Protein composition of human prespliceosomes isolated by a tobramycin affinity-selection method

Abstract

Detailed knowledge of the composition and structure of the spliceosome and its assembly intermediates is a prerequisite for understanding the complex process of pre-mRNA splicing. To this end, we have developed a tobramycin affinity-selection method that is generally applicable for the purification of native RNP complexes. By using this method, we have isolated human prespliceosomes that are ideally suited for both biochemical and structural studies. MS identified >70 prespliceosome-associated proteins, including nearly all known U1 and U2 snRNP proteins, and expected non-snRNP splicing factors. In addition, the DEAD-box protein p68, RNA helicase A, and a number of proteins that appear to perform multiple functions in the cell, such as YB-1 and TLS, were detected. Several previously uncharacterized proteins of unknown function were also identified, suggesting that they play a role in splicing and potentially act during prespliceosome assembly. These data provide insight into the complexity of the splicing machinery at an early stage of its assembly.

Fig 1.

(A) Purification strategy. (B) Matrix-bound, aptamer-tagged pre-mRNA is efficiently spliced. Solid-phase splicing was performed for the indicated times at 30°C. RNA was recovered after elution (lanes 2–4) or from the reaction supernatant (lanes 5–7; note that three times more supernatant than eluate was assayed), analyzed by denaturing PAGE, and visualized by autoradiography. Lane 1, input pre-mRNA. The positions of the pre-mRNA and splicing intermediates/products are indicated on the right.

Fig 2.

(A) RNA and protein content of splicing complexes eluted with tobramycin. Solid-phase splicing was performed for 45 min at 30°C in the presence (lanes 1 and 3) or absence (lanes 2 and 4) of pre-mRNA, and the complexes were subsequently eluted with tobramycin. RNA and protein were recovered, analyzed by PAGE, and visualized by silver staining. (B) Glycerol gradient analysis of affinity-purified spliceosomal complexes. Naked pre-mRNA (•) or complexes formed after splicing for 15 min at 4°C (○) or for 45 min at 30°C (▴) were subjected to 10–30% glycerol gradient centrifugation, and the percent of ³²P-labeled pre-mRNA in each fraction was determined by Cherenkov counting. (C) RNA profile of the glycerol gradient fractionated, 45-min splicing complexes. RNA was recovered from odd-numbered fractions and analyzed as in A. PRE, pre-mRNA; *, contaminating RNAs.

Fig 3.

Characterization of RNA–RNA interactions in gradient-fractionated spliceosomal complexes by psoralen crosslinking. Affinity-selected complexes were fractionated on a 10–30% glycerol gradient, and crosslinking was performed with even-numbered gradient fractions (as indicated above each lane) or ≈2% of unfractionated eluate (E lanes). In the left-most E lane, the eluate was UV-irradiated in the absence of psoralen to control for nonspecific crosslinking. RNA was transferred to a nylon membrane that was hybridized sequentially with ³²P-labeled probes specific for pre-mRNA (A) U2 snRNA (B) and U1 snRNA (C). The identity of the crosslinked species is indicated on the right. AMT, psoralen; PRE, pre-mRNA; intPRE, internally crosslinked pre-mRNA.

Fig 4.

Protein content of the glycerol gradient fractionated, 45-min splicing complexes (A), or of pooled gradient fractions 11–13 (B). Protein was recovered from odd-numbered or pooled fractions and analyzed as in Fig. 2.