Detection and characterization of a 3' untranslated region ribonucleoprotein complex associated with human alpha-globin mRNA stability.
The highly stable nature of globin mRNA is of central importance to erythroid cell differentiation. We have previously identified cytidine-rich (C-rich) segments in the human alpha-globin mRNA 3' untranslated region (alpha-3'UTR) which are critical in the maintenance of mRNA stability in transfected erythroid cells. In the present studies, we have detected trans-acting factors which interact with these cis elements to mediate this stabilizing function. A sequence-specific ribonucleoprotein (RNP) complex is assembled after incubation of the alpha-3'UTR with a variety of cytosolic extracts. This so-called alpha-complex is sequence specific and is not formed on the 3'UTR of either beta-globin or growth hormone mRNAs. Furthermore, base substitutions within the C-rich stretches which destabilize alpha-globin mRNA in vivo result in a parallel disruption of the alpha-complex in vitro. Competition studies with a series of homoribopolymers reveals a striking sensitivity of alpha-complex formation to poly(C), suggesting the presence of a poly(C)-binding activity within the alpha-complex. Three predominant proteins are isolated by alpha-3'UTR affinity chromatography. One of these binds directly to poly(C). This cytosolic poly(C)-binding protein is distinct from previously described nuclear poly(C)-binding heterogeneous nuclear RNPs and is necessary but not sufficient for alpha-complex formation. These data suggest that a messenger RNP complex formed by interaction of defined segments within the alpha-3'UTR with a limited number of cytosolic proteins, including a potentially novel poly(C)-binding protein, is of functional importance in establishing high-level stability of alpha-globin mRNA.
Wang
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Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia 19104-6145.
Kiledjian
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Weiss
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Liebhaber
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D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002846:Chromatography, Affinity; D017931:DNA Primers; D005347:Fibroblasts; D005914:Globins / Q000096:biosynthesis* / Q000235:genetics; D013006:Growth Hormone / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D017354:Point Mutation; D011066:Poly C / Q000378:metabolism; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000302:isolation & purification / Q000378:metabolism*; D013698:Templates, Genetic; D014162:Transfection
D017931:DNA Primers; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D011066:Poly C; D013006:Growth Hormone; D005914:Globins