Complex protein interactions within the human polyadenylation machinery identify a novel component

Abstract

Polyadenylation of mRNA precursors is a two-step reaction requiring multiple protein factors. Cleavage stimulation factor (CstF) is a heterotrimer necessary for the first step, endonucleolytic cleavage, and it plays an important role in determining the efficiency of polyadenylation. Although a considerable amount is known about the RNA binding properties of CstF, the protein-protein interactions required for its assembly and function are poorly understood. We therefore first identified regions of the CstF subunits, CstF-77, CstF-64, and CstF-50, required for interaction with each other. Unexpectedly, small regions of two of the subunits participate in multiple interactions. In CstF-77, a proline-rich domain is necessary not only for binding both other subunits but also for self-association, an interaction consistent with genetic studies in Drosophila. In CstF-64, a small region, highly conserved in metazoa, is responsible for interactions with two proteins, CstF-77 and symplekin, a nuclear protein of previously unknown function. Intriguingly, symplekin has significant similarity to a yeast protein, PTA1, that is a component of the yeast polyadenylation machinery. We show that multiple factors, including CstF, cleavage-polyadenylation specificity factor, and symplekin, can be isolated from cells as part of a large complex. These and other data suggest that symplekin may function in assembly of the polyadenylation machinery.

FIG. 1

Structural domains of CstF-64 and CstF-50 required for association with CstF-77. (A) The hinge domain of CstF-64 is necessary for association with CstF-77. Purified CstF was resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and either stained with India ink (Pro, lane 1) or probed with ³⁵S-labeled wild-type (wt, lane 2) or mutant (deletions 1 to 10, lanes 3 to 7 and 9 to 13) CstF-64. ³⁵S-labeled luciferase (C, lane 8) was used as a negative control. Positions of protein size markers and CstF-77 are indicated on the left and right, respectively. Diagrams of wild-type and mutant CstF-64 proteins are shown at the bottom. (B) The WD-40 motif of CstF-50 is required for association with CstF-77. Purified CstF was transferred onto a nitrocellulose membrane and stained with India ink (Pro, lane 13) or probed with ³⁵S-labeled wild-type (wt, lanes 2 and 8) or mutant (deletions 1 to 8, lanes 3 to 6 and 9 to 12) CstF-50. ³⁵S-labeled brome mosaic virus proteins (C1, lane 1) and luciferase (C2, lane 7) were used as negative controls. Diagrams of wild-type and mutant CstF-50 proteins are shown at the bottom.

FIG. 2

The C terminus of CstF-77 is necessary for its association with other CstF subunits. Purified CstF was transferred onto a nitrocellulose membrane and probed with ³⁵S-labeled wild-type (wt, lane 1) or mutant (deletions 1 to 12, lanes 2 to 13) CstF-77 as in Fig. 1. Diagrams of wild-type and mutant CstF-77 proteins are shown at the bottom.

FIG. 3

The hinge domain of CstF-64 is necessary and sufficient for its binding to a 135-kDa protein. (A) The hinge domain of CstF-64 is necessary for binding to a 135-kDa nuclear protein. The (NH₄)₂SO₄ fraction (20 to 40% saturation) derived from HeLa cell nuclear extract (lanes 1 and 2) and a CFI- and CFII-containing fraction obtained by Mono Q chromatography (lanes 3 to 15) were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with ³⁵S-labeled wild-type (wt, lanes 2 and 4) or mutant (deletions 1 to 10, lanes 5 to 9 and 11 to 15) CstF-64. ³⁵S-labeled brome mosaic virus proteins (C1, lane 3) and luciferase (C2, lanes 1 and 10) were used as negative controls. Diagrams of wild-type and mutant CstF-64 proteins are shown at the bottom. (B) The hinge domain of CstF-64 is sufficient for binding the 135-kDa protein. Proteins in the (NH₄)₂SO₄ fraction used in panel A were transferred onto a nitrocellulose membrane and probed with ³²P-labeled GST (lane 3) or GST fusion (lanes 1 and 2) proteins.

FIG. 4

Structure of symplekin and similarity to yeast PTA1. (A) Symplekin-I and symplekin-II have a common amino acid sequence. They are identical up to amino acid residue 964 but diverge thereafter, apparently due to alternative splicing (AS). Putative nuclear localization signals (NLS) are indicated by arrowheads. The region homologous to yeast PTA1 (box) and the sizes of the proteins are also shown. (B) Symplekin has extensive similarity with yeast PTA1. Amino acid (a.a.) sequences of symplekin (top) and PTA1 (bottom) are optimally aligned according to the FASTA program, and identical (lines) and similar (dots) residues are shown in bold type. Similarities are defined as I = L = V = M, Y = F = W, K = R, D = E, S = T, and Q = N. Amino acid residues are numbered on the right.

FIG. 5

Both symplekin-I and symplekin-II interact with CstF-64. Symplekin-I (Sym-I) and symplekin-II (Sym-II), CstF-77 (77K), and CstF-64 (64K) mRNAs were translated in vitro alone or in combination in the presence of [³⁵S]methionine and immunoprecipitated with an anti-CstF-64 MAb. Then 25% of the input in vitro translation mixtures (I, odd-numbered lanes) and 50% of the immunoprecipitated proteins (P, even-numbered lanes) were resolved by SDS-PAGE and visualized by autoradiography.

FIG. 6

Symplekin-I inhibits association between CstF-77 and CstF-64. CstF-77 and CstF-64 mRNAs were translated in vitro in the absence (lanes 1 and 2) or presence of increasing concentrations of symplekin-I (lanes 3 to 12). Then 10% of the in vitro translation mixtures (I, odd-numbered lanes) and 30% of the immunoprecipitated proteins (P, even-numbered lanes) were resolved by SDS-PAGE.

FIG. 7

Symplekin-I exists in a complex containing both CstF and CPSF. (A) Immunoaffinity purification of a polyadenylation complex under mild conditions. The CSF-containing fraction obtained by Superose 6 column chromatography was subjected to immunoaffinity purification using an anti-CstF-64 MAb in the presence of 50 mM (NH₄)₂SO₄ but no NP-40. Total starting material (T) (lanes 1 and 4) and flowthrough (F) (lanes 2 and 5) and bound (B) (lanes 3 and 6) fractions were loaded on an SDS–10% polyacrylamide gel and stained with silver (lanes 1 to 3) or probed with anti-CstF-64, anti-CPSF-160, or anti-symplekin antibody (lanes 4 to 6). (B) Immunoaffinity purification of a polyadenylation complex under stringent conditions. The CSF-containing fraction was subjected to immunoaffinity purification using an anti-polyomavirus large T (αPy-lgT; lanes 1 to 3 and 7 to 9) or anti-CstF-64 (αCstF-64; lanes 4 to 6 and 10 to 12) MAb in the presence of 150 mM (NH₄)₂SO₄ and 0.05% NP-40. Total starting material (T), (lanes 1, 4, 7, and 10) and flowthrough (F) (lanes 2, 5, 8, and 11) and bound (B) (lanes 3, 6, 9, and 12) fractions were loaded on an SDS–7.5% polyacrylamide gel and stained with silver (lanes 1 to 6) or probed with anti-CstF-64, anti-CPSF-160, anti-CPSF-100/73, or anti-symplekin antibody (lanes 7 to 12).