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. 2001 Oct;21(19):6406-17.
doi: 10.1128/MCB.21.19.6406-6417.2001.

Domains in human splicing factors SF3a60 and SF3a66 required for binding to SF3a120, assembly of the 17S U2 snRNP, and prespliceosome formation

D Nesic  1 , A Krämer
Affiliations

Domains in human splicing factors SF3a60 and SF3a66 required for binding to SF3a120, assembly of the 17S U2 snRNP, and prespliceosome formation

D Nesic et al. Mol Cell Biol. 2001 Oct.

Abstract

The active 17S U2 small nuclear ribonucleoprotein particle (snRNP), which binds to the intron branch site during the formation of the prespliceosome, is assembled in vitro by sequential interactions of the essential splicing factors SF3b and SF3a with the 12S U2 snRNP. We have analyzed the function of individual subunits of human SF3a (SF3a60, SF3a66, and SF3a120) by testing recombinant proteins, expressed in insect cells, in various in vitro assays. The recombinant subunits readily form the SF3a heterotrimer, where SF3a60 and SF3a66 interact with SF3a120, but not with each other. All SF3a subunits are essential for the formation of the mature 17S U2 snRNP and the prespliceosome. Single subunits engage in interactions with the 15S U2 snRNP (consisting of the 12S U2 snRNP and SF3b), and SF3a60 appears to play a major role in recruiting SF3a120 into the U2 particle. Analysis of functional domains in SF3a60 and SF3a66 identified interaction sites for SF3a120 in their N-terminal portions. C(2)H(2)-type zinc finger domains mediate the integration of SF3a60 and SF3a66 into the U2 snRNP, and we propose a model in which protein-protein interactions between the zinc finger domains and the Sm proteins, common to all spliceosomal snRNPs, contribute to the assembly of the 17S U2 snRNP. Finally, we demonstrate that all domains required for interactions within the SF3a heterotrimer and the formation of the 17S U2 snRNP are also necessary to assemble the prespliceosome.

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Figures

FIG. 1
FIG. 1
Schematic representation of the structure of recombinant SF3a60 and SF3a66 proteins. Recombinant SF3a60 (A) and SF3a66 (B) proteins carried N-terminal His6 tags derived from the expression vector. Amino acids present in or deleted from the recombinant proteins are given in parentheses behind the names of the proteins. Conserved regions are indicated above the diagrams and numbered according to the amino acids in full-length SF3a60 and SF3a66.
FIG. 2
FIG. 2
The N terminus of SF3a60 is required for binding to SF3a120. Recombinant SF3a60 proteins, as indicated above the figure, were separated by SDS-PAGE and transferred to nitrocellulose. The membrane was incubated with in vitro-translated [35S]methionine-labeled SF3a120 (TNT 3a120), and bound SF3a120 was visualized by autoradiography (top). Input proteins separated in a parallel gel were visualized by Western blotting with antipolyhistidine antibodies (bottom). The migration of molecular mass standards (in kilodaltons) is indicated to the left of each panel.
FIG. 3
FIG. 3
Amino acids 35 to 107 of SF3a60 are sufficient for its incorporation into SF3a. (A) Coimmunoprecipitation assay. Recombinant SF3a60 proteins were separated by SDS-PAGE and stained with silver (left panel). Identical amounts of the proteins were precipitated with mAb66 bound to protein A-Sepharose in the presence or absence of SF3a66 and/or SF3a120 as indicated above the figure. Bound proteins were separated by SDS-PAGE and visualized by silver staining (right panel). The positions of SF3a120, SF3a66, and the IgG heavy and light chains are indicated. Arrows point to 3a60-N2 and -N3, which bound inefficiently to SF3a120, and to 3a60Δ1 migrating just above the IgG heavy chain. The migration of molecular mass standards (in kilodaltons) is indicated to the left of each panel. (B) GST pull-down. Recombinant SF3a proteins (as indicated above the figure) were either loaded directly onto a SDS-polyacrylamide gel or incubated with GST-SF3a66 and SF3a120. Before elution of bound proteins, the gluthathione agarose was washed with buffer containing 100 or 400 mM NaCl as indicated. Arrows indicate full-length SF3a60, 3a60-N1, and 3a60-N2. The positions of SF3a120 and GST-SF3a66 are shown on the right, and the migration of molecular mass standards (in kilodaltons) is indicated on the left.
FIG. 4
FIG. 4
The N-terminal half of SF3a66 is required and sufficient for binding to SF3a120. The recombinant SF3a66 proteins indicated above the figure were separated by SDS-PAGE and transferred to nitrocellulose. The membrane was incubated with in vitro-translated [35S]methionine-labeled SF3a120 (see Fig. 2) and bound SF3a120 was visualized by autoradiography (top). Input proteins separated in a parallel gel were visualized by Western blotting with antipolyhistidine antibodies (bottom). The migration of molecular mass standards (in kilodaltons) is indicated to the left of each panel.
FIG. 5
FIG. 5
The zinc finger domains in SF3a60 and SF3a66 are required for their integration into the active 17S U2 snRNP. (A) SF3a purified from HeLa cells (+), buffer (−), or recombinant SF3a subunits, as indicated above the figure, were incubated under splicing conditions with partially purified 15S U2 snRNP in the presence of a 5′-end-labeled oligoribonucleotide complementary to the 5′ end of U2 snRNA. RNP complexes were separated in a native 4% polyacrylamide gel at room temperature and exposed to X-ray film. The positions of the 15S and 17S U2 snRNPs are indicated to the left. (B) Recombinant SF3a subunits were incubated with the 15S U2 snRNP in the combinations shown above the figure and subjected to immunoprecipitation with anti-SF3b155 antibodies coupled to protein G-Sepharose (lanes 6 to 14). Precipitation of the 17S U2 snRNP served as a control (lane 15). Input (lanes 1 to 5) and bound proteins were separated by SDS-PAGE and stained with silver. The positions of SF3b155, SF3b145, and SF3b130 are indicated by a bracket labeled 3b. Recombinant SF3a proteins and SF3a subunits associated with the 17S U2 snRNP are indicated by arrows and lines, respectively. The migration of molecular mass standards (in kilodaltons) is indicated to the left. (C) Derivatives of SF3a60 were incubated with the 15S U2 snRNP and recombinant full-length SF3a66 and SF3a120 as indicated above the figure. The analysis of RNP complexes was performed as in panel A, except that the complexes were resolved at 4°C. (D) Derivatives of SF3a66 were incubated with the 15S U2 snRNP and recombinant full-length SF3a60 and SF3a120 as indicated above the figure. The analysis of RNP complexes was performed as in panel A.
FIG. 6
FIG. 6
The N-terminal regions and the zinc finger domains of SF3a60 and SF3a66 are essential for prespliceosome formation. The assembly of presplicing complex A was tested in the presence of buffer (−), HeLa cell nuclear extract (NE), or fractions containing partially purified SF1, U1 snRNP, the 12S U2 snRNP, U2AF, and SF3b. HeLa cell SF3a or recombinant proteins were added as indicated. Splicing complexes were separated in a native 4% polyacrylamide gel. The positions of pre-mRNA, complex H, presplicing complex A, and splicing complexes B and C are indicated on the left.
FIG. 7
FIG. 7
Recombinant SF3a66 and SF3a120 bind RNA. Full-length SF3a60, SF3a66, and SF3a120 (A) or full-length and mutant SF3a66 proteins (B) were UV cross-linked to synthetic [α-32P]UTP-labeled RNA, treated with RNases A and T1, and separated by SDS-PAGE. The gel was dried and exposed to X-ray film. The positions of SF3a66 and SF3a120 are shown. Asterisks indicate binding of unrelated proteins. Molecular mass standards (in kilodaltons) are shown on the left.
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