The constitutive transport element (CTE) of the type D retroviruses promotes nuclear export of unspliced viral RNAs apparently by recruiting host factor(s) required for export of cellular messenger RNAs. Here, we report the identification of TAP as the cellular factor that specifically binds to wild-type CTE but not to export-deficient CTE mutants. Microinjection experiments performed in Xenopus oocytes demonstrate that TAP directly stimulates CTE-dependent export. Furthermore, TAP overcomes the mRNA export block caused by the presence of saturating amounts of CTE RNA. Thus, TAP, like its yeast homolog Mex67p, is a bona fide mRNA nuclear export mediator. TAP is the second cellular RNA binding protein shown to be directly involved in the export of its target RNA.
The nuclear export of the unspliced type D retrovirus mRNA depends on the cis-acting constitutive transport RNA element (CTE) that has been shown to interact with the human TAP (hTAP) protein promoting the export of the CTE-containing mRNAs. We report here that hTAP is a 619-amino-acid protein extending the previously identified protein by another 60 residues at the N terminus and that hTAP shares high homology with the predicted rat and mouse TAP proteins. We found that hTAP is a nuclear protein that accumulates in the nuclear rim and the nucleoplasm. We further demonstrated that hTAP is able to shuttle between the nucleus and the cytoplasm. Identification of the signals responsible for nuclear import (NLS) and export (NES) revealed that they are distinct but partially overlapping. NLS and NES of hTAP are active transferable signals that do not share similarities with known elements. The C-terminal portion contributes further to hTAP's nuclear retention and contains a signal(s) for nuclear rim association. Taken together, our data show that hTAP is a dynamic protein capable of bidirectional trafficking across the nuclear envelope. These data further support hTAP's role as an export factor of the CTE-containing mRNAs.Posttranscriptional regulation is an essential regulatory step of many retroviruses and is necessary for virus production. This key regulatory step mediates the export of the unspliced, fulllength viral RNA, which requires the interaction of viral and/or cellular factors. This controlled export of the viral RNA to the cytoplasm ensures the availability of the genomic RNA for packaging into the progeny virions and the production of the Gag/Pol polyproteins. Among the best-studied export systems are those used by the simian type D retroviruses (SRV/D) and the lentiviruses, such as human immunodeficiency virus type 1 (HIV-1) (for reviews see references 6, 14, and 28).SRV/D expression is controlled by the essential cis-acting constitutive transport element (CTE) (5,12,54,63). The SRV/D CTE (11, 55) and a related CTE-like element in a murine intracisternal A-particle retroelement (54) fold into an extended RNA stem-loop structure containing two conserved internal loops and an AAGA bulge. These loops and the bulge, the spacing of the loops within the RNA element, as well as the overall secondary structure of the element, have been shown to be essential features for CTE function (11,55). Recently, we showed that the human TAP protein (hTAP) binds specifically to these internal loops and promotes nucleocytoplasmic transport of the CTE-containing intron lariat from the Xenopus oocyte nucleus (22). TAP had previously been identified as a factor binding to Tip, a herpesvirus saimiri protein responsible for cell transformation (60). The role of hTAP interaction with Tip is still unclear.Whereas the SRV/D retroviruses have been proposed to utilize the cellular hTAP protein to export their unspliced mRNA (22), HIV-1 uses the viral Rev protein to promote the transport of the Rev responsive element (RRE)-containing...
In the absence of the viral regulatory protein Rev, the human immunodeficiency virus type 1 gaglpol and env mRNAs are inefficiently expressed, since nucleocytoplasmic transport, stability, and polysomal loading are impaired. It has been suggested that splicing is necessary for Rev function and that the low expression of the unspliced and intermediate spliced mRNAs in the absence of Rev is associated with specific splice sites. Previous studies identified distinct RNA elements within the gagipol region responsible for low expression that are not associated with splice sites. Here we study the determinants for Rev dependence of the authentic env mRNA. We demonstrate that upon removal of all the utilized splice sites, the env mRNA is still Rev dependent and Rev responsive for expression in human cells. We have identified several regions within the env mRNA that inhibit expression of a gag-env hybrid mRNA. Elimination of one of these elements, located within the
It was previously shown that a 240-nucleotide (nt) RNA element (cis-acting transactivation element [CTE]) located between the env gene and the 3 long terminal repeat of simian retrovirus type 1 (SRV-1) can functionally replace posttranscriptional activation directed by Rev and the Rev-responsive element (RRE) when inserted into a Rev-and RRE-deficient molecular clone of human immunodeficiency virus type 1, resulting in efficient virus replication. Here, we analyze the molecular and structural requirements for function of this RNA element. Deletion mutagenesis demonstrated that the core element spans 173 nt. SRV-2 and Mason-Pfizer monkey virus have highly homologous elements, which function similarly when inserted into the Rev/RRE-deficient human immunodeficiency virus type 1. Computer prediction indicated that the core CTEs of all three viruses have similar extensive secondary structures. Mutagenesis of the SRV-1 CTE revealed that both sequence and secondary structure are essential for function. Nuclease probing of the SRV-1 CTE further supported the genetic analysis and confirmed the predicted structural features of the RNA element. Sequence analysis of the 240-nt SRV-1 CTE, after continuous long-term propagation of the Rev-independent viruses, revealed that the genetically defined core element remained unchanged, while regions outside the core CTE underwent deletions or duplications. These data further support our in vitro mutagenesis data and demonstrate the importance of the sequence and structure of the SRV-1 CTE for appropriate function.
We have isolated and characterized the cDNA and genomic DNA coding for a phenoloxidase, laccase I, previously purified from culture supernatant of the newly isolated ligninolytic basidiomycete PM1 (CECT 2971). A cDNA library from basidiomycete PM1 was constructed, and laccase-encoding cDNAs were identified by screening with antiserum raised against the purified enzyme. The lac1 gene coding for the laccase was identified in a partial genomic library by using the isolated cDNA as a probe. Nucleotide sequence determination of the full-length cDNA revealed an open reading frame of 1,551 bp encoding a polypeptide of 517 amino acid residues with a putative signal peptide of 21 amino acid residues. Ten small introns interrupted the genomic DNA. A single 1.8-kb transcript mRNA was detected by Northern (RNA) blot analysis, and its 5' end maps to a position 51 bp upstream from the site of initiation of protein synthesis. Eukaryotic regulatory sequences, CAAT and TATA, were observed in the 5' flanking region, which also contains sequences similar to those of copper-regulated proteins. Comparative analysis of the predicted amino acid sequence showed that basidiomycete PM1 laccase I had great similarity to the laccases from Coriolus versicolor, Coriolus hirsutus, and Phlebia radiata.
en relación con las ciencias naturales nos permite explorar detenidamente los procesos de generación, circulación y gestión de conocimiento científico-tecnológico en un contexto particularmente convulso de la historia reciente de España, el final de la dictadura de Franco. Con un enfoque centrado en las relaciones entre los seres humanos y su entorno, el trabajo multidimensional de Rodríguez de la Fuente, como cetrero, naturalista, activista y comunicador, aportó un excepcional componente científico-técnico a los procesos de transformación social, política y cultural de España en esos años. El objetivo de este trabajo es mostrar cómo este componente científico-técnico estaba frecuentemente ligado, y entre otros aspectos, a la definición y contextualización de España y su patrimonio natural tanto dentro de sus fronteras como en el escenario internacional.paLabras cLave: medios de comunicación; patrimonio natural; ciencias naturales; conservación; franquismo; imagen de España; Félix Rodríguez de la Fuente.abstract: Félix Rodríguez de la Fuente's huge media output concerning the natural sciences allows us to thoroughly explore the processes of generation, circulation and management of scientific-technological knowledge in a particularly turbulent context of Spain's recent history, the end of Franco's dictatorship.With an approach focused on the relations between human beings and their environment, Rodríguez de la Fuente's multidimensional work, as a falconer, naturalist, activist and broadcaster, contributed an exceptional scientific-technical component to the processes of social, political and cultural transformation of Spain in those years. The aim of this paper is to show how that scientific-technical element was often linked, among other aspects, to the definition and contextualization of Spain and its natural heritage both within its borders and in the international scene.
The gene bgaA encoding an alkaline endo-beta-1,3-1,4-glucanase (lichenase) from an alkalophilic Bacillus sp. strain N137, isolated in our laboratory, was cloned and expressed from its own promoter in Escherichia coli. The nucleotide sequence of a 1,416-bp DNA fragment containing bgaA was determined and revealed an open reading frame of 828 nucleotides. The deduced protein sequence consists of 276 amino acids and has a 31-amino-acid putative signal peptide which is functional in E. coli, in which the BgaA protein is located mainly in the periplasmic space. The lichenase activity of BgaA is stable between pH 6 and 12, it shows optimal activity at a temperature between 60 and 70 degrees C, and it retains 65% of its activity after incubation at 70 degrees C for 1 h. This protein is similar to some other lichenases from Bacillus species such as B. amyloliquefaciens, B. brevis, B. licheniformis, B. macerans, B. polymyxa, and B. subtilis. However, it has a lysine-rich region at the carboxy terminus which is not found in any other published lichenase sequence and might be implicated in the unusual biochemical properties of this enzyme. The location of the mRNA 5' end was determined by primer extension and corresponds to nucleotide 235. A typical Bacillus sigma A promoter precedes the transcription start site.
Human immunodeficiency virus type 1 (HIV-1) replication depends on the posttranscriptional regulation by the viral Rev protein and can be replaced with the posttranscriptional RNA control element (CTE) of the type D simian retroviruses. We have identified a sequence which shares only nucleotide sequences of the internal loops and secondary structure with the CTE and which is part of a novel murine intracisternal-A particle (IAP) retroelement, inserted within the transcribed mouse osteocalcin-related gene. This sequence, named CTE IAP , can replace the Rev-mediated regulation of HIV-1, hence it is a posttranscriptional regulatory element. Related elements have been identified in other IAPs. These results suggest that insertional mutagenesis can affect gene expression by providing a functional posttranscriptional control element. The CTE IAP and CTEs of the type D simian retroviruses represent a novel class of RNA elements characterized by unique sequences within the internal loops which are predicted to represent the interaction site with cellular factor(s). These findings suggest that such elements may be involved in posttranscriptional regulation of cellular mRNAs.
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