U1A inhibits cleavage at the immunoglobulin M heavy-chain secretory poly(A) site by binding between the two downstream GU-rich regions

Abstract

The immunoglobulin M heavy-chain locus contains two poly(A) sites which are alternatively expressed during B-cell differentiation. Despite its promoter proximal location, the secretory poly(A) site is not expressed in undifferentiated cells. Crucial to the activation of the secretory poly(A) site during B-cell differentiation are changes in the binding of cleavage stimulatory factor 64K to GU-rich elements downstream of the poly(A) site. What regulates this change is not understood. The secretory poly(A) site contains two downstream GU-rich regions separated by a 29-nucleotide sequence. Both GU-rich regions are necessary for binding of the specific cleavage-polyadenylation complex. We demonstrate here that U1A binds two (AUGCN(1-3)C) motifs within the 29-nucleotide sequence and inhibits the binding of cleavage stimulatory factor 64K and cleavage at the secretory poly(A) site.

FIG. 1.

Schematic model of the Ig secretory poly(A) site. (A) The genetic organization of the IgM heavy chain and its alternative processing to a secretory or a membrane form of mRNA. (B) The location of the secretory poly(A) site and relative location of the 5′ splice site, the U1A binding motifs, the hexanucleotide sequence, and the downstream GU-rich regions. Numbers indicate the positions referred to in the text. (C) A comparison of the AUGCN_1-3C sequences for the 2s, 4s, 8s, ds1, and ds2 U1A binding sites.

FIG. 2.

U1A protects AUGCN_1-3C sequences downstream of the cleavage site between the two GU-rich regions. (A) 3′ end-labeled RNA. The indicated amounts of U1A were allowed to bind the RNA before partial RNase T1 digestion. Samples were treated with proteinase K before PAGE. Sites of RNase T1 digestion are labeled with position numbers according to the mouse IgM sequence with accession number V00818. The downstream footprint is labeled with a line, the positions of the two AUGCN_1-3C sequences are indicated with asterisks. The protected bands are indicated with open symbols, and those used as comparison are indicated with solid symbols. Symbols relate to quantitation in panel B. (B) Quantitation of the bands marked with symbols in panel A by phosphorimager analysis. Results were normalized to zero U1A. Symbols refer to bands marked with equivalent symbols in panel A. (C) Schematic diagram of predicted RNA structure of the poly(A) site and the downstream region (position 1949 to 2085). The position of the RNase T1 cuts are indicated with arrows of the size proportional to the intensity of the cut. Positions of RNase T1 cuts quantitated in panel B are indicated with the equivalent symbol. The AUGCN_1-3C motifs are indicated with brackets and labeled with asterisks. The positions of the GU-rich sequences are indicated by arrows and shaded.

FIG. 3.

Inclusion of sequences downstream of the secretory poly(A) site results in the formation of two extra U1A(IgM) complexes. EMSAs using recombinant U1A and uniformly radiolabeled RNA spanning the indicated sequences. IgM(U1A) complexes are labeled with arrows and numbers representing the number of U1A molecules in each complex.

FIG. 4.

Mutations in either or both of the downstream AUGCN_1-3C sequences reduced U1A binding to the secretory poly(A) site in the context of intact upstream U1A binding sites. EMSAs used recombinant U1A and uniformly radiolabeled RNA containing the indicated mutation. (A) Mutation of the downstream U1A binding sites results in fewer IgM(U1A) complexes. Bands representing the unbound substrate (IgM) and the IgM(U1A) complexes are indicated with an arrow or bracket, respectively. The number of complexes formed [(U1A)N] are indicated below each lane. wt, wild type. (B) A phosphorimager quantitation of the bands in panel A. Bars represent pixels (arbitrary units) for each of the five complexes. The number of U1A molecules in the complex is indicated on the left. Each set of bar graphs appears directly below the lane it represents.

FIG. 5.

U1A binding between the downstream GU-rich regions inhibits secretory poly(A) site expression in vivo. Luciferase constructs containing the wild-type secretory poly(A) site from position 1790 to 2085 or that containing single (mut ds1 and mut ds2) or double mutations (mut ds12) in the downstream U1A binding sites were transfected into HeLa cells, and luciferase activity was measured 24 h later. Bars represent the mean of triplicates ± SE. Activity was expressed as a percentage of the wild type (wt).

FIG. 6.

U1A binding between the two GU-rich regions inhibits CstF64K binding. (A) CstF64K UV cross-linking assays. Uniformly radiolabeled RNA was incubated with recombinant GSTCstF64KRBD (2.5 μM) and increasing concentrations of U1A (as indicated), also expressed as a molar ratio of U1A to GSTCstF64KRBD. Products were cross-linked with UV light, subjected to RNase A digestion, and run on SDS-12% PAGE. wt, wild type. (B) Phosphorimager quantitation of panel A. CstF64K cross-link, results expressed as a percentage of CstF64K binding without added U1A; U1A cross-linking, results expressed a percentage of 500 nM U1A binding the wild-type substrate (lane 4, U1A cross-link). Solid circles, wild type; solid squares, IgM 1790-2085 mut 248; open circles, IgM 1790-2085 mut ds12; open squares, IgM 1790-2085 mut 248 ds12. Data are means of triplicates from three separate cross-linkings ± SE.

FIG. 7.

U1A binding between the two GU-rich regions inhibits cleavage at the secretory poly(A) site. Uniformly radiolabeled RNA was incubated with increasing concentrations of recombinant U1A and HeLa cell extracts for 2 h at 30°C. After proteinase K digestion, phenol-chloroform extraction, and ethanol precipitation, products were run on 8% denaturing PAGE. The full-length RNA and cleavage products are indicated on the right. The controls were an extended hexanucleotide mutant spanning position 1790 to 2085 which did not cleave in HeLa cell extracts (lane 2) and a precleaved RNA substrate (position 1790 to 1998) as a marker of the correct position for the cleaved product (lane 3). Lanes 4 and 10 are input RNA without nuclear extracts. wt, wild type. (B) Phosphorimager quantitation of panel A. Results are expressed as percent cleavage. Solid squares are the quantitation of the wild type (lanes 5 to 9) with increasing concentrations of recombinant U1A; open circles represent the same for the substrate containing mutations in the downstream U1A (lanes 11 to 15). Data are means of triplicates from three separate cleavage assays ± SE.