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Protein composition and electron microscopy structure of affinity-purified human spliceosomal B complexes isolated under physiological conditions - PubMed

Protein composition and electron microscopy structure of affinity-purified human spliceosomal B complexes isolated under physiological conditions

Jochen Deckert et al. Mol Cell Biol. 2006 Jul.

Abstract

The spliceosomal B complex is the substrate that undergoes catalytic activation leading to catalysis of pre-mRNA splicing. Previous characterization of this complex was performed in the presence of heparin, which dissociates less stably associated components. To obtain a more comprehensive inventory of the B complex proteome, we isolated this complex under low-stringency conditions using two independent methods. MS2 affinity-selected B complexes supported splicing when incubated in nuclear extract depleted of snRNPs. Mass spectrometry identified over 110 proteins in both independently purified B complex preparations, including approximately 50 non-snRNP proteins not previously found in the spliceosomal A complex. Unexpectedly, the heteromeric hPrp19/CDC5 complex and 10 additional hPrp19/CDC5-related proteins were detected, indicating that they are recruited prior to spliceosome activation. Electron microscopy studies revealed that MS2 affinity-selected B complexes exhibit a rhombic shape with a maximum dimension of 420 A and are structurally more homogeneous than B complexes treated with heparin. These data provide novel insights into the composition and structure of the spliceosome just prior to its catalytic activation and suggest a potential role in activation for proteins recruited at this stage. Furthermore, the spliceosomal complexes isolated here are well suited for complementation studies with purified proteins to dissect factor requirements for spliceosome activation and splicing catalysis.

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Figures

FIG. 1.
FIG. 1.

Schematic diagram of the MS2 affinity purification strategy used to isolate human spliceosomal B complexes. MINX pre-mRNA tagged at its 3′ end with three RNA binding sites of the MS2 coat protein was preincubated with a fusion protein of the MS2 coat protein and MBP (step I). Subsequently, nuclear extract was added and spliceosomes were allowed to form for 8 min (step II). Complexes were then fractionated by size on a linear 10 to 30% glycerol gradient (step III). Spliceosomal B complexes from the 40S peak fractions were affinity selected using amylose beads and subsequently eluted with maltose (step IV).

FIG. 2.
FIG. 2.

Characterization of MS2 affinity-selected B complexes. (A) In vitro splicing kinetics of 32P-labeled MS2-tagged MINX pre-mRNA. Splicing was performed in HeLa cell nuclear extract for 0 to 20 min as indicated above each lane. RNA was recovered and separated on an 8.3 M urea-10% polyacrylamide gel. The 32P-labeled pre-mRNA and splicing intermediates or products were detected by autoradiography, and their positions are indicated on the right; note that the straight line represents debranched, excised intron. The lengths (in base pairs) of a size marker (3′ end-labeled MspI cleaved pBR322) (NEB) are indicated on the left. (B) RNA composition of MS2 affinity-purified B complexes. Spliceosomal complexes were isolated as described in Fig. 1, and RNA was recovered from 20% of the MS2 eluate (lanes 2 and 4). For comparison, RNA was also isolated from 0.2% of the input splicing reaction (lanes 1 and 3). RNA was separated on an 8.3 M urea-10% polyacrylamide gel and visualized by staining with silver (lanes 1 and 2) or by autoradiography (lanes 3 and 4). The positions of the pre-mRNA, splicing intermediates or products, and/or snRNAs are indicated on the right and left. The band indicated by an asterisk is a pre-mRNA degradation product.

FIG. 3.
FIG. 3.

MS2 affinity-selected spliceosomal B complexes catalyze splicing in nuclear extracts depleted of snRNPs. MS2 affinity-selected spliceosomal B complex was incubated for the indicated times (0 to 90 min) in HeLa nuclear extract from which U2 snRNPs (A) or U4/U6 snRNPs (B) were depleted using biotinylated 2′-OMe oligonucleotides complementary to the U2 or U6 snRNA. (C) Naked 32P-labeled MS2-tagged MINX pre-mRNA (MS2-pre-mRNA) (lanes 1 to 6) or MS2 affinity-selected spliceosomal B complex (B complex) (lanes 7 to 12) was incubated from 0 to 90 min under splicing conditions in the presence of MN-treated HeLa nuclear extract. (D) Naked 32P-labeled MS2-tagged MINX pre-mRNA (MS2-pre-mRNA) (lanes 1 to 6) or MS2 affinity-selected spliceosomal B complex (B complex) (lanes 7 to 12) was incubated for 0 to 90 min (as indicated above each lane) under splicing conditions together with an equimolar amount of untagged, naked 32P-labeled MINX pre-mRNA in the presence of MN-nuclear extract. In each case, RNA was recovered and analyzed as described in the legend of Fig. 2A. The positions of the intron 3′ exon intermediate, pre-mRNA, spliced out intron, spliced mRNA, and 5′ exon intermediate are indicated on the right. NX, nuclear extract.

FIG. 4.
FIG. 4.

Purification strategy and RNA composition of B complexes purified by tobramycin affinity selection coupled with anti-61K immunoaffinity purification. (A) Schematic diagram of the two-step tobramycin-immunoaffinity purification strategy used to isolate human spliceosomal B complexes. Pre-mRNA tagged at its 3′ end with the tobramycin RNA aptamer was bound to a tobramycin matrix (step I). Spliceosomal complexes were allowed to form for 60 min by incubating under splicing conditions in the presence of nuclear extract (step II). After elution with tobramycin (step III), precatalytic spliceosomal B complexes were isolated by immunoaffinity selection using anti-peptide antibodies directed against the U4/U6-61K (hPrp31) protein (step IV). Spliceosomal B complexes were subsequently eluted with cognate peptide (step V). (B) RNA composition of tobramycin-immunoaffinity purified B complexes. RNA was recovered from 12% of the anti-61K/hPrp31 eluate (lanes 2 and 4) and also isolated from 0.2% of the input material used for binding to the tobramycin matrix (lanes 1 and 3). RNA was separated on an 8.3 M urea-10% polyacrylamide gel and visualized by staining with silver (lanes 1 and 2) or by autoradiography (autorad; lanes 3 and 4). The positions of the pre-mRNA, splicing intermediates or products, and/or snRNAs are indicated on the right and left. The band indicated by an asterisk is a pre-mRNA degradation product. Tob, tobramycin; NHS, N-hydroxysuccinimide.

FIG. 5.
FIG. 5.

EM of the native spliceosomal complex B. (A) CCD image of negatively stained complex BΔU1. Particles were purified as previously described (7), adsorbed onto a carbon film, and sandwiched beneath another carbon film in 2% (wt/vol) uranyl formate. The images were taken at a magnification of ×27,500 under low-dose conditions. Bar, 1,500 Å. (B) CCD image of negatively stained complex B. Particles were adsorbed onto a carbon film and sandwiched beneath another carbon film in 2% (wt/vol) uranyl formate. The images were taken at a magnification of ×122,000 under low-dose conditions. Bar, 1,500 Å. (C) A gallery of individual class averages of the native B complex obtained by averaging ∼20 individual raw images. Bar, 200 Å.

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References

    1. Ajuh, P., B. Kuster, K. Panov, J. C. Zomerdijk, M. Mann, and A. I. Lamond. 2000. Functional analysis of the human CDC5L complex and identification of its components by mass spectrometry. EMBO J. 19:6569-6581. - PMC - PubMed
    1. Azubel, M., S. G. Wolf, J. Sperling, and R. Sperling. 2004. Three-dimensional structure of the native spliceosome by cryo-electron microscopy. Mol. Cell 15:833-839. - PubMed
    1. Ben Yehuda, S., I. Dix, C. S. Russell, S. Levy, J. D. Beggs, and M. Kupiec. 1998. Identification and functional analysis of hPRP17, the human homologue of the PRP17/CDC40 yeast gene involved in splicing and cell cycle control. RNA 4:1304-1312. - PMC - PubMed
    1. Bennett, M., S. Michaud, J. Kingston, and R. Reed. 1992. Protein components specifically associated with prespliceosome and spliceosome complexes. Genes Dev. 6:1986-2000. - PubMed
    1. Blanton, S., A. Srinivasan, and B. C. Rymond. 1992. PRP38 encodes a yeast protein required for pre-mRNA splicing and maintenance of stable U6 small nuclear RNA levels. Mol. Cell. Biol. 12:3939-3947. - PMC - PubMed

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