Identification of cellular mRNA targets for RNA-binding protein Sam68.
Sam68 (Src-associated in mitosis, 68 kDa), a nuclear RNA-binding protein, has been postulated to play a role in cell-growth control as a modulator of signal transduction and activation of RNA metabolism. Although Sam68 was demonstrated to bind to the UAAA sequences in synthetic oligoribonucleotides and poly(U) homopolymers in vitro, the legitimate cellular mRNA target remained unclear. By using the differential display and cDNA-representational difference analysis techniques, followed by reverse transcription polymerase chain reaction of RNAs co-immunoprecipitated with Sam68 from a HeLa cell lysate, we identified 10 mRNA species that bind in vivo to Sam68 in an RNA-binding domain-dependent manner. Among them, the mRNA species for hnRNP A2/B1 and beta-actin were found to bind prominently in vivo as well as in vitro, suggesting the possible involvement of Sam68 in the post- transcriptional regulation of these genes. Mapping of the Sam68-binding sequence revealed that Sam68 associates with these mRNAs through different nucleotide motifs, UAAA for hnRNP A2/B1 mRNA and UUUUUU for beta-actin mRNA, and that both binding sequences must reside in a loop structure for recognition by Sam68. The results indicated that Sam68 recognizes both the UAAA motif and poly(U) sequences in vivo for binding to cellular target mRNAs.
Itoh
Michiyasu
M
Department of Microbiology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi, Osaka 570-8506, Japan.
Haga
Izumi
I
Li
Qing-Hua
QH
Fujisawa
Jun-ichi
J
D020413:3' Untranslated Regions / Q000235:genetics / Q000378:metabolism; D000199:Actins / Q000235:genetics; D048868:Adaptor Proteins, Signal Transducing; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D020413:3' Untranslated Regions; D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D034441:Heterogeneous-Nuclear Ribonucleoproteins; C489559:KHDRBS1 protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase