number was quantitated by plasmid standard curve. Abundance relative to 18s  or hGAPDH  as the reference gene confirmed the presence of significantly more hCAREx7 transcripts than hCAREx8.RNA was isolated from primary airway cultures greater than 2 weeks old or primary lung tissue using TRIzol with the Pure Link RNA kit (Invitrogen). cDNA was synthesized using RT2 EZ First Strand Kit (SA Biosciences). Primers and probes for the seventh or eighth exon of 3 Darkroom .Cells from two 100mm plates were placed on ice, washed once with ice cold PBS, and lysed with lysis buffer  leupeptin, aprotinin, pepstatin, and 1mM phenylmethylsulfonyl fluoride) by rocking at 4°C. Cells were scraped, sonicated 5 times and spun in a microcentrifuge at full speed for 10 minutes. For co-immunoprecipitation, supernatant was incubated with the indicated Ab with rotation at 4°C overnight. Protein A or G conjugated sepharose  was added for 1-2 hours followed by a wash with lysis buffer, 10% lysis buffer in TBS , and TBS. Beads were suspended in loading buffer  and proteins were separated by SDS-poly acrylamide gel electrophoresis. Gels were transferred to a polyvinylidene difluoride membrane , blocked with 5% BSA, washed, probed with primary Ab as indicated, followed by washing and incubation with protein A or G conjugated HRP . Bands were detected with ECL reagents  and imaged on the EpiChemiFigure S1Endogenously expressed CAREx8  in polarized human airway epithelia localizes above ZO-1  and co-localizes with the apical protein ezrin . Sections are shown in X-Y (A-F) or X-Z (G) axes. Confocal microscopy (60x oil immersion).(9.08 MB TIF)Click here for additional data file.Figure S2Quantitative RT-PCR primers for CAREx7 or CAREx8 are specific. Primary airway were either mock transduced or transduced with adenovirus carrying the gene for CAREx7 or CAREx8. RNA was isolated 36 hours later and subjected to isoform specific quantitative RT-PCR. Under mock conditions there was more endogenous CAREx7 than CAREx8. Epithelia transduced with CAREx7 or CAREx8 showed increased trascript levels but did not increase transcript levels of the other isoform.(2.91 MB TIF)Click here for additional data file.Figure S3Expression of exogenous CAREx8 in polarized human airway epithelia mediates five-fold greater Ad-β-Gal gene transfer than endogenous expression . *p<0.0001 Ad-CAREx8 vs. Mock/Ad-β-Gal or Mock/no virus. p = 0.03 Mock/Ad-β-Gal vs. Mock/no virus.(2.50 MB TIF)Click here for additional data file.