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.
The majority of pre-mRNAs in mammalian cells are processed in the nucleus by 7-methylguanosine (m 7 GpppG) cap formation on the 5Ј end and splicing and polyadenylation on the 3Ј end. The biochemistry of each of these individual processing reactions has been extensively studied (9,10,13,16,27,29). These studies suggest that in the cell, the processing of an mRNA is highly coordinated and that regulatory interactions must occur between the specialized factors which mediate each individual processing reaction. Such coordination has been suggested in the exon definition model proposed by Berget (2).According to the exon definition model (Fig. 1), mammalian interior exons, because of their small size (average of 137 nucleotides), are defined when the splicing factors locate a pair of closely spaced splice sites in exonic polarity (3Ј splice siteexon sequences-5Ј splice site). This localization of splice sites allows exon definition by the binding of U1 and U2 small nuclear ribonucleoproteins (snRNPs) and associated factors (2). However, the two terminal exons require a different mechanism given that the first exon has a cap structure instead of a 3Ј splice site and the last exon has a polyadenylation signal instead of a 5Ј splice site.The mechanism for the processing of the first exon has been suggested by the work of Izaurralde et al. (9), which showed that an m 7 GpppG cap and a nuclear cap-binding complex (CBC; an 80-kDa and a 20-kDa cap-binding protein) were essential for efficient in vitro removal of the adjacent intron, using a simple one-intron substrate. These data suggest that an early stage of spliceosome assembly, possibly the formation of the commitment complex, is blocked by either the use of a cap analog (to which CBC does not bind) or the depletion of the CBC from splicing extracts. These data suggest that the CBC, in association with the m 7 GpppG cap, may interact with components of the commitment complex (Fig. 1), providing a means to define the exon or to activate the progression to full spliceosome assembly. This effect of the cap and the CBC appears to affect only the removal of the first intron. Previous in vitro experiments using a two-intron system indicated that changing the m 7 GpppG cap to an adenosine inhibited removal of the first intron but had little or no effect on the removal of the second (22).Several lines of experimentation have suggested that processing of the last exon involves interaction with splicing components at the 3Ј splice site and the polyadenylation complex (Fig. 1). Such interactions have been suggested by experiments using a coupled in vitro splicing and polyadenylation system (19,21). These data showed that mutations in the polyadenylation signal which inhibited polyadenylation also caused a decrease in the efficiency of splicing, i.e., removal of the last intron. Likewise, mutations in the 3Ј splice site of the last exon, which inhibit splicing, caused inhibition of polyadenylation. Analogous transfection experiments have provided similar results in vivo (4,17,18). ...
Polyadenylation and splicing are highly coordinated on substrate RNAs capable of coupled polyadenylation and splicing. Individual elements of both splicing and polyadenylation signals are required for the in vitro coupling of the processing reactions. In order to understand more about the coupling mechanism, we examined specific protein-RNA complexes formed on RNA substrates, which undergo coupled splicing and polyadenylation. We hypothesized that formation of a coupling complex would be adversely affected by mutations of either splicing or polyadenylation elements known to be required for coupling. We defined three specific complexes (A C , A C , and B C ) that form rapidly on a coupled splicing and polyadenylation substrate, well before the appearance of spliced and/or polyadenylated products. The A C complex is formed by 30 s after mixing, the A C complex is formed between 1 and 2 min after mixing, and the B C complex is formed by 2 to 3 min after mixing. A C is a precursor of A C , and the A C and/or A C complex is a precursor of B C . Of the three complexes, B C appears to be a true coupling complex in that its formation was consistently diminished by mutations or experimental conditions known to disrupt coupling. The characteristics of the A C complex suggest that it is analogous to the spliceosomal A complex, which forms on splicing-only substrates. Formation of the A C complex is dependent on the polypyrimidine tract. The transition from A C to A C appears to require an intact 3-splice site. Formation of the B C complex requires both splicing elements and the polyadenylation signal. A unique polyadenylation-specific complex formed rapidly on substrates containing only the polyadenylation signal. This complex, like the A C complex, formed very transiently on the coupled splicing and polyadenylation substrate; we suggest that these two complexes coordinate, resulting in the B C complex. We also suggest a model in which the coupling mechanism may act as a dominant checkpoint in which aberrant definition of one exon overrides the normal processing at surrounding wild-type sites.
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