A splicing alteration of 4.1R pre-mRNA generates 2 protein isoforms with distinct assembly to spindle poles in mitotic cells.
The C-terminal region of erythroid cytoskeletal protein 4.1R, encoded by exons 20 and 21, contains a binding site for nuclear mitotic apparatus protein (NuMA), a protein needed for the formation and stabilization of the mitotic spindle. We have previously described a splicing mutation of 4.1R that yields 2 isoforms: One, CO.1, lacks most of exon 20-encoded peptide and carries a missense C-terminal sequence. The other, CO.2, lacks exon 20-encoded C-terminal sequence, but retains the normal exon 21-encoded C-terminal sequence. Knowing that both shortened proteins are expressed in red cells and assemble to the membrane skeleton, we asked whether they would ensure 4.1R mitotic function in dividing cells. We show here that CO.2, but not CO.1, assembles to spindle poles, and colocalizes with NuMA in erythroid and lymphoid mutated cells, but none of these isoforms interact with NuMA in vitro. In microtubule-destabilizing conditions, again only CO.2 localizes to the centrosomes. These data suggest that the stability of 4.1R association with centrosomes requires an intact C-terminal end, either for a proper conformation of the protein, for a direct binding to an unknown centrosome-cytoskeletal network, or for both. We also found that 4.1G, a ubiquitous homolog of 4.1R, is present in mutated as well as control cells and that its C-terminal region binds efficiently to NuMA, suggesting that in fact mitotic spindles host a mixture of the two 4.1 family members. These findings led to the postulate that the coexpression at the spindle poles of 2 related proteins, 4.1R and 4.1G, might reflect a functional redundancy in mitotic cells.
Delhommeau
François
F
Institut National de la Santé et de la Recherche Médicale, U 473 and U 488, Le Kremlin-Bicêtre, France.
Vasseur-Godbillon
Corinne
C
Leclerc
Philippe
P
Schischmanoff
Pierre-Olivier
PO
Croisille
Laure
L
Rince
Patricia
P
Morinière
Madeleine
M
Benz
Edward J
EJ
Tchernia
Gil
G
Tamagnini
Gabriel
G
Ribeiro
Leticia
L
Delaunay
Jean
J
Baklouti
Faouzi
F
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001483:Base Sequence; D002455:Cell Division / Q000502:physiology; D018385:Centrosome / Q000502:physiology; D003598:Cytoskeletal Proteins; D017931:DNA Primers; D006412:Hematopoietic Stem Cells / Q000502:physiology; D006801:Humans; D008565:Membrane Proteins; D008938:Mitosis / Q000235:genetics*; D008969:Molecular Sequence Data; D009479:Neuropeptides; D020033:Protein Isoforms / Q000235:genetics; D011506:Proteins / Q000235:genetics*; D012322:RNA Precursors / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D008941:Spindle Apparatus / Q000235:genetics*
D003598:Cytoskeletal Proteins; D017931:DNA Primers; D008565:Membrane Proteins; D009479:Neuropeptides; D020033:Protein Isoforms; D011506:Proteins; D012322:RNA Precursors; D011994:Recombinant Proteins; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1