English   español  
Por favor, use este identificador para citar o enlazar a este item: http://hdl.handle.net/10261/8338
Compartir / Impacto:
Estadísticas
Add this article to your Mendeley library MendeleyBASE
Ver citas en Google académico
Visualizar otros formatos: MARC | Dublin Core | RDF | ORE | MODS | METS | DIDL
Exportar otros formatos: Exportar EndNote (RIS)Exportar EndNote (RIS)Exportar EndNote (RIS)
Título

Mecanismos implicados en la generación de isoformas de la proteína 4.1R

Autor Lospitao, Eva Pilar
DirectorCorreas, Isabel
Palabras clave Síntesis de proteínas
Fecha de publicación 2007
EditorUniversidad Autónoma de Madrid
ResumenRed blood cell protein 4.1, 4.1R, is an extreme variation on the theme of isoform multiplicity and involvement of alternative pre-mRNA splicing events for the generation of 4.1R protein diversity has been widely explored. Two types of 4.1R isoforms varying in N-terminal extensions are originated by alternative splicing events involving exon 2’. This exon encompasses translation initiation site AUG1 and hence 4.1R mRNAs containing exon 2’ regulate expression of longer (135 kDa) isoforms of 4.1R protein. By contrast, 4.1R mRNAs excluding exon 2’ allow expression of shorter (80 kDa) isoforms, the synthesis of which is initiated at the AUG2 translation initiation site comprised in exon 4. The current study reports that 4.1R mRNAs containing exons 2’ and 4 regulate expression of both longer and shorter 4.1R isoforms. Analysis of in vitro expression of a set of 4.1R cDNAs containing exon 2’ showed an unexpected result: that two proteins 4.1R, longer (∼135-kDa) and shorter (∼80-kDa), were synthesized. Mutational studies indicated that the shorter protein 4.1R was not a proteolytic product of the longer one but a product synthesized from the downstream AUG2 site. Results of further experiments showed that the sequence 5’-upstream of exon 4 was essential for the use of the AUG2 as internal initiation site of translation. When this sequence was introduced in a bicistronic vector, it directed the synthesis of the second cistron even when a hairpin structure was added 5’- upstream of the first cistron and only one mRNA species was detected by Northern blot analysis. In vivo expression of this set of 4.1R cDNAs confirmed that shorter and longer 4.1R isoforms were generated but not when the 4.1R sequence was introduced in a promoterless vector. These results indicate that the 5’ region upstream of exon 4 contains an internal ribosome entry site (IRES) element which directs the synthesis of 80-kDa 4.1R isoforms from mRNAs containing exon 2’. These data show that generation of 4.1R isoforms is also regulated at the translational level. The current study identified a set of 4.1R cDNAs coding a new group of isoforms lacking the carboxy-terminal domain (CTD) characteristic of all 4.1R proteins identified so far. An alternative poly-A signal present in the sequence between exons 17 and 18 of the EPB41 gene and the inclusion of twenty seven nucleotides comprising a stop codon regulated the production of this set of 4.1R isoforms lacking the CTD. Generation of an antiserum that specifically recognized this set of isoforms as well as transfection experiments using the isolated 4.1R cDNAs have allowed to determine the localization of these isoforms within the cell. Together, our data implicate two mechanisms not previously described as involved in the regulation of 4.1R expression: internal translation and alternative polyadenilation with the inclusion of a premature stop codon.
Descripción Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura 20-06-2007
URI http://hdl.handle.net/10261/8338
Aparece en las colecciones: (CBM) Tesis
Ficheros en este ítem:
Fichero Descripción Tamaño Formato  
Eva Pilar Lospitao Ruiz.pdf4,3 MBAdobe PDFVista previa
Visualizar/Abrir
Mostrar el registro completo
 


NOTA: Los ítems de Digital.CSIC están protegidos por copyright, con todos los derechos reservados, a menos que se indique lo contrario.