Recognition of RNA branch point sequences by the KH domain of splicing factor 1 (mammalian branch point binding protein) in a splicing factor complex - PubMed (original) (raw)

Recognition of RNA branch point sequences by the KH domain of splicing factor 1 (mammalian branch point binding protein) in a splicing factor complex

H Peled-Zehavi et al. Mol Cell Biol. 2001 Aug.

Abstract

Mammalian splicing factor 1 (SF1; also mammalian branch point binding protein [mBBP]; hereafter SF1/mBBP) specifically recognizes the seven-nucleotide branch point sequence (BPS) located at 3' splice sites and participates in the assembly of early spliceosomal complexes. SF1/mBBP utilizes a "maxi-K homology" (maxi-KH) domain for recognition of the single-stranded BPS and requires a cooperative interaction with splicing factor U2AF65 bound to an adjacent polypyrimidine tract (PPT) for high-affinity binding. To investigate how the KH domain of SF1/mBBP recognizes the BPS in conjunction with U2AF and possibly other proteins, we constructed a transcriptional reporter system utilizing human immunodeficiency virus type 1 Tat fusion proteins and examined the RNA-binding specificity of the complex using KH domain and RNA-binding site mutants. We first established that SF1/mBBP and U2AF cooperatively assemble in our reporter system at RNA sites composed of the BPS, PPT, and AG dinucleotide found at 3' splice sites, with endogenous proteins assembled along with the Tat fusions. We next found that the activities of the Tat fusion proteins on different BPS variants correlated well with the known splicing efficiencies of the variants, supporting a model in which the SF1/mBBP-BPS interaction helps determine splicing efficiency prior to the U2 snRNP-BPS interaction. Finally, the likely RNA-binding surface of the maxi-KH domain was identified by mutagenesis and appears similar to that used by "simple" KH domains, involving residues from two putative alpha helices, a highly conserved loop, and parts of a beta sheet. Using a homology model constructed from the cocrystal structure of a Nova KH domain-RNA complex (Lewis et al., Cell 100:323-332, 2000), we propose a plausible arrangement for SF1/mBBP-U2AF complexes assembled at 3' splice sites.

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Figures

FIG. 1

(A) Schematic diagrams of the Tat fusion expressor and RNA reporter plasmids. The expressor plasmid encodes HIV-1 Tat residues 1 to 72, followed by a linker of three glycines and the relevant fusion, expressed from a simian virus 40 early promoter (PSV40). The reporter plasmid utilizes a modified HIV-1 LTR to drive CAT expression, with the BPS-PPT-AG sequence replacing the TAR site at the 5′ end of the mRNA. (B) Domain organization of wild-type SF1/mBBP and U2AF65 (top) and the Tat fusion proteins (bottom). The maxi-KH domain with flanking QUA1 and QUA2 regions characteristic of STAR proteins, the Zn knuckle, and the proline-rich region of SF1/mBBP and the RS domain and three RNP domains of U2AF65 are indicated. Numbers refer to amino acid positions in the proteins. The Tat fusions contain amino acids 1 to 72 of Tat followed by the glycine linker. (C) The configuration of the BPS, PPT, and AG dinucleotide at canonical 3′ splice sites (ss) (top) and the sequences of the BPS reporter and mutants (bottom) are shown. BPS variants indicate the single nucleotide substitutions used at each position.

FIG. 2

Activation of the BPS reporter by Tat-fused SF1/mBBP and Tat-fused U2AF65. (A) Titration of the Tat-fused SF1/mBBP expressor plasmid (filled circles) and unfused Tat (open circles) on the BPS reporter. Tat-expressing and BPS reporter plasmids were cotransfected into HeLa cells, and CAT activities were measured after 44 h. (Insets) CAT assays, with expressor plasmid amounts (nanograms) indicated. Fold activation was determined relative to the activity of the reporter alone. (B) Titration of the Tat-fused U2AF65 expressor plasmid (filled squares) and unfused Tat (open circles) on the BPS reporter. (C) Titration of the Tat-fused SF1/mBBP (filled circles), Tat-fused U2AF65 (filled squares), and unfused Tat (open circles) expressor plasmids on the HIV-1 TAR reporter. All fusions contain the RNA-binding domain of Tat and thus are able to activate transcription via the TAR element. Relative activities of the fusion proteins on the TAR reporter were used to normalize for fusion protein expression levels.

FIG. 3

Activation by the Tat fusion proteins requires RNA-protein interactions at the BPS, PPT, and AG dinucleotide. (A) Activation of the BPS reporter and mutants by Tat-fused SF1/mBBP (gray bars) and Tat-fused SF1/mBBPΔN (white bars). Tat-expressing (5 ng) and BPS reporter (100 ng) plasmids were cotransfected into HeLa cells, and CAT activities were measured after 44 h. Percent activation was calculated by normalizing to the activation level of the Tat-fused SF1/mBBP-BPS reporter interaction, and standard deviations (bars) were calculated as described in Materials and Methods. (B) Activation of the BPS reporter and mutants by Tat-fused U2AF65 (gray bars) and Tat-fused U2AF65Δ95-138 (white bars). Activities were normalized to the activation level of the Tat-fused U2AF65-BPS reporter interaction.

FIG. 4

Activation of BPS variant reporters by Tat-fused SF1/mBBP (A) and Tat-fused U2AF65 (B). Percentages of activation and standard deviations (bars) were calculated as for Fig. 3, with activities normalized to the Tat-fused SF1/mBBP-BPS reporter and Tat-fused U2AF65-BPS reporter interactions, respectively. BPS substitutions are highlighted.

FIG. 5

Activation of the BPS reporter by Tat-fused SF1/mBBP mutants. (A) Sequence alignment of the maxi-KH domain of SF1/mBBP and selected KH domains from vigilin (vig; domain 6), Nova-2 (domain 3), Nova-1 (domain 3), and hFMR-1 (domain 1). Secondary structure elements were assigned based on the X-ray and NMR structures of these individual domains (, ; Musco et al., letter). B and H, β-sheet and α-helical residues, respectively, which are defined clearly in all structures; lowercase letters, residues that can be assigned to a secondary structure in only one or two structures. Residues considered to be part of the hydrophobic core and therefore not chosen for mutation are shaded. Numbers refer to the SF1/mBBP sequence, and residues marked with an asterisk were mutated to alanines. (B) Averaged NMR structure of the sixth KH domain from vigilin (PDB file 1VIH [39]), shown from two different faces. Circles, approximate locations of amino acids chosen for mutagenesis, assuming that SF1/mBBP adopts a similar fold; yellow circles, positions that decrease activity by at least twofold (see panel C); white circles, positions that have little or no effect. (C) Activities of the Tat-fused SF1/mBBP mutants normalized to the activity of the wild-type (wt) protein with standard deviations (bars) calculated as for Fig. 3. The line corresponds to a twofold decrease in activity. The predicted corresponding units of secondary structures are indicated.

FIG. 6

(A) Sequence alignment of SF1/mBBP, BBP from Saccharomyces cerevisiae (yBBP), and KH domain 3 from Nova-2. Black boxes, identical residues; shaded boxes, conserved residues. The corresponding units of secondary structure are indicated. An extra C-terminal region is shown, compared to the sequence shown in Fig. 5A; this region corresponds to part of the Nova-2 structure. Asterisks and numbers, positions in SF1/mBBP important for binding based on the mutagenesis data (Fig. 5); minuses, positions where mutations have little or no effect. The numbering of Nova-2 residues discussed in the text (and not shown) corresponds to that described for the cocrystal structure (35). The region corresponding to the beginning of QUA2 is indicated. (B) Structure of the Nova-2 KH domain, taken from the cocrystal structure of the protein-RNA complex (35). Amino acids involved in RNA binding are shown in yellow. (C) Structure of the Nova-2 KH domain complexed to RNA (35), with the RNA tetranucleotide specifically recognized by the protein shown in yellow. The 5′ and 3′ ends of the RNA are indicated. (D) Homology model of SF1/mBBP maxi-KH domain, based on the structure of the RNA-bound Nova-2 domain and the alignment shown in panel A. The approximate locations of residues important for RNA binding, as defined by mutagenesis, are shown in yellow. The large β1/α1 and β2/β3 loops found in the SF1/mBBP maxi-KH domain were positioned arbitrarily by the homology modeling. (E) Proposed RNA-binding orientation of the SF1/mBBP-U2AF complex, based on similarities to the Nova-2 protein-RNA complex (see Discussion). The schematic drawing is not intended to indicate the relative orientations of the U2AF65 and U2AF35 subunits or of the RS domain of U2AF65, which also may contact the RNA (56).

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Recognition of RNA branch point sequences by the KH domain of splicing factor 1 (mammalian branch point binding protein) in a splicing factor complex - PubMed (original) (raw)