An internal ribosome entry site element directs the synthesis of the 80 kDa isoforms of protein 4.1R

<p>Abstract</p> <p>Background</p> <p>In red blood cells, protein 4.1 (4.1R) is an 80 kDa protein that stabilizes the spectrin-actin network and anchors it to the plasma membrane through its FERM domain. While the expression pattern of 4.1R in mature red cells is relatively simple, a rather complex array of 4.1R protein isoforms varying in N-terminal extensions, internal sequences and subcellular locations has been identified in nucleated cells. Among these, 135 kDa and 80 kDa isoforms have different N-terminal extensions and are expressed either from AUG1- or AUG2-containing mRNAs, respectively. These two types of mRNAs, varying solely by presence/absence of 17 nucleotides (nt) which contain the AUG1 codon, are produced by alternative splicing of the 4.1R pre-mRNA. It is unknown whether the 699 nt region comprised between AUG1 and AUG2, kept as a 5' untranslated region in AUG2-containing mRNAs, plays a role on 4.1R mRNA translation.</p> <p>Results</p> <p>By analyzing the <it>in vitro </it>expression of a panel of naturally occurring 4.1R cDNAs, we observed that all AUG1/AUG2-containing cDNAs gave rise to both long, 135 kDa, and short, 80 kDa, 4.1R isoforms. More importantly, similar results were also observed in cells transfected with this set of 4.1R cDNAs. Mutational studies indicated that the short isoforms were not proteolytic products of the long isoforms but products synthesized from AUG2. The presence of a cryptic promoter in the 4.1R cDNA sequence was also discounted. When a 583 nt sequence comprised between AUG1 and AUG2 was introduced into bicistronic vectors it directed protein expression from the second cistron. This was also the case when ribosome scanning was abolished by introduction of a stable hairpin at the 5' region of the first cistron. Deletion analysis of the 583 nt sequence indicated that nucleotides 170 to 368 are essential for expression of the second cistron. The polypyrimidine tract-binding protein bound to the 583 nt active sequence but not to an inactive 3'-fragment of 149 nucleotides.</p> <p>Conclusion</p> <p>Our study is the first demonstration of an internal ribosome entry site as a mechanism ensuring the production of 80 kDa isoforms of protein 4.1R. This mechanism might also account for the generation of 60 kDa isoforms of 4.1R from a downstream AUG3. Our results reveal an additional level of control to 4.1R gene expression pathways and will contribute to the understanding of the biology of proteins 4.1R and their homologues, comprising an ample family of proteins involved in cytoskeletal organization.</p

Higher COVID-19 pneumonia risk associated with anti-IFN-α than with anti-IFN-ω auto-Abs in children

We found that 19 (10.4%) of 183 unvaccinated children hospitalized for COVID-19 pneumonia had autoantibodies (auto-Abs) neutralizing type I IFNs (IFN-alpha 2 in 10 patients: IFN-alpha 2 only in three, IFN-alpha 2 plus IFN-omega in five, and IFN-alpha 2, IFN-omega plus IFN-beta in two; IFN-omega only in nine patients). Seven children (3.8%) had Abs neutralizing at least 10 ng/ml of one IFN, whereas the other 12 (6.6%) had Abs neutralizing only 100 pg/ml. The auto-Abs neutralized both unglycosylated and glycosylated IFNs. We also detected auto-Abs neutralizing 100 pg/ml IFN-alpha 2 in 4 of 2,267 uninfected children (0.2%) and auto-Abs neutralizing IFN-omega in 45 children (2%). The odds ratios (ORs) for life-threatening COVID-19 pneumonia were, therefore, higher for auto-Abs neutralizing IFN-alpha 2 only (OR [95% CI] = 67.6 [5.7-9,196.6]) than for auto-Abs neutralizing IFN-. only (OR [95% CI] = 2.6 [1.2-5.3]). ORs were also higher for auto-Abs neutralizing high concentrations (OR [95% CI] = 12.9 [4.6-35.9]) than for those neutralizing low concentrations (OR [95% CI] = 5.5 [3.1-9.6]) of IFN-omega and/or IFN-alpha 2

4.1R Proteins Associate with Interphase Microtubules in Human T Cells

Red blood cell protein 4.1 (4.1R) is an 80-kDa protein that stabilizes the spectrin-actin network and anchors it to the plasma membrane. To contribute to the characterization of functional roles and partners of specific nonerythroid 4.1R isoforms, we analyzed 4.1R in human T cells and found that endogenous 4.1R was distributed to the microtubule network. Transfection experiments of T cell 4.1R cDNAs in conjunction with confocal microscopy analysis revealed the colocalization of exogenous 4.1R isoforms with the tubulin skeleton. Biochemical analyses using Taxol® (paclitaxel)-polymerized microtubules from stably transfected T cells confirmed the association of the exogenous 4.1R proteins with microtubules. Consistent with this, endogenous 4.1R immunoreactive proteins were also detected in the microtubule-containing fraction. In vitro binding assays using glutathione S-transferase-4.1R fusion proteins showed that a constitutive domain of the 4.1R molecule, one that is therefore present in all 4.1R isoforms, is responsible for the association with tubulin. A 22-amino acid sequence comprised in this domain and containing heptad repeats of leucine residues was essential for tubulin binding. Furthermore, ectopic expression of 4.1R in COS-7 cells provoked microtubule disorganization. Our results suggest an involvement of 4.1R in interphase microtubule architecture and support the hypothesis that some 4.1R functional activities are cell type-regulated.</p

An Alternative Domain Containing a Leucine-rich Sequence Regulates Nuclear Cytoplasmic Localization of Protein 4.1R

In red blood cells, protein 4.1 (4.1R) is an 80-kDa protein that stabilizes the spectrin-actin network and anchors it to the plasma membrane. The picture is more complex in nucleated cells, in which many 4.1R isoforms, varying in size and intracellular location, have been identified. To contribute to the characterization of signals involved in differential intracellular localization of 4.1R, we have analyzed the role the exon 5-encoded sequence plays in 4.1R distribution. We show that exon 5 encodes a leucine-rich sequence that shares key features with nuclear export signals (NESs). This sequence adopts the topology employed for NESs of other proteins and conserves two hydrophobic residues that are shown to be critical for NES function. A 4.1R isoform expressing the leucine-rich sequence binds to the export receptor CRM1 in a RanGTP-dependent fashion, whereas this does not occur in a mutant whose two conserved hydrophobic residues are substituted. These two residues are also essential for 4.1R intracellular distribution, because the 4.1R protein containing the leucine-rich sequence localizes in the cytoplasm, whereas the mutant protein predominantly accumulates in the nucleus. We hypothesize that the leucine-rich sequence in 4.1R controls distribution and concomitantly function of a specific set of 4.1R isoforms.</p

The N-terminal 209-aa domain of high molecular- weight 4.1R isoforms abrogates 4.1R targeting to the nucleus

An extensive repertoire of protein 4.1R isoforms is predominantly generated by alternative pre-mRNA splicing and differential usage of two translation initiation sites. The usage of the most upstream ATG (ATG-1) generates isoforms containing N-terminal extensions of up to 209 aa compared with those translated from the downstream ATG (ATG-2). To characterize nonerythroid 4.1R proteins translated from ATG-1 and analyze their intracellular localization, we cloned 4.1R cDNAs containing this translation initiation site. Six different clones were isolated from the nucleated human MOLT-4 T-cell line by reverse transcriptase–PCR techniques. Transient expression of the six ATG-1-translated 4.1R isoforms tagged with a c-Myc epitope revealed that all of them predominantly distributed to the plasma membrane and the endoplasmic reticulum. Staining of MOLT-4 cell plasma membranes but not nuclei was also observed by immunofluorescence microscopy by using an antibody specific to the N-terminal extension. Consistent with this, the antibody reacted with a major endogenous protein of ≈145 kDa present in nonnuclear but absent from nuclear fractions prepared from MOLT-4 cells. Because these data suggested that ATG-1-translated 4.1R isoforms were predominantly excluded from the nucleus, we fused the 209-aa domain to nuclear 4.1R isoforms encoded from ATG-2 and observed that this domain inhibited their nuclear targeting. All these results indicate that the N-terminal domain of ATG-1-translated 4.1R isoforms plays a pivotal role in differential targeting of proteins 4.1R

Informe de la Campaña “CAIBEX-I”: Shelf-ocean exchanges in the Canaries-Iberia

36 páginas, 29 figuras, 1 tablaCAIBEX es un proyecto del Plan Nacional financiado por el Ministerio de Ciencia e Innovación (CTM2007- 66408)

Caibex-I: basic hydrographic and chemical data

Este dataset está compuesto por 2 archivos, de los cuales el primero es el conjunto de datos con 191 análisis de muestras de agua de temperatura, salinidad, oxígeno, nutrientes, pH, alcalinidad y clorofila, y el otro (Readme.txt) incluye una pequeña descripción de las variables calculadasEl proyecto CAIBEX es un estudio coordinado para comparar la dinámica y actividad biogeoquímica contrastada que se da entre la zona costera y el océano adyacente en la zona de estudio. Es un programa de observación y modelización multidisciplinar que abarca diferentes escalas espaciales y temporales, incluyendo un estudio del papel de los filamentos en el afloramiento estival del caladero de Cabo Silleiro, Galicia (CAIBEX I) y el afloramiento perenne del caladero de Cabo Guir, Marruecos (CAIBEX III) con especial referencia a sus repercusiones en el reclutamiento/dispersión de paralarvas de pulpo común y a las transformaciones biogeoquímicas experimentadas por el material biogénico producido en la zona costera. La campaña CAIBEX I, realizada a bordo del buque Sarmiento de Gamboa, está enfocada al estudio de las estructuras y procesos a mesoescala del sistema de Cabo Silleiro, y tiene como meta principal la definición de la estructura tri-dimensional física, biológica y biogeoquímica de un filamento de afloramiento y su evolución en el tiempo como función del forzamiento por el viento. Para alcanzar estos objetivos, el plan de campaña se planteó tres modos de operación: 1) un mapeado rápido al principio y al fin de la campaña para localizar el filamento y observar los cambios en el contexto general durante el estudio; 2) varios experimentos de deriva en el filamento para ver el desarrollo de una parcela de agua mientras se traslada de la costa al océano; 3) cortes repetidos a través del filamento para estudiar su desarrollo in situ. En cada estación se realizaron perfiles verticales de temperatura, salinidad y oxígeno disuelto con un SBE911plus CTD. Se realizaron tiradas con una roseta con botellas de 10 L de PVC Niskin para obtener muestras de agua para análisis oxígeno disuelto y nutrientes inorgánicos disueltos. El oxígeno disuelto se determinó por titulación potenciométrica de Winkler. El error estándar analítico estimado fue de 1 μmol/kg. Las muestras de nutrientes se determinaron mediante análisis de flujo segmentado con el autoanalizador Alliance Futura siguiendo a Grashoff et al. (1999) con algunas mejoras (Mouriño y Fraga 1985). Los límites de detección fueron 0,02 μmol/L para nitritos, 0,05 μmol/L para nitratos, amonio y silicato y 0,01 μmol/L para fosfatos. El pH del agua de mar se midió espectrofotométricamente siguiendo a Clayton y Byrne (1993) aplicándose una adición de 0,0047 (DelValls & Dickson, 1998). Las muestras de pH (escala de concentración total de hidrógeno, 25°C) se recogieron directamente en cubetas ópticas cilíndricas de 100 mm de longitud en las que se realizan directamente las medidas. La precisión fue 0,003 unidades de pH. Las muestras de alcalinidad total (TA) se recogieron en frascos de vidrio de 500 ml. El TA se determinó por titulación a pH 4,4 con HCl, según el método potenciométrico de Pérez y Fraga (1987) con una precisión de ±2 micromol/kgCSIC1 data csv ‘29AH200901706_hy1.csv’ file and 1 readme.txt filePeer reviewe