A novel cytoplasm-specific mRNP assembles on the 3′UTR of β-globin mRNA. (A) Site-specific assembly of a cytoplasm-restricted β-complex in vitro. A [³²P]-labeled RNA, corresponding to the polyadenylated human β-globin 3′UTR, was incubated with nuclear (Nuc) or cytoplasmic (Cyt) extract prepared from K562 cells, and RNase T1-resistant mRNPs resolved on a native polyacrylamide gel. Parallel reactions were supplemented with a 15-nt competitor DNA (WT₁₅) corresponding to a previously identified regulatory region of the β-globin 3′UTR (panel B), either in the sense (S) or control antisense (AS) orientation. An arrow indicates the β-complex. (B) Composition of the 132-nt β-globin 3′UTR. The positions of the 15-nt regulatory region (WT₁₅), the translation stop codon (TAA), and the poly(A) tail are indicated. (C) The β-complex assembles in erythroid-induced CD34⁺ cells. A [³²P]-labeled RNA, corresponding to the polyadenylated human β-globin 3′UTR, was incubated with cytoplasmic extract prepared from K562 cells, uninduced human CD34⁺ cells (U; day 5), or erythroid-induced CD34⁺ cells (I; day 17). Reactions were amended with WT₁₅ competitor DNA in either the sense (S) or antisense (AS) orientation. An arrow indicates the β-complex. (D) The β-complex is restricted to the cytoplasm of erythroid precursor cells. EMSA analyses were conducted on a [³²P]-labeled RNA using nuclear and cytoplasmic extracts prepared from erythroid-induced CD34⁺ cells after 12 days of culture. Sense (S) or antisense (AS) WT₁₅ DNA competitor was added as indicated. An arrow denotes the β-complex.