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. 2008 Jan;28(2):849-62.
doi: 10.1128/MCB.01410-07. Epub 2007 Oct 29.

Functional coupling of last-intron splicing and 3'-end processing to transcription in vitro: the poly(A) signal couples to splicing before committing to cleavage

Frank Rigo  1 , Harold G Martinson
Affiliations

Functional coupling of last-intron splicing and 3'-end processing to transcription in vitro: the poly(A) signal couples to splicing before committing to cleavage

Frank Rigo et al. Mol Cell Biol. 2008 Jan.

Abstract

We have developed an in vitro transcription system, using HeLa nuclear extract, that supports not only efficient splicing of a multiexon transcript but also efficient cleavage and polyadenylation. In this system, both last-intron splicing and cleavage/polyadenylation are functionally coupled to transcription via the tether of nascent RNA that extends from the terminal exon to the transcribing polymerase downstream. Communication between the 3' splice site and the poly(A) site across the terminal exon is established within minutes of their transcription, and multiple steps leading up to 3'-end processing of this exon can be distinguished. First, the 3' splice site establishes connections to enhance 3'-end processing, while the nascent 3'-end processing apparatus makes reciprocal functional connections to enhance splicing. Then, commitment to poly(A) site cleavage itself occurs and the connections of the 3'-end processing apparatus to the transcribing polymerase are strengthened. Finally, the chemical steps in the processing of the terminal exon take place, beginning with poly(A) site cleavage, continuing with polyadenylation of the 3' end, and then finishing with splicing of the last intron.

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Figures

FIG. 1.
FIG. 1.
Coupled transcription, splicing, and 3′-end processing. (A) Oligo(dT) selection to visualize polyadenylated species. For each time point, 25% of the RNA from a fourfold transcription reaction was set aside as the input and the remainder was subjected to a single round of oligo(dT) selection using a poly(A) Purist MAG kit (Ambion). (B) Use of 3′-dATP to visualize clearly the spliced species. The same construct, pβΔ-L, was used for both panel A and panel B.
FIG. 2.
FIG. 2.
Connections between splicing and 3′-end processing. (A) The poly(A) signal enhances second-intron splicing. The percent splicing was calculated as the sum of bands 3 and 4 (graph 1), bands 2 and 4 (graph 2), or band 2 alone (graph 3) expressed as a percentage of the sum of all the bands (i.e., 1 through 4). The averages and ranges for two independent experiments are shown. The isolated RNA was postcut at the poly(A) site by using RNase H and oligonucleotide 5. The cutting efficiency for lanes 1 to 5 was 72% ± 2% (average ± standard deviation), but that for lane 6 was only 50%, which exaggerates the impression of RNA loss for this lane. RNA recovery at 60 min compared to 10 min for all data is summarized in panel A and Fig. S2A in the supplemental material was 64% ± 9% (average ± standard deviation). (B) The 3′ splice site of the second intron enhances 3′-end processing. No RNase H was used in this experiment. The percent poly(A) site cleavage was calculated as the ratio of RNA that is poly(A) site cleaved (all four species in the gel) to the total of this cleaved RNA plus all uncleaved RNA that is longer than the unspliced precursor. (C) First-intron splicing requires second-exon definition. No RNase H was used in this experiment.
FIG. 3.
FIG. 3.
Poly(A) site cleavage is inhibited by cutting the tether, but cleavage at the poly(A) site is not required for the enhanced second-intron splicing conferred by the poly(A) signal. (A) Cartoon of the core poly(A) site cleavage complex showing the RNA tether, together with diagrams of the construct and the transcription protocol used. (Cartoon adapted from references and with permission.) (B and C) These experiments were all done using extract A, but for the experiment corresponding to panel C, the protocol for extract B was used. The precutting oligonucleotide (77 oligo) directs RNase H (72) to cut the tether during transcription within the vector sequence predominantly at a position about 77 nucleotides downstream of the SV40 or β-globin poly(A) cleavage sites. The control oligonucleotide (lanes 1 to 5) is the complement to 77 oligo. Splicing of the RNase H-cut RNA in lanes 6 to 15 is quantitated in Fig. S5 in the supplemental material.
FIG. 4.
FIG. 4.
A tether is required for efficient second-intron splicing when the β-globin poly(A) signal defines the terminal exon. (A) This experiment was done as described in the legend to Fig. 3C except that transcripts were postcut at the poly(A) site with RNase H by using oligonucleotide 7 before loading onto the gel. (B and C) Quantitations are as described in the legend to Fig. 2A. Averages and ranges are for two independent experiments, including the experiment shown in panel A. wt, wild type; mt, mutant; 77, 77 oligo.
FIG. 5.
FIG. 5.
RNase H cutting has much less effect on second-intron splicing when the SV40 late poly(A) signal defines the terminal exon. (A) This experiment was done as described in the legend to Fig. 3B except that transcripts were postcut at the poly(A) site with RNase H by using oligonucleotide 5 before loading onto the gel. (B and C) Quantitations are as described in the legend to Fig. 2A. wt, wild type; mt, mutant; 77, 77 oligo.
FIG. 6.
FIG. 6.
Coupling of transcription to terminal exon definition. (A) Experimentally determined functional connections among transcription, splicing and poly(A) site cleavage in this in vitro system. (B) Cartoon of terminal exon definition in the context of the transcription complex. Only the core factors required for poly(A) site cleavage and terminal exon definition are shown. The factors at the core of the presumptive exon definition complex are labeled in red. The diagram accommodates the following known protein-protein and protein-RNA interactions: U2AF with CFIm (48); U2 snRNP with CPSF (36); CFIIm and CstF with the CTD, with CPSF, and with each other (18, 22, 52, 59, 75); CFIm with CFIIm and CPSF (18, 63, 71); and U2AF, U2 snRNP, CFIm, CPSF and CstF with the RNA (7, 9, 71, 77). The placement of U2AF on the CTD of the polymerase is arbitrary but is based on the fact that U2AF has been shown to bind tightly to the polymerase (61, 69) except when the polymerase is isolated by use of a CTD-binding antibody which might displace the U2AF (16). SR proteins are not shown.
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