A1 Binding Sites Research Articles

Calculated vibrational properties of pigments in protein binding sites 2: Semiquinones in photosynthetic proteins

[QA– – QA] Fourier transform infrared difference spectra have previously been obtained using purple bacterial reaction centers from Rhodobacter sphaeroides with unlabeled, 18O and 13C isotope labeled phylloquinone (PhQ, also known as vitamin K1) incorporated into the QA protein binding site (Breton, (1997), Proc. Natl. Acad. Sci. USA94 11318–11323). The nature of the bands in these spectra and the isotope induced band shifts are poorly understood, especially for the phyllosemiquinone anion (PhQ–) state. To aid in the interpretation of the bands in these experimental spectra, ONIOM type QM/MM vibrational frequency calculations were undertaken. Calculations were also undertaken for PhQ– in solution. Surprisingly, both sets of calculated spectra are similar and agree well with the experimental spectra. This similarity suggests pigment-protein interactions do not perturb the electronic structure of the semiquinone in the QA binding site. This is not found to be the case for the neutral PhQ species in the same protein binding site. PhQ also occupies the A1 protein binding site in photosystem I, and the vibrational properties of PhQ– in the QA and A1 binding sites are compared and shown to exhibit considerable differences. These differences probably arise because of changes in the degree of asymmetry of hydrogen bonding of PhQ– in the A1 and QA binding sites.

TIMP1 intron 3 retention is a marker of colon cancer progression controlled by hnRNPA1.

We previously reported a 40-transcripts signature marking the normal mucosa to colorectal adenocarcinoma transition. Eight of these mRNAs also showed splicing alterations, including a specific intron 3 retention in tissue metalloprotease inhibitor I (TIMP1), which decreased during the early steps of colorectal cancer progression. To decipher the mechanism of intron 3 retention/splicing, we first searched for putative RNA binding protein binding sites onto the TIMP1 sequence. We identified potential serine arginine rich splicing factor 1 (SRSF1) and heterogeneous nuclear RiboNucleoProtein A1 (hnRNPA1) binding sites at the end of intron 3 and the beginning of exon 4, respectively. RNA immunoprecipitation showed that hnRNPA1, but not SRSF1 could bind to the corresponding region in TIMP1 pre-mRNA in live cells. Furthermore, using a TIMP1-based ex vivo minigene approach, together with a plasmon resonance in vitro RNA binding assay, we confirmed that hnRNPA1 could indeed bind to wild type TIMP1 exon 4 pre-mRNA and control TMP1 intron 3 splicing, the interaction being abolished in presence of a mutant sequence that disrupted this site. These results indicated that hnRNPA1, upon binding to TIMP1 exon 4, was a positive regulator of intron 3 splicing. We propose that this TIMP1-intron 3 + transcript belongs to the class of nuclear transcripts with "detained" introns, an abundant molecular class, including in cancer.

Global identification of hnRNP A1 binding sites for SSO-based splicing modulation.

BackgroundMany pathogenic genetic variants have been shown to disrupt mRNA splicing. Besides splice mutations in the well-conserved splice sites, mutations in splicing regulatory elements (SREs) may deregulate splicing and cause disease. A promising therapeutic approach is to compensate for this deregulation by blocking other SREs with splice-switching oligonucleotides (SSOs). However, the location and sequence of most SREs are not well known.ResultsHere, we used individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) to establish an in vivo binding map for the key splicing regulatory factor hnRNP A1 and to generate an hnRNP A1 consensus binding motif. We find that hnRNP A1 binding in proximal introns may be important for repressing exons. We show that inclusion of the alternative cassette exon 3 in SKA2 can be significantly increased by SSO-based treatment which blocks an iCLIP-identified hnRNP A1 binding site immediately downstream of the 5’ splice site. Because pseudoexons are well suited as models for constitutive exons which have been inactivated by pathogenic mutations in SREs, we used a pseudoexon in MTRR as a model and showed that an iCLIP-identified hnRNP A1 binding site downstream of the 5′ splice site can be blocked by SSOs to activate the exon.ConclusionsThe hnRNP A1 binding map can be used to identify potential targets for SSO-based therapy. Moreover, together with the hnRNP A1 consensus binding motif, the binding map may be used to predict whether disease-associated mutations and SNPs affect hnRNP A1 binding and eventually mRNA splicing.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0279-9) contains supplementary material, which is available to authorized users.

Hypoxia is a modifier of SMN2 splicing and disease severity in a severe SMA mouse model

Spinal muscular atrophy (SMA) is a progressive neurodegenerative disease associated with low levels of the essential survival motor neuron (SMN) protein. Reduced levels of SMN is due to the loss of the SMN1 gene and inefficient splicing of the SMN2 gene caused by a C>T mutation in exon 7. Global analysis of the severe SMNΔ7 SMA mouse model revealed altered splicing and increased levels of the hypoxia-inducible transcript, Hif3alpha, at late stages of disease progression. Severe SMA patients also develop respiratory deficiency during disease progression. We sought to evaluate whether hypoxia was capable of altering SMN2 exon 7 splicing and whether increased oxygenation could modulate disease in a severe SMA mouse model. Hypoxia treatment in cell culture increased SMN2 exon 7 skipping and reduced SMN protein levels. Concordantly, the treatment of SMNΔ7 mice with hyperoxia treatment increased the inclusion of SMN2 exon 7 in skeletal muscles and resulted in improved motor function. Transfection splicing assays of SMN minigenes under hypoxia revealed that hypoxia-induced skipping is dependent on poor exon definition due to the SMN2 C>T mutation and suboptimal 5' splice site. Hypoxia treatment in cell culture led to increased hnRNP A1 and Sam68 levels. Mutation of hnRNP A1-binding sites prevented hypoxia-induced skipping of SMN exon 7 and was found to bind both hnRNP A1 and Sam68. These results implicate hypoxic stress as a modulator of SMN2 exon 7 splicing in disease progression and a coordinated regulation by hnRNP A1 and Sam68 as modifiers of hypoxia-induced skipping of SMN exon 7.

Modulation of A1 adenosine receptor expression by cell aggregation and long‐term activation of A2a receptors in cultures of avian retinal cells: involvement of the cyclic AMP/PKA pathway

The expression of A1 and A2a adenosine receptors is developmentally regulated in the chick retina, but little is known about the factors important for this regulation. Here, we show that cell aggregation and cAMP analogs promote a dramatic increase in A1 receptor expression. Importantly, a long-term stimulation of A2a receptors also promotes an increase of A1 receptor expression accompanied by a down-regulation of A2a receptors. Chick embryo retina cultures grown in the form of aggregates or dispersed cells accumulate cAMP when stimulated with dopamine or the adenosine agonist 2-chloroadenosine. However, inhibition of dopamine-dependent cAMP accumulation by 2-chloroadenosine was observed in aggregate cultures but not in dispersed cell cultures. Accordingly, A1 receptor binding sites were detected in aggregate cultures, but were low or absent from dispersed cell cultures. Interestingly, an increase of A1 binding sites was detected when dispersed cell cultures were treated for 5 days with permeable cAMP analogs, the adenylyl cyclase activator forskolin or A2a receptor agonists. Although a significant amount of A1 receptor protein was detected in dispersed cell cultures by western blot or immunocytochemistry, the long-term stimulation of A2a receptors also promoted an increase of the A1 receptor protein and mRNA, indicating that A2a receptors and cAMP were regulating transcription and/or translation of A1 receptors. We also found an increase of A1 receptors in locations in or near the membrane after treatment with A2a agonist. The long-term stimulation of retinal explants with A2a agonist also promoted an increase of A1 receptor protein. The results indicate that A2a receptors and the cAMP-dependent protein kinase pathway are involved in the regulation of A1 receptor expression during retinal development.

Pentylenetetrazole-induced seizures affect binding site densities for GABA, glutamate and adenosine receptors in the rat brain

Pentylenetetrazole (PTZ) is a convulsant used to model epileptic seizures in rats. In the PTZ-model, altered heat shock protein 27 (HSP-27) expression highlights seizure-affected astrocytes, which play an important role in glutamate and GABA metabolism. This raises the question whether impaired neurotransmitter metabolism leads to an imbalance in neurotransmitter receptor expression. Consequently, we investigated the effects of seizures on the densities of seven different neurotransmitter receptors in rats which were repeatedly treated with PTZ (40 mg/kg) over a period of 14 days. Quantitative in vitro receptor autoradiography was used to measure the regional binding site densities of the glutamate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and N-methyl-d-aspartate (NMDA) receptors, the adenosine receptor type 1 (A1), which is part of the system controlling glutamate release, and the γ-aminobutyric acid (GABA) receptors GABAA and GABAB as well as the GABAA-associated benzodiazepine (BZ) binding sites in each rat. Our results demonstrate altered receptor densities in brain regions of PTZ-treated animals, including the HSP-27 expressing foci (i.e. amygdala, piriform and entorhinal cortex, dentate gyrus). A general decrease of kainate receptor densities was observed together with an increase of NMDA binding sites in the hippocampus, the somatosensory, piriform and the entorhinal cortices. Furthermore, A1 binding sites were decreased in the amygdala and hippocampal CA1 region (CA1), while BZ binding sites were increased in the dentate gyrus and CA1. Our data demonstrate the impact of PTZ induced seizures on the densities of kainate, NMDA, A1 and BZ binding sites in epileptic brain. These changes are not restricted to regions showing glial impairment. Thus, an altered balance between different excitatory (NMDA) and modulatory receptors (A1, BZ binding sites, kainate) shows a much wider regional distribution than that of glial HSP-27 expression, indicating that receptor changes are not following the glial stress responses, but may precede the HSP-27 expression.

Inhibition of HPV-16 L1 expression from L1 cDNAs correlates with the presence of hnRNP A1 binding sites in the L1 coding region

The human papillomavirus type 16 (HPV-16) L1 capsid protein is very poorly expressed from cDNA expression plasmids transiently transfected into mammalian cells. The results described herein demonstrate that inhibition of HPV-16 L1 expression from L1 cDNAs correlates with the presence of splicing regulatory sequences in the L1 coding region. This inhibitory effect correlates with the binding of hnRNP A1 to the RNA elements. Similar to unutilised splice sites that may retain mRNAs in the nucleus, regulatory splicing RNA elements may also inhibit gene expression in the absence of splicing. The results presented here explain the inefficient expression of HPV-16 L1 protein from the wild type L1 cDNA expression plasmids in mammalian cells. These results may be of general interest since alteration of RNA sequences to prevent unwanted RNA-protein interactions may increase expression of many different genes in transient transfections or after plasmid uptake in DNA vaccination approaches.

First-in-Human Evaluation of Anti–von Willebrand Factor Therapeutic Aptamer ARC1779 in Healthy Volunteers

ARC1779 is a therapeutic aptamer antagonist of the A1 domain of von Willebrand Factor (vWF), the ligand for receptor glycoprotein 1b on platelets. ARC1779 is being developed as a novel antithrombotic agent for use in patients with acute coronary syndromes. This was a randomized, double-blind, placebo-controlled study in 47 healthy volunteers of doses of ARC1779 from 0.05 to 1.0 mg/kg. Pharmacodynamic effects were measured by an ELISA for free vWF A1 binding sites and by a platelet function analyzer. In terms of pharmacokinetics, the concentration-time profile of ARC1779 appeared monophasic. The observed concentration and area under the curve were dose proportional. The mean apparent elimination half-life was approximately 2 hours, and mean residence time was approximately 3 hours. The mean apparent volumes of distribution (at steady state and during terminal phase) were approximately one half the blood volume, suggesting that ARC1779 distribution is in the central compartment. The mean clearance ranged from approximately 10% to approximately 21% of the glomerular filtration rate, suggesting that renal filtration may not be a major mechanism of clearance of ARC1779. Inhibition of vWF A1 binding activity was achieved with an EC(90) value of 2.0 mug/mL (151 nmol/L) and of platelet function with an EC(90) value of 2.6 mug/mL (196 nmol/L). ARC1779 was generally well tolerated, and no bleeding was observed. Adverse events tended to be minor and not dose related. This is the first-in-human evaluation of a novel aptamer antagonist of vWF. ARC1779 produced dose- and concentration-dependent inhibition of vWF activity and platelet function with duration of effect suitable for the intended clinical use in acute coronary syndromes.

Abstract 3218: Pharmacokinetics, Pharmacodynamics, and Safety of an Anti-von Willebrand Factor Therapeutic Aptamer, ARC1779, in Healthy Volunteers

Background: The prominent role played by vWF in arterial thrombogenesis suggests that vWF inhibition may offer an effective adjunct therapy to PCI in ACS patients. ARC1779 is a PEG-conjugated aptamer that blocks platelet activation through inhibition of vWF A1 domain binding to platelet receptor GPIb. Design: This was an ascending-dose, double-blind, placebo-controlled study in 47 healthy volunteers at doses of 0 (placebo, n = 6) or 0.05 to 1.0 mg/kg ARC1779 (n = 41) given via IV push, “slow bolus” IV infusion over 15 minutes, or “slow bolus” followed by 4-hour IV infusion. PK parameters were estimated from plasma ARC1779 concentrations determined with a validated assay. PD effects were measured by an ELISA for free vWF A1 binding sites and by a platelet function analyzer, the PFA-100 ® . PK: The concentration-time profiles for ARC1779 after IV push or slow bolus appeared monophasic, though the terminal phase may not have been fully captured. The C max and AUC values were dose-proportional. The highest exposure was observed after 1.0 mg/kg slow bolus, with mean C max of 21.15 μg/mL and AUC (0-∞) of 80.92 μ g·hr/mL. The mean apparent elimination half-life (t 1/2β ) was ~2 hours and mean residence time (MRT) was ~3 hours. The mean apparent volumes of distribution (V z and V ss ) were ~1/2 of the blood volume, suggesting that ARC1779 distribution is in the central compartment. The mean clearance (CL) values ranged from ~10% to 21% of GRF, suggesting that renal filtration may not be a major mechanism of clearance of ARC1779. PD: Inhibition of vWF A1 binding was achieved in a dose- and concentration-dependent manner, with respective EC 50 and EC 90 values of 0.22 μ g/mL (17 nM) and 1.98 μg/mL (151 nM). Platelet function inhibition (PFA-100 ® closure time) was achieved, with respective EC 50 and EC 90 values of 0.75 μ g/mL (57 nM) and 2.57 μg/mL (196 nM). vWF activity returned in a dose- and concentration-dependent manner. Safety: ARC1779 was generally well tolerated and no bleeding was observed. Adverse events tended to be minor and not dose related. One volunteer had a hypersensitivity reaction to IV push administration, but no such reactions occurred at higher doses given by slow bolus or infusion. Conclusion: The PK, PD and safety profile of ARC1779 supports its therapeutic potential for use in ACS.

Chronic buspirone treatment normalizes open field behavior in olfactory bulbectomized rats: Assessment with a quantitative autoradiographic evaluation of the 5-HT 1A binding sites

The olfactory bulbectomized (OBX) rat model of depression has been widely used in studies on the behavioral and neurochemical aspects of human depression. The objective of the present investigation was to assess open field (OF) activity and the brain regional 5-HT 1A receptor densities of the sham operated (SHX) and OBX rats treated with saline (SHX-SAL, OBX-SAL), and either 10 mg/(kg day) (SHX-B10, OBX-B10) or 20 mg/(kg day) (SHX-B20, OBX-B20) of buspirone for 14 days, delivered by a subcutaneous osmotic minipump. Adult Sprague–Dawley rats were used for this experiment. The surgery was performed on the first day of the experiment and the rats were randomly assigned to either the SHX or OBX groups. The results of the OF tests were organized in eight groups. Following 14 days of treatment and the final OF tests, the rats were sacrificed and the brains were used for 5-HT 1A receptor autoradiography using [ 3H]8-OH-DPAT. The data showed that the OF activities, 14 days following surgery, in the OBX rats were significantly elevated when compared to the SHX rats. In the OBX rats, only the 14-day treatment with 20 mg/(kg day) of buspirone normalized the elevated OF activity, the same dose shown previously to be needed for the normalization of the regional 5-HT synthesis. A significant reduction in the number of 5-HT 1A receptor sites was found in most brain regions in the OBX rats when compared to the SHX rats. Data also show that the regional density of the 5-HT 1A receptors in OBX-SAL treated rats is lower than that of the SHX-SAL rats. The 14-day treatment with either 10 or 20 mg/(kg day) of buspirone reduced the 5-HT 1A receptors in most brain regions of the SHX rats, without an obvious dose-dependent effect of the buspirone. The comparison between the OBX-B20 and control (SHX-B20) rats suggests that the buspirone treatment resulted in a regional balance in the 5-HT 1A sites. A dose dependent reduction in the density of 5-HT 1A sites was observed in the sham rats, but the buspirone treatment had very little effect on the density of the 5-HT 1A receptors in the OBX rats. From these observations, we conclude that the antidepressant effects of buspirone in the OBX rat model of depression are likely mediated through the fine tuning of the regional imbalance of 5-HT 1A receptors with even increases of about 20% in some limbic regions. The data suggest that the neurochemical effects of antidepressants should be studied in animal models of depression rather than in normal rats.

HnRNP A1 functions with specificity in repression of SMN2 exon 7 splicing

Homozygous deletion or mutation of the survival of motor neuron 1 gene (SMN1) causes spinal muscular atrophy. SMN1 has been duplicated in humans to create SMN2, which produces a low level of functional SMN protein. However, most SMN2 transcripts lack exon 7, resulting in a non-functional protein. A single nucleotide difference near the 5' end of exon 7 largely accounts for SMN2 exon 7 skipping, an effect that has been attributed to loss of an exonic splicing enhancer (ESE) dependent on the SR protein splicing factor ASF/SF2 or to the creation of an exonic splicing silencer (ESS) element that functions by binding of the splicing repressor hnRNP A1. Our earlier experiments favored the latter mechanism and here we provide further evidence supporting the ESS model. We demonstrate that the striking effect of hnRNP A1 depletion on SMN2 exon 7 splicing is specific, as hnRNP A1 depletion has little or no effect on other inefficient splicing events tested, and ASF/SF2 depletion does not affect SMN1/2 splicing. By two different methods, we find a strong and specific interaction of hnRNPA1 with SMN2 exon 7 and only weak and equivalent interactions between ASF/SF2 and other SR proteins with the 5' ends of SMN1 and SMN2 exon 7. Finally, we describe two disease-related exon-skipping mutations that create hnRNP A1 binding sites, but show that splicing can be restored only modestly or not at all by hnRNP A1 depletion. Together our results provide strong support for the idea that SMN2 exon 7 splicing is repressed by an hnRNPA1-dependent ESS, but also indicate that creation of such elements is context-dependent.

Biochemical and NMR Study on the Competition between Proteins SC35, SRp40, and Heterogeneous Nuclear Ribonucleoprotein A1 at the HIV-1 Tat Exon 2 Splicing Site

The human immunodeficiency virus, type 1, Tat protein plays a key role in virus multiplication. Because of its apoptotic property, its production is highly controlled. It depends upon the A3 splicing site utilization. A key control of site A3 activity is the ESS2 splicing silencer, which is located within the long stem-loop structure 3 (SLS3), far downstream from site A3. Here, by enzymatic footprints, we demonstrate the presence of several heterogeneous nuclear ribonucleoprotein (hnRNP) A1-binding sites on SLS3 and show the importance of the C-terminal Gly domain of hnRNP A1 in the formation of stable complexes containing several hnRNP A1 molecules bound on SLS3. Mutations in each of the UAG triplets in ESS2 strongly reduce the overall hnRNP A1 binding, showing the central role of ESS2 in hnRNP A1 assembly on SLS2-SLS3. Using NMR spectroscopy, we demonstrate the direct interaction of ESS2 with the RNA recognition motifs domains of hnRNP A1. This interaction has limited effect on the RNA two-dimensional structure. The SR proteins SC35 and SRp40 were found previously to be strong activators of site A3 utilization. By enzymatic and chemical footprints, we delineate their respective binding sites on SLS2 and SLS3 and find a strong similarity between the hnRNP A1-, SC35-, and SRp40-binding sites. The strongest SC35-binding site only has a modest contribution to site A3 activation. Hence, the main role of SR proteins at site A3 is to counteract hnRNP A1 binding on ESS2 and ESE2. Indeed, we found that ESE2 has inhibitory properties because of its ability to bind hnRNP A1.

Ontogeny of 5-HT 1A receptor expression in the developing hippocampus

Serotonin (5-HT) has long been implicated in a number of neurodevelopmental processes including neuronal cell division, migration, neurite outgrowth, and synapse formation. However, relatively little is known about how these effects are mediated during normal brain development in vivo and the identity of the receptor subtypes involved in mediating these effects. In recent years, a number of pharmacological studies have suggested a role for the serotonin 1A (5HT 1A) receptor subtype in mediating the developmental effects of 5-HT in the hippocampus. These studies, however, have been difficult to interpret due to lack of information regarding the expression and distribution of 5HT 1A in the developing brain and hippocampus in particular. In the current study, specific anti-5-HT 1A antibodies, developed in our laboratory [F.C. Zhou, T.D. Patel, D. Swartz, Y. Xu, M.R. Kelley, Production and characterization of an anti-serotonin 1A receptor antibody which detects functional 5-HT 1A binding sites, Brain Res Mol Brain Res, 69 (1999) 186–201], were utilized to map the ontogeny and distribution of the 5HT 1A receptor protein in the developing rat hippocampus through embryonic and early postnatal life. This is the first such study of 5-HT 1A expression in the developing rat brain. Our findings revealed that expression of the 5HT 1A receptor emerges during the initial stages of embryonic hippocampal development. Remarkably, most if not all hippocampal neurons begin to express 5HT 1A shortly upon completion of their terminal mitosis. We found that 5HT 1A is initially concentrated around the cell bodies and later becomes more sparsely distributed along the dendrites after the neurons have matured. In addition to postmitotic neurons, we have observed that S100 and GFAP positive glia transiently express 5HT 1A during early postnatal development of the hippocampus. These findings demonstrate that the 5-HT 1A receptor is positioned to mediate developmental effects of serotonin in the hippocampus. Furthermore, the temporal patterns of expression suggest a role for 5-HT 1A in postmitotic events such as neuronal migration, neurite outgrowth, and phenotypic differentiation.

Recruitment of a Foreign Quinone into the A1 Site of Photosystem I: CHARACTERIZATION OF A menB rubA DOUBLE DELETION MUTANT IN SYNECHOCOCCUS SP. PCC 7002 DEVOID OF FX, FA, AND FB AND CONTAINING PLASTOQUINONE OR EXCHANGED 9,10-ANTHRAQUINONE

A photosystem I (PS I) complex containing plastoquinone-9 (PQ-9) but devoid of F(X), F(B), and F(A) was isolated and characterized from a mutant strain of Synechococcus sp. PCC 7002 in which the menB and rubA genes were insertionally inactivated. In isolated PS I trimers, the decay of P700+ measured in the near-IR and the decay of A1- measured in the near-UV were found to be biphasic, with (averaged) room temperature lifetimes of 12 and 350 micros. The decay-associated spectra of both kinetic phases are characteristic of the oxidized minus reduced difference spectrum of a semiquinone, consistent with charge recombination between P700+ and PQ-9-. The amplitude of the flash-induced absorbance changes in both the near-IR and the near-UV show that approximately one-half of the A1 binding sites are either empty or nonfunctional. A spin-polarized chlorophyll triplet is observed by time-resolved EPR, and it is attributed to the 3P700 product of P700+A0- charge recombination via the T0 spin level in those PS I complexes that do not contain a functional quinone. In those A1 sites that are occupied, the P700+Q- polarization pattern indicates that PQ-9 is oriented in a similar manner to that in the menB mutant. When excess 9,10-anthraquinone is added in vitro, it displaces PQ-9 and occupies the A1 binding site more readily than in the menB mutant. This can be explained by a greater accessibility to the A1 site in the menB rubA mutant due to the absence of F(X) and the stromal ridge polypeptides. The relatively low binding affinity of 9,10-anthraquinone allows it to be readily removed from the A1 site by washing. However, all A1 sites are shown to bind napthoquinones with high affinity and thus are proven to be functionally competent in quinone binding. The ability to readily displace PQ-9 from the A1 site makes the menB rubA mutant ideal for introducing novel quinones, particularly anthraquinones, into PS I.

Identification of the human rhinovirus serotype 1A binding site on the murine low-density lipoprotein receptor by using human-mouse receptor chimeras.

Human rhinovirus serotype 1A (HRV1A) binds more strongly to the mouse low-density lipoprotein receptor (LDLR) than to the human homologue (M. Reithmayer, A. Reischl, L. Snyers, and D. Blaas, J. Virol. 76:6957-6965, 2002). Here, we used this fact to determine the binding site of HRV1A by replacing selected ligand binding modules of the human receptor with the corresponding ligand binding modules of the mouse receptor. The chimeric proteins were expressed in mouse fibroblasts deficient in endogenous LDLR and LDLR-related protein, both used by minor group HRVs for cell entry. Binding was assessed by virus overlay blots, by immunofluorescence microscopy, and by measuring cell attachment of radiolabeled virus. Replacement of ligand binding repeat 5 of the human LDLR with the corresponding mouse sequence resulted in a substantial increase in HRV1A binding, whereas substitution of repeats 3 and 4 was without effect. Replacement of human receptor repeats 1 and 2 with the murine homologues also increased virus binding. Finally, murine receptor modules 1, 2, and 5 simultaneously introduced into the human receptor resulted in HRV1A binding indistinguishable from mouse wild-type receptor. Thus, repeats 1 and/or 2 and repeat 5 are involved in HRV1A attachment. Changing CDGGPD in the acidic cluster of module 5 in the human receptor to CDGEAD present in the mouse receptor led to substantially increased binding of HRV1A, indicating an important role of the glutamate residue in HRV1A recognition.

SC35 and Heterogeneous Nuclear Ribonucleoprotein A/B Proteins Bind to a Juxtaposed Exonic Splicing Enhancer/Exonic Splicing Silencer Element to Regulate HIV-1 tat Exon 2 Splicing

Splicing of the human immunodeficiency virus, type 1, primary transcript is highly regulated. Maintaining the proper equilibrium among spliced, unspliced, and partially spliced isoforms is essential for the replication of the virus. Here we characterize a complex cis-acting element located in tat exon 2 that is required for the splicing regulation of the upstream intron. An exonic splicing enhancer (ESE) and an exonic splicing silencer (ESS) are both located within the regulatory element. Heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins bind the ESS to repress splicing, whereas the SR protein SC35 binds the ESE to activate it. We show that the SC35 and the hnRNP A1 binding sites overlap within the juxtaposed ESE/ESS. We propose that hnRNP A1 binding to the ESS inhibits splicing of the upstream intron by directly masking the SC35 binding site.

Photoperiodic regulation of vasopressin receptor binding in female Syrian hamsters

During long “summer-like” photoperiods, female Syrian hamsters display a regular 4-day estrous cycle. However, during short “winter-like” photoperiods (<12.5 h of light/day) hamsters become anestrus. Short photoperiod exposure eliminates reproductive behavior but social behaviors such as aggression and scent marking continue to be displayed. In long photoperiods, the types and intensity of social behaviors change as a function of the estrous cycle. For example, aggression and scent marking tend to occur at higher levels on diestrus 1 and diestrus 2 than on proestrus or estrus. Aggression and scent marking may be regulated, at least in part, by changes in the density of arginine vasopressin-V 1a receptors (V 1aR). In Experiment 1, it was hypothesized that the density of V 1aR would change across the estrous cycle in several subcortical regions implicated in the regulation of aggression and scent marking. In Experiment 2, it was hypothesized that exposure to short photoperiod would alter the density of V 1aR in several regions involved in the regulation of social behavior. Interestingly, there were no dramatic changes in V 1aR binding across the estrous cycle within any of the neuroanatomical areas measured. However, in hamsters housed in short photoperiod, there were lower levels of V 1aR binding within the medial preoptic nucleus (MPN), medial preoptic area (MPO), lateral hypothalamus (LH), central nucleus of the amygdala (Ce) and bed nucleus of the stria terminalis (BST) than in hamsters housed in long photoperiod. These data suggest that photoperiodic mechanisms can alter the density of V 1aR in subset of V 1a binding sites thought to be involved in the regulation of social behaviors in female hamsters.

Syntaxin 1A inhibits GABA flux, efflux, and exchange mediated by the rat brain GABA transporter GAT1.

GABA transporters control extracellular GABA levels by coupling transmitter uptake to the sodium and chloride cotransport. The rat brain GABA transporter GAT1 and other members of this family are regulated by direct interactions with syntaxin 1A, a protein involved in vesicle docking and in the regulation of several ion channels and transporters. We have shown previously that syntaxin 1A exerts its effects on GAT1 by decreasing the net uptake of GABA and its associated ions through interactions with aspartic acid residues in the N-terminal tail of GAT1. This reduction in net uptake could be caused by many steps in the transport cycle, including substrate binding, substrate flux, substrate efflux, or reorientation of the unliganded transporter. To address this question, we performed GABA flux assays, measured flux- and efflux-associated ion currents, and assessed GABA exchange in multiple experimental systems expressing syntaxin 1A and wild-type GAT1 or GAT1 mutants. Syntaxin 1A caused similar reductions in forward and reverse transport that did not involve changes in apparent transport affinities for sodium, chloride, or GABA. The syntaxin 1A-mediated reduction in GABA flux and efflux was mimicked by mutations in GAT1 at the syntaxin 1A binding site. The binding site GAT1 mutant also caused a reduction in exchange. These data suggest that syntaxin 1A exerts its effects, directly or indirectly, on GAT1 function through interactions with GAT1's N-terminal tail and that the inhibition occurs at a step in the translocation process after substrate binding but which involves both unidirectional transport and transmitter exchange.

Control of HIV-1 env RNA splicing and transport: investigating the role of hnRNP A1 in exon splicing silencer (ESS3a) function

The control of HIV-1 viral RNA splicing and transport plays an important role in the successful replication of the virus. Previous studies have identified both an exon splicing enhancer (ESE) and a bipartite exon splicing silencer (ESS3a and ESS3b) within the terminal exon of HIV-1 that are involved in modulating both splicing and Rev-mediated export of viral RNA. To define the mechanism of ESS3a function, experiments were carried out to better define the cis and trans components required for ESS3a activity. Mutations throughout the 30-nt element resulted in partial loss of ESS function. Combining mutations was found to have an additive effect, suggesting the presence of multiple binding sites. Analysis of interacting factors identified hnRNP A1 as one component of the complex that modulates ESS3a activity. However, subsequent binding analyses determined that hnRNP A1 interacts with only one portion of ESS3a, suggesting the involvement of another host factor. Parallel analysis of the effect of the mutations on Rev-mediated export determined that there is not a direct correlation between the effect of the mutations on splicing and RNA transport. Consistent with this hypothesis, replacement of ESS3a with consensus hnRNP A1 binding sites was found to be insufficient to block Rev-mediated RNA export.

HnRNP A1 controls HIV-1 mRNA splicing through cooperative binding to intron and exon splicing silencers in the context of a conserved secondary structure.

The removal of the second intron in the HIV-1 rev/tat pre-mRNAs, which involves the joining of splice site SD4 to SA7, is inhibited by hnRNP A1 by a mechanism that requires the intronic splicing silencer (ISS) and the exon splicing silencer (ESS3). In this study, we have determined the RNA secondary structure and the hnRNP A1 binding sites within the 3' splice site region by phylogenetic comparison and chemical/enzymatic probing. A biochemical characterization of the RNA/protein complexes demonstrates that hnRNP A1 binds specifically to primarily three sites, the ISS, a novel UAG motif in the exon splicing enhancer (ESE) and the ESS3 element, which are all situated in experimentally supported stem loop structures. A mutational analysis of the ISS region revealed that the core hnRNP A1 binding site directly overlaps with a major branchpoint used in splicing to SA7, thereby providing a direct explanation for the inhibition of U2 snRNP association with the pre-mRNA by hnRNP A1. Binding of hnRNP A1 to the ISS core site is inhibited by RNA structure but strongly stimulated by the exonic silencer, ESS3. Moreover, the ISS also stimulate binding of hnRNP A1 to the exonic splicing regulators ESS3 and the ESE. Our results suggest a model where a network is formed between hnRNP A1 molecules situated at discrete sites in the intron and exon and that these interactions preclude the recognition of essential splicing signals including the branch point.