The evolutionarily conserved single-copy gene for murine Tpr encodes one prevalent isoform in somatic cells and lacks paralogs in higher eukaryotes.
Vertebrate Tpr and its probable homologs in insects and yeast are heptad repeat-dominated nuclear proteins of M(r) 195,000 to M(r) 267,000 the functions of which are still largely unknown. Whereas two homologs exist in Saccharomyces cerevisiae, it has remained uncertain whether metazoans possess different paralogs or isoforms of Tpr that might explain controversial reports on the subcellular localization of this protein. To address these possibilities, we first determined the sequence and structure of the murine tpr gene, revealing a TATA box-less gene of approximately 57 kb and 52 exons. Southern hybridization of genomic DNA and radiation hybrid mapping showed that murine tpr exists as a single-copy gene on chromosome 1; RNA blotting analyses and EST (expressed sequence tag) database mining revealed that its expression results in only one major mRNA in embryonic and most adult tissues. Accordingly, novel antibodies against the N- and C-terminus of Tpr identified the full-length protein as the major translation product in different somatic cell types; reinvestigation of Tpr localization by confocal microscopy corroborated a predominant localization at the nuclear pore complexes in these cells. Antibody specificity and reliability of Tpr localization was demonstrated by post-transcriptional tpr gene silencing using siRNAs that eliminated the Tpr signal at the nuclear periphery but did not affect intranuclear staining of Tpr-unrelated proteins. Finally, we defined several sequence and structural features that characterize Tpr polypeptides in different species and used these as a guideline to search whole-genome sequence databases for putative paralogs of Tpr in higher eukaryotes. This approach resulted in identification of the Tpr orthologs in Arabidopsis thaliana and Caenorhabditis elegans, but also in the realization that no further paralogs of Tpr exist in several metazoan model organisms or in humans. In summary, these results reveal Tpr to be a unique protein localized at the nuclear periphery of the somatic cell in mammals.
Kuznetsov
Nikolai V
NV
Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.
Sandblad
Linda
L
Hase
Manuela E
ME
Hunziker
Andreas
A
Hergt
Michaela
M
Cordes
Volker C
VC
D000818:Animals; D002874:Chromosome Mapping; D017124:Conserved Sequence / Q000235:genetics*; D003360:Cosmids; D005057:Eukaryotic Cells; D019143:Evolution, Molecular; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000737:chemistry / Q000502:physiology*; D010802:Phylogeny; D020033:Protein Isoforms / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000145:classification / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; C073597:TPR protein, human