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Title

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

AuthorsLospitao, Eva Pilar
AdvisorCorreas, Isabel
KeywordsSíntesis de proteínas
Issue Date2007
PublisherUniversidad Autónoma de Madrid
AbstractRed 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.
DescriptionTesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura 20-06-2007
URIhttp://hdl.handle.net/10261/8338
Appears in Collections:(CBM) Tesis
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