Functional spliceosomal A complexes can be assembled in vitro in the absence of a penta-snRNP

Abstract

Two different models currently exist for the assembly pathway of the spliceosome, namely, the traditional model, in which spliceosomal snRNPs associate in a stepwise, ordered manner with the pre-mRNA, and the holospliceosome model, in which all spliceosomal snRNPs preassemble into a penta-snRNP complex. Here we have tested whether the spliceosomal A complex, which contains solely U1 and U2 snRNPs bound to pre-mRNA, is a functional, bona fide assembly intermediate. Significantly, A complexes affinity-purified from nuclear extract depleted of U4/U6 snRNPs (and thus unable to form a penta-snRNP) supported pre-mRNA splicing in nuclear extract depleted of U2 snRNPs, whereas naked pre-mRNA did not. Mixing experiments with purified A complexes and naked pre-mRNA additionally confirmed that under these conditions, A complexes do not form de novo. Thus, our studies demonstrate that holospliceosome formation is not a prerequisite for generating catalytically active spliceosomes and that, at least in vitro, the U1 and U2 snRNPs can functionally associate with the pre-mRNA, prior to and independent of the tri-snRNP. The ability to isolate functional spliceosomal A complexes paves the way to study in detail subsequent spliceosome assembly steps using purified components.

FIGURE 1.

Characterization of spliceosomal A complexes purified via tobramycin affinity selection followed by immunoaffinity selection and gradient centrifugation. Complexes were separated on a 10%–30% glycerol gradient. RNA was recovered from each gradient fraction (as indicated above), analyzed by denaturing PAGE, and visualized by silver staining (upper panel) or by autoradiography (lower panel). The identity of the various RNAs is indicated on the right. Contaminating high molecular weight RNAs are indicated by an asterisk. “PRE” indicates the pre-mRNA. 30S and 50S correspond to migration positions of E. coli ribosomal subunits in parallel gradients.

FIGURE 2.

Double affinity-purified A complexes are functional. (A) RNA composition of U2 snRNP-depleted (lane 2) and mock-depleted (lane 1) extracts. RNA was separated by denaturing PAGE and detected by staining with ethidium bromide. The identity of the various RNAs is indicated on the left; 5.8S rRNA is indicated by an asterisk. (B) In vitro splicing of mock- vs. U2-depleted extract. An equimolar mixture of ³²P-labeled tobramycin aptamer-tagged MINX pre-mRNA (MINX-To) and MINX pre-mRNA (lanes 1,2,7,8), MINX pre-mRNA alone (lanes 3,4,9,10), or MINX-To pre-mRNA alone (lanes 5,6,11,12) were incubated under splicing conditions for 3 h at 30°C in the presence of U2-depleted nuclear extract (lanes 1,3,5), U2-depleted extract complemented with purified 12S U2 snRNP (lanes 2,4,6), mock-depleted extract (lanes 7,9,11), or mock-depleted extract plus purified 12S U2 snRNP (lanes 8,10,12). RNA was recovered and analyzed by denaturing PAGE and visualized by autoradiography. The positions of the pre-mRNAs, the splicing intermediates, and the products are indicated on the right. (C) Splicing activity of purified A complexes. Purified spliceosomal A complexes from gradient fraction 10 (lanes 1,2) or an equimolar mixture of the purified A complex and naked, untagged MINX pre-mRNA (lane 3) were incubated in the presence of U2-depleted extract for 3 h at 0°C (lane 1) or at 30°C (lanes 2,3). RNA was analyzed as in B. Similar results were obtained with A complexes from gradient fractions 9, 11, and 12.

FIGURE 3.

U4/U6-depleted nuclear extract supports the formation of only spliceosomal A complexes. (A) RNA composition of U4/U6-depleted (lane 2) and mock-depleted (lane 1) extracts. RNA was analyzed as in Figure 2A. (B) Spliceosome assembly in U4/U6-depleted extract (lanes 1–7) and mock-depleted extract (lanes 8–14). Spliceosomal complexes were analyzed on a native gel at the indicated times. The positions of the H, A, and B complexes are indicated on the right. (C) Spliceosome assembly in mock- vs. U4/U6-depleted extract. Spliceosomal complexes were allowed to form under splicing conditions at 30°C for 20 min in U4/U6-depleted nuclear extract (triangles) and mock-depleted extract (circles). Subsequently, the complexes were separated on a 10%–30% glycerol gradient. The distribution of the radioactivity across each gradient is shown. Peaks were assigned as A or B complex based on their migration behavior according to Figure 1; the position of H complexes was verified independently (data not shown).

FIGURE 4.

Spliceosomal A complexes purified from U4/U6-depleted nuclear extract are functional. (A) RNA composition of spliceosomal complexes purified from U4/U6-depleted nuclear extract. RNA was analyzed as in Figure 2A and visualized by silver staining (upper panel) or by autoradiography (lower panel). (B) Splicing activity of A complexes purified from U4/U6-depleted extract. Purified spliceosomal A complexes (lanes 1,2) or an equimolar mixture of the purified A complex and naked, untagged MINX pre-mRNA (lanes 3,4) were incubated in the presence of U2-depleted extract for 3 h at 0°C (lane 1) or at 30°C (lanes 2,3), or for 3 h at 30°C in U2-depleted extract complemented with 12S U2 snRNPs (lane 4). RNA was analyzed as in Figure 2B.