Utilization of splicing elements and polyadenylation signal elements in the coupling of polyadenylation and last-intron removal

Abstract

Polyadenylation (PA) is the process by which the 3' ends of most mammalian mRNAs are formed. In nature, PA is highly coordinated, or coupled, with splicing. In mammalian systems, the most compelling mechanistic model for coupling arises from data supporting exon definition (2, 34, 37). We have examined the roles of individual functional components of splicing and PA signals in the coupling process by using an in vitro splicing and PA reaction with a synthetic pre-mRNA substrate containing an adenovirus splicing cassette and the simian virus 40 late PA signal. The effects of individually mutating splicing elements and PA elements in this substrate were determined. We found that mutation of the polypyrimidine tract and the 3' splice site significantly reduced PA efficiency and that mutation of the AAUAAA and the downstream elements of the PA signal decreased splicing efficiency, suggesting that these elements are the most significant for the coupling of splicing and PA. Although mutation of the upstream elements (USEs) of the PA signal dramatically decreased PA, splicing was only modestly affected, suggesting that USEs modestly affect coupling. Mutation of the 5' splice site in the presence of a viable polypyrimidine tract and the 3' splice site had no effect on PA, suggesting no effect of this element on coupling. However, our data also suggest that a site for U1 snRNP binding (e.g., a 5' splice site) within the last exon can negatively effect both PA and splicing; hence, a 5' splice site-like sequence in this position appears to be a modulator of coupling. In addition, we show that the RNA-protein complex formed to define an exon may inhibit processing if the definition of an adjacent exon fails. This finding indicates a mechanism for monitoring the appropriate definition of exons and for allowing only pre-mRNAs with successfully defined exons to be processed.

FIG. 1

Linear diagram of the SVLPA signal. Above the diagram are the SV40 nucleotide numbers corresponding to the SVLPA signal. Elements shown are as follows: AAUAAA, the consensus hexonucleotide; An, the site for cleavage and PA; USEs, indicated by three black boxes representing the homologous AUUUGURA core elements; DSEs, indicated by three white boxes representing the GU-rich, G-rich, and U-rich elements; and U1BS, the U1 snRNP binding site. All elements are described in the text.

FIG. 2

MXSVL splicing and PA substrate. At the top of the figure is a detailed map of the MXSVL substrate described in the text. Specific elements of the splice cassette are the 5′ splice site (5′), the polypyrimidine tract (PPT), and the 3′ splice site (3′). Specific elements of the SVLPA signal are the same as those shown in Fig. 1. Below the top diagram are simpler diagrams showing the WT Sp/PA substrate and a series of similar substrates with mutations in the various splicing and PA elements. Each is described in the text. In addition, LSMs were made across the DSEs of the PA signal; the positions of mutations DM1 to DM5 (1 to 5) are shown. Also shown is the WT PA only substrate, which we have used in our previous studies of the SVLPA signal. Adeno, adenovirus; nts., nucleotides.

FIG. 3

Products of the splicing and PA reaction carried out with the MXSVL (WT Sp/PA) substrate. Lanes: Sub., migration position of the intact MXSVL (WT Sp/PA) substrate (Sub.); Prods., products (Prods.) of the splicing and PA reaction carried out under coupling conditions (see Materials and Methods). The major products are S−A+, S+A−, and S+A+.

FIG. 4

Quantitation of the splicing and PA products from the MXSVL (WT Sp/PA) substrate in the presence of increasing Mg²⁺ concentrations. The amounts of each splicing and PA product were quantitated from data similar to those shown in Fig. 3. These values were converted to the percentage of input substrate and shown as a bar graph. (A) Level of total PA (Total) as well as levels of the PA products S+A+ and S−A+. (B) Level of total splicing (Total) as well as levels of the splicing products S+A+ and S+A−. The error of the data shown was ±2 to 4%. WT, wild type.

FIG. 5

Analysis of total PA (A) and total splicing (B) of wild-type and mutant MXSVL substrates in the presence of increasing Mg²⁺ concentrations. The substrates analyzed are those shown in Fig. 2. Details are given in the text. The error of the data shown was ±2 to 4%.

FIG. 6

Sequence of the downstream region of the SVLPA signal and positions of LSMs. The AAUAAA and the three DSEs (GU rich, G rich, and U rich) are shown in larger letters. The positions of LSMs DM1 to DM5 are indicated.

FIG. 7

Total splicing and total PA of MXSVL substrates containing LSMs in the DSEs of the PA signal. Mutations DM1 to DM5 (Fig. 6) were introduced into the MXSVL substrate (see the DSM LSMs substrates in Fig. 2) and analyzed for in vitro splicing and PA under coupling conditions (1.5 mM Mg²⁺). Total splicing and total PA were quantitated as a percentage of processing obtained with the WT Sp/PA substrate (WT). The error of the data was ±2 to 4%.

FIG. 8

Model for how the failure to properly define an exon may cause the definition complex on the previous exon to inhibit processing. WT, wild type.