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


RESULTS
By analyzing the in vitro 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.


CONCLUSIONS
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.
Lospitao
Eva
E
Departamento de Biología Molecular, Universidad Autónoma de Madrid y Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas, Nicolás Cabrera, Madrid, Spain. elospitao@cbm.uam.es
Pérez-Ferreiro
Carmen M
CM
Gosálbez
Altea
A
Alonso
Miguel A
MA
Correas
Isabel
I
D024506:5' Flanking Region; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003598:Cytoskeletal Proteins / Q000096:biosynthesis* / Q000235:genetics*; D005786:Gene Expression Regulation; D005796:Genes / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics*; D038941:Polypyrimidine Tract-Binding Protein / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics*; D020033:Protein Isoforms
D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D020033:Protein Isoforms; C028841:erythrocyte membrane band 4.1 protein; D038941:Polypyrimidine Tract-Binding Protein