Identification, molecular cloning, expression and chromosome mapping of a family of transformation upregulated hnRNP-K proteins derived by alternative splicing.
Acidic nuclear proteins (M(r) between 64,000 and 66,000; pI 4.9 to 5.5) that are highly upregulated in transformed cells and that belong to the hnRNP-K family have been identified using a monoclonal antibody (mAB B4B6) that distinguish between quiescent and proliferating human keratinocytes. The family, which is composed of four major proteins (hnRNPs-K A, B, C and D) and their modified forms, is present in similar overall levels in quiescent and proliferating normal keratinocytes although clear differences were observed in the levels of some of the individual variants. Immunofluorescence staining of proliferating normal keratinocytes with mAB B4B6 showed that about 40% of the keratinocytes, corresponding mainly to G1 and to half of the cells in S-phase, reacted with the antibody depicting a dotted, nucleoplasmic staining that excluded the nucleolus. Only 3 to 4% of the quiescent keratinocytes reacted with the antibody while simian virus 40 (SV40) transformed keratinocytes (K14) stained constitutively throughout the cell cycle. Using mAB B4B6 as a probe we cloned a cDNA coding for one member of the family (hnRNP-K B) and this was used to screen for additional family members. Sequencing of the positive clones revealed four different cDNAs, all resulting from alternative splicing of a common primary transcript of a gene that mapped to chromosome 9. Expression of the cDNAs in the vaccinia virus system confirmed their identity as hnRNPs-K A, B, C and D and showed that their modified forms are phosphorylated. All four hnRNPs bound poly(rC) on NorthWestern blots, although the more acidic of the phosphorylated forms, did so at a much reduced level. hnRNP-K has been implicated in pre-mRNA metabolism of transcripts containing cytidine-rich sequences and our results point towards a role during cell cycle progression.
Dejgaard
K
K
Institute of Medical Biochemistry, University of Aarhus, Denmark.
Leffers
H
H
Rasmussen
H H
HH
Madsen
P
P
Kruse
T A
TA
Gesser
B
B
Nielsen
H
H
Celis
J E
JE
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002455:Cell Division; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D002874:Chromosome Mapping; D002899:Chromosomes, Human, Pair 9; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D015870:Gene Expression; D005786:Gene Expression Regulation; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008969:Molecular Sequence Data; D012322:RNA Precursors / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000096:biosynthesis; D012261:Ribonucleoproteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D012867:Skin / Q000166:cytology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins