Abstract
We isolated cDNAs that encode a 77-kDa peptide similar to repeats 10-16 of beta-spectrins. Its gene localizes to human chromosome 19q13.13-q13.2 and mouse chromosome 7, at 7.5 centimorgans. A 289-kDa isoform, similar to full-length beta-spectrins, was partially assembled from sequences in the human genomic DNA data base and completely cloned and sequenced. RNA transcripts are seen predominantly in the brain, and Western analysis shows a major peptide that migrates as a 72-kDa band. This new gene, spectrin betaIV, thus encodes a full-length minor isoform (SpbetaIVSigma1) and a truncated major isoform (SpbetaIVSigma5). Immunostaining of cells shows a micropunctate pattern in the cytoplasm and nucleus. In mesenchymal stem cells, the staining concentrates at nuclear dots that stain positively for the promyelocytic leukemia protein (PML). Expression of SpbetaIVSigma5 fused to green fluorescence protein in cells produces nuclear dots that include all PML bodies, which double in number in transfected cells. Deletion analysis shows that partial repeats 10 and 16 of SpbetaIVSigma5 are necessary for nuclear dot formation. Immunostaining of whole-mount nuclear matrices reveals diffuse positivity with accentuation at PML bodies. Spectrin betaIV is the first beta-spectrin associated with a subnuclear structure and may be part of a nuclear scaffold to which gene regulatory machinery binds.
Highlights
Spectrin is an important component of the membrane skeleton attached to the inner leaf of the lipid bilayer of plasma membranes
Spectrin IV resides in the cytoplasm, where it may attach to vesicles, and in the nucleus, where it associates with PML2 bodies and the nuclear matrix
Comparison between the cDNA and genomic sequence of the spectrin-like gene reveals an exon-intron structure identical to that of spectrin I gene [38], with 36 exons that span Ͼ145 kb, except that exon 31, which contains most of the unique threerepeat domain, is longer in size. These results indicate that the new spectrin-like gene is a bona fide -spectrin gene, which we designate as spectrin IV and which has both a full-length isoform (SpIV⌺1; GenBank accession number AF311855) and a truncated isoform represented by clone N164/N155 (SpIV⌺5; GenBank accession number AF311856)
Summary
Spectrin is an important component of the membrane skeleton attached to the inner leaf of the lipid bilayer of plasma membranes. We have previously described spectrin III (see footnote 1 for nomenclature), which associates with the Golgi and intracellular vesicles [9]. GFP, green fluorescence protein; MDCK, Madin-Darby canine kidney cells; MSCs, mesenchymal stem cells; N164/N155, a contig of overlapping spectrin IV clones N164 and N155; NR, non-repeat segment of. While this manuscript was in revision, Berghs et al [23] independently described spectrin IV and four of its isoforms: IV⌺1–IV⌺4. The 77-kDa isoform (IV⌺5) described here is the major isoform of spectrin IV It was not reported by Berghs et al and could not have been detected with the antibodies they employed [23]
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