Polyadenylation releases mRNA from RNA polymerase II in a process that is licensed by splicing

Abstract

When transcription is coupled to pre-mRNA processing in HeLa nuclear extracts nascent transcripts become attached to RNA polymerase II during assembly of the cleavage/polyadenylation apparatus (CPA), and are not released even after cleavage at the poly(A) site. Here we show that these cleaved transcripts are anchored to the polymerase at their 3' ends by the CPA or, when introns are present, by the larger 3'-terminal exon definition complex (EDC), which consists of splicing factors complexed with the CPA. Poly(A) addition releases the RNA from the polymerase when the RNA is anchored only by the CPA. When anchored by the EDC, poly(A) addition remains a requirement, but it triggers release only after being licensed by splicing. The process by which RNA must first be attached to the polymerase by the EDC, and then can only be released following dual inputs from splicing and polyadenylation, provides an obvious opportunity for surveillance as the RNA enters the transport pathway.

FIGURE 1.

Cartoons of the presumptive 3′-terminal EDC and of the CPA. (A) Assembly of the 3′-terminal EDC (Berget 1995; Sharma et al. 2008). Only the core factors required for poly(A) site cleavage and 3′-terminal exon definition are labeled. The CPA is shown in green, and the splicing factors that interact with the CPA, and presumably combine with it to form the 3′-terminal EDC, are shown in yellow. RNA polymerase II is shown in blue. The diagram accommodates the following known protein–protein and protein–RNA interactions: CFI_m with U2AF (Millevoi et al. 2006); CPSF with U2 snRNP (Kyburz et al. 2006); CFII_m and CstF with the CTD, with CPSF and with each other (Murthy and Manley 1995; de Vries et al. 2000; Fong and Bentley, 2001; Qu et al. 2006; Zhang and Gilmour, 2006); CFI_m with CFII_m and CPSF (Ruegsegger et al. 1996; de Vries et al. 2000; Venkataraman et al. 2005); and U2AF, U2 snRNP, CFI_m, CPSF, and CstF with the RNA (Zhao et al. 1999; Black, 2003; Brown and Gilmartin, 2003; Venkataraman et al. 2005). The placement of U2AF on the CTD of RNA polymerase II is arbitrary but is based on the fact that U2AF has been shown to bind tightly to the polymerase (Robert et al. 2002; Ujvari and Luse, 2004) except when the polymerase is isolated by use of a CTD-binding antibody which might displace the U2AF from the CTD (Das et al. 2007). SR proteins are not shown. (B) The EDC after poly(A) site cleavage. Poly(A) polymerase (PAP) is shown in this panel, interacting with CPSF (Kaufmann et al. 2004). PAP also interacts with U2AF (Vagner et al. 2000) and CFI_m (Kim and Lee, 2001), but these interactions are not shown because of difficulties of depiction in two dimensions. The DNA oligo used for RNaseH-cutting in Figure 2B and later experiments is shown. (C) The CPA after poly(A) site cleavage.

FIGURE 2.

EDC-mediated attachment of poly(A)site-cleaved RNA to RNA polymerase II. (A) The plasmid construct and the outline of the experimental protocol (Rigo et al. 2005). (B) Pull-down of poly(A)site-cleaved RNA (Method 1). The DNA oligo for RNaseH cutting was CCATCTTCTGCCAGG (5′ → 3′) at 1 ng/μL. The concentration of this oligo was chosen so that only a small proportion of the transcripts would be cut by RNaseH at this upstream location. For lanes 1 and 2 the polymerase antibody used was N-20, lot L1407. Lanes 7 and 8 are from a different experiment using antibody from lot I0706. For lanes 1, 2, 5, and 6 the average and standard deviation are given for the experiment shown plus two additional independent experiments. For lanes 3 and 4 the average and range are given, for this and one additional independent experiment. For lanes 7 and 8 the average and range are for duplicates within the same experiment. The lane 7 and 8 data are not included in the average for lanes 1 and 2. (C) The amount of RNAPII and CstF pulled down concomitant with poly(A)site-cleaved RNA. The experiment was carried out as for part B except that each reaction was scaled up by twofold and then split in half for the RNA or the Western analysis. For simplicity, only the poly(A)site-cleaved RNA and the relevant protein bands are shown.

FIGURE 3.

Both the EDC and the CPA attach poly(A)site-cleaved RNA to the polymerase, and polyadenylation does not release the EDC-attached RNA. (A) Pull-down (Method 2) of EDC-attached poly(A)site-cleaved RNA that either is or is not polyadenylated. Band 1 in lanes 3 and 4 was quantitated after subtracting the background appearing at the same position in lanes 1 and 2, respectively. The averages and ranges for two independent experiments are shown. (B) Pull-down of poly(A)site-cleaved RNA (Method 3) that either has or does not have a 3′ splice site. For lanes 3 and 4 a plasmid construct with a mutant 3′ splice site (AG → ct) was used. The averages and ranges for two independent experiments are shown.

FIGURE 4.

Polyadenylation releases RNA whose only attachment to the polymerase is through the CPA. Pull-down Method 1 was used. A construct with a mutant 3′ splice site (AG → ct) was used. The RNA in lanes 3 and 4 was oligo(dT) selected prior to loading on the gel.

FIGURE 5.

Splicing of EDC-attached, nonpolyadenylated RNA has little effect on release from the polymerase. Pull-down Method 2 was used. The average and range for two independent experiments is shown.

FIGURE 6.

Splicing releases EDC-attached, polyadenylated RNA from the polymerase. Pull-down Method 2 was used. The average and range for two independent experiments is shown.

FIGURE 7.

Splicing remodels the EDC. Pull-down Method 2 was used. The spliced/unspliced pull-down efficiencies were calculated as an average of the two ways in which the splicing of each intron can occur, as illustrated in the table. For lanes 1 and 2 the average and range for two independent experiments is given. Lanes 5 and 6 are from a different experiment.

FIGURE 8.

Polyadenylation releases EDC-attached mRNA from the polymerase in a process that must be licensed by splicing. The upper pathway in the model shows release from CPA attachment being triggered by polyadenylation. The lower pathway depicts splicing factors constraining the CPA and preventing this release until the splicing factors themselves are ejected by splicing.