Characterization of a protein complex containing spliceosomal proteins SAPs 49, 130, 145, and 155 - PubMed (original) (raw)

Comparative Study

Characterization of a protein complex containing spliceosomal proteins SAPs 49, 130, 145, and 155

B K Das et al. Mol Cell Biol. 1999 Oct.

Abstract

SF3b is a U2 snRNP-associated protein complex essential for spliceosome assembly. Although evidence that SF3b contains the spliceosomal proteins SAPs 49, 130, 145, and 155 has accumulated, a protein-mediated association between all of these proteins has yet to be directly demonstrated. Here we report the isolation of a cDNA encoding SAP 130, which completes the cloning of the putative SF3b complex proteins. Using antibodies to SAP 130 and other putative SF3b components, we showed that SAPs 130, 145, and 155 are present in a protein complex in nuclear extracts and that these proteins associate with one another in purified U2 snRNP. Moreover, SAPs 155 and 130 interact with each other (directly or indirectly) within this complex, and SAPs 49 and 145 are known to interact directly with each other. Thus, together with prior work, our studies indicate that SAPs 49, 130, 145, and 155 are indeed components of SF3b. The Saccharomyces cerevisiae homologs of SAPs 49 and 145 are encoded by essential genes. We show here that the S. cerevisiae homologs of SAPs 130 and 155 (scSAP 130/RSE1 and scSAP 155, respectively) are also essential. Recently, the SF3b proteins were found in purified U12 snRNP, which functionally substitutes for U2 snRNP in the minor spliceosome. This high level of conservation, together with the prior observation that the SF3b proteins interact with pre-mRNA very close to the branch site, suggest that the SF3b complex plays a critical role near or at the spliceosome catalytic core.

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Figures

FIG. 1

FIG. 1

Predicted amino acid sequence of SAP 130 cDNA. The amino acid sequence predicted from the human SAP 130 cDNA sequence is shown. The two peptide sequences obtained from microsequencing are underlined once and twice, and the peptide used for making antibodies is boxed.

FIG. 2

FIG. 2

SAP 130 antibodies detect a single ∼130-kDa band in nuclear extract and spliceosomal complexes. (A) The in vitro translation product of the SAP 130 cDNA comigrates with a protein in the nuclear extract that is detected by SAP 130 antibodies. (B) 32P-labeled adenovirus major late pre-mRNA was incubated under splicing conditions for the times indicated and then total RNA was fractionated on a 15% denaturing gel. Splicing intermediate and products are indicated. (C) SAP 130 antibodies were used to probe a Western blot of purified spliceosomal complexes assembled for the indicated times under splicing conditions. IVT, in vitro translation.

FIG. 3

FIG. 3

SAPs 130, 145, and 155 are part of a protein complex in nuclear extracts. (A) The indicated antibodies were used for immunoprecipitations from total nuclear extracts (NE). Total RNA was fractionated on an 8% denaturing gel. The snRNAs and tRNA are indicated. (B) An aliquot of each immunoprecipitate was fractionated on an SDS–6% polyacrylamide gel, and then Western blots were probed with the indicated antibodies (α). (C) Same as panel B except that nuclear extract was incubated with 2 μl of RNase A (10 mg/ml) prior to the immunoprecipitations.

FIG. 4

FIG. 4

SAPs 130 and 155 interact with each other in the protein complex. (A) Antibodies to the U2 snRNP protein B" were used to immunoprecipitate U2 snRNP from nuclear extract. Total proteins were eluted with RNase A and fractionated on an SDS–9% polyacrylamide gel. The abundant SAPs are indicated and were identified on two-dimensional gels and on Western blots (panel B and data not shown). The asterisk designates a band of unknown identity. However, it is unlikely to be a component of SF3b since it does not coimmunoprecipitate with SF3b antibodies (data not shown). (B) The eluate from panel A was used for immunoprecipitations with the antibodies (α) indicated on the top of each lane, and Western blots were probed with the antibodies indicated to the left of each blot. (C) Same as panel A except that proteins were eluted with 1 M urea. (D) The urea eluate was used for immunoprecipitations with the SAP 155 and SAP 145 antibodies. The bound (IP) and unbound (FT) proteins were fractionated on an SDS–6% polyacrylamide gel, and Western blots were probed with the indicated antibodies.

FIG. 5

FIG. 5

Alignment of SAP 130 with scSAP 130 and human DDB. The alignment was done by the Clustal method (DNASTAR Inc.). Residues that are identical in hsSAP 130 and the other two proteins are shown in white on black. The overall identities of scSAP 130 and hsDDB to hsSAP 130 are 27 and 20%, respectively.

FIG. 6

FIG. 6

The scSAP 130 and scSAP 155 genes are essential in S. cerevisiae. (Panel I) (A) Structure of scSAP 130 and scSAP 130Δ::LEU alleles. The sizes of _Alw_NI and _Stu_I restriction fragments are indicated. The probe is shown below the region with which it hybridizes. (B) Southern analysis of total genomic DNA from a normal diploid strain (WT) and diploid strains transformed with the scSAP 130Δ::LEU allele (lanes 1 and 2). The sizes (in kilobases) of markers and restriction fragments are shown. (C) Tetrad analysis. The scSAP 130Δ::LEU strain was sporulated, and 13 individual tetrads were dissected. (D) Viable spores are Leu−. Fifteen viable spores were patched onto a plate lacking leucine. The diploid strain was patched as a control. (Panel II) (A) Structure of SAP 155 and scSAP 155Δ::LEU alleles. The sizes of the _Stu_I and _Xho_I restriction fragments used to identify each allele are indicated. (B) Southern analysis of total genomic DNA from a normal diploid yeast strain (wt) and diploid strains transformed with the scSAP 155Δ::LEU allele (lanes 1 through 5). The sizes (in kilobases) of the markers and restriction fragments are shown. The differences in band intensities between lanes is most likely due to different levels of DNA loaded on the gel. (C) Tetrad analysis. The scSAP 155Δ::LEU strain was sporulated, and 15 individual tetrads were dissected. (D) Viable spores are Leu−. Eighteen viable spores were patched onto a plate lacking leucine. The diploid strain was patched as a control.

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References

    1. Behrens S E, Tyc K, Kastner B, Reichelt J, Luhrmann R. Small nuclear ribonucleoprotein (RNP) U2 contains numerous additional proteins and has a bipartite RNP structure under splicing conditions. Mol Cell Biol. 1993;13:307–319. - PMC - PubMed
    1. Bennett M, Michaud S, Kingston J, Reed R. Protein components specifically associated with prespliceosome and spliceosome complexes. Genes Dev. 1992;6:1986–2000. - PubMed
    1. Bennett M, Reed R. Correspondence between a mammalian spliceosome component and an essential yeast splicing factor. Science. 1993;262:105–108. - PubMed
    1. Bochnig P, Reuter R, Bringmann P, Luhrmann R. A monoclonal antibody against 2,2,7-trimethylguanosine that reacts with intact, class U, small nuclear ribonucleoproteins as well as with 7-methylguanosine-capped RNAs. Eur J Biochem. 1987;168:461–467. - PubMed
    1. Brosi R, Groning K, Behrens S E, Luhrmann R, Kramer A. Interaction of mammalian splicing factor SF3a with U2 snRNP and relation of its 60-kD subunit to yeast PRP9. Science. 1993;262:102–105. - PubMed

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