2024-03-29T08:55:59Zhttp://digital.csic.es/dspace-oai/requestoai:digital.csic.es:10261/83382016-02-16T04:04:55Zcom_10261_79com_10261_1col_10261_836
2008-11-07T13:14:47Z
urn:hdl:10261/8338
Mecanismos implicados en la generación de isoformas de la proteína 4.1R
Lospitao, Eva Pilar
Correas, Isabel
Síntesis de proteínas
Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura 20-06-2007
Red 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.
2008-11-07T13:14:47Z
2008-11-07T13:14:47Z
2007
tesis doctoral
http://hdl.handle.net/10261/8338
spa
openAccess
Universidad Autónoma de Madrid