Phosphorylation of spliceosomal protein SAP 155 coupled with splicing catalysis

Abstract

The U2 snRNP component SAP 155 contacts pre-mRNA on both sides of the branch site early in spliceosome assembly and is therefore positioned near or at the spliceosome catalytic center. We have isolated a cDNA encoding human SAP 155 and identified its highly related Saccharomyces cerevisiae homolog (50% identity). The carboxy-terminal two-thirds of SAP 155 shows the highest conservation and is remarkably similar to the regulatory subunit A of the phosphatase PP2A. Significantly, SAP 155 is phosphorylated concomitant with or just after catalytic step one, making this the first example of a protein modification tightly regulated with splicing catalysis.

Figure 1

(A) The amino acid sequence predicted from the human SAP 155 cDNA sequence. The RWDETP repeats at the amino terminus are aligned for convenience. These repeats and the TPGX repeats are indicated in boldface type, and the TPGH repeats are also underlined. The PP2A-like repeats are aligned according to the consensus for PP2A. This consensus is shown below the SAP 155 sequence. The underlined amino acids indicate those that match the consensus or are similar in type (hydrophobic or charged) to the consensus residue. The brackets indicate the 82-amino-acid region that is missing from the ΔSAP 155 cDNA (see Materials and Methods). (B) SAP 155 homologs are shown aligned with human SAP 155. Identities are indicated in white on black. ceSAP 155, spSAP 155, and scSAP 155 correspond to the C. elegans, S. pombe, and S. cerevisiae proteins, respectively.

Figure 1

(A) The amino acid sequence predicted from the human SAP 155 cDNA sequence. The RWDETP repeats at the amino terminus are aligned for convenience. These repeats and the TPGX repeats are indicated in boldface type, and the TPGH repeats are also underlined. The PP2A-like repeats are aligned according to the consensus for PP2A. This consensus is shown below the SAP 155 sequence. The underlined amino acids indicate those that match the consensus or are similar in type (hydrophobic or charged) to the consensus residue. The brackets indicate the 82-amino-acid region that is missing from the ΔSAP 155 cDNA (see Materials and Methods). (B) SAP 155 homologs are shown aligned with human SAP 155. Identities are indicated in white on black. ceSAP 155, spSAP 155, and scSAP 155 correspond to the C. elegans, S. pombe, and S. cerevisiae proteins, respectively.

Figure 2

SAP 155 is phosphorylated in the C complex assembled on ΔAG pre-mRNA. (A) Schematic of ΔAG AdML pre-mRNA; (B) Western blot of A/B (lane 1) and C (lane 2) complexes probed with SAP 155 antibodies; (C) Western blot of the C complex alone (lane 1), incubated in buffer (lane 2), or incubated in buffer and phosphatase (CIP) (lane 3). The asterisk indicates the novel band detected in the C complex. (D) The C complex was assembled under standard splicing conditions (lane 1) or in a reaction containing [γ-³²P]ATP (lane 2). An aliquot of the radiolabeled C complex was treated with CIP (lane 3). The autoradiograph (right) and Western blot (left) probed with SAP 155 antibodies are shown. The arrow indicates the 200-kD protein that is phosphorylated and also weakly detected by the antisera.

Figure 2

SAP 155 is phosphorylated in the C complex assembled on ΔAG pre-mRNA. (A) Schematic of ΔAG AdML pre-mRNA; (B) Western blot of A/B (lane 1) and C (lane 2) complexes probed with SAP 155 antibodies; (C) Western blot of the C complex alone (lane 1), incubated in buffer (lane 2), or incubated in buffer and phosphatase (CIP) (lane 3). The asterisk indicates the novel band detected in the C complex. (D) The C complex was assembled under standard splicing conditions (lane 1) or in a reaction containing [γ-³²P]ATP (lane 2). An aliquot of the radiolabeled C complex was treated with CIP (lane 3). The autoradiograph (right) and Western blot (left) probed with SAP 155 antibodies are shown. The arrow indicates the 200-kD protein that is phosphorylated and also weakly detected by the antisera.

Figure 3

SAP 155-P cross-links on both sides of the branch site. A schematic of ΔAG AdML pre-mRNA showing the sequence of the 3′ portion of the intron. (+5 and −6) The guanosine residues that were ³²P-labeled. The GG at the 3′ ss and BPS are shown in uppercase letters; the branch-site adenosine is underlined. Proteins were UV cross-linked in the H (lanes 1,4), A/B (lanes 2,5), and C (lanes 3,6) complexes assembled on pre-mRNAs labeled at the +5 (lanes 1–3) or −6 (lanes 4–6) sites. (ori) The gel origin. SAP 155 and SAP 62 were identified on two-dimensional gels (data not shown).

Figure 4

Phosphorylation of SAP 155 occurs concomitant with splicing catalysis. Wild-type AdML (A–C) or tropomyosin (D,E) pre-mRNA was incubated under splicing conditions for the times indicated. Total RNA (A,D) or protein from spliceosomal complexes (B,C,E) was analyzed. Western blots were probed with SAP 155 (B,E) or SAP 130 (C) antisera. The bands below SAP 155 may be breakdown products.

Figure 5

SAP 155-P is not detected in nuclear extracts incubated under splicing conditions. Shown is Western analysis of purified spliceosomal complexes assembled on AdML pre-mRNA (60- and 75-min time points) or total NE incubated as indicated. SAP 155 antibodies were used as a probe.