Mutation of the AAUAAA Polyadenylation Signal Depresses in Vitro Splicing of Proximal but Not Distal Introns

Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Mutation of the AAUAAA polyadenylation signal depresses in vitro splicing of proximal but not distal introns Maho Niwa and Susan M. Berget Marrs McClean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030 USA To investigate the relationship between splicing and polyadenylation during the production of vertebrate mRNAs, we examined the effect of mutation of a poly(A) site on splicing of upstream introns. Mutation of the AAUAAA polyadenylation consensus sequence inhibited in vitro splicing of an upstream intron. The magnitude of the depression depended on the magnesium concentration. Dependence of splicing on polyadenylation signals suggests the existence of interaction between polyadenylation and splicing factors. In multi-intron precursor RNAs containing duplicated splice sites, mutation of the poly(A) site inhibited removal of the last intron, but not the removal of introns farther upstream. Inhibition of removal of only the last intron suggests segmental recognition of multi-exon precursor RNAs and is consistent with previous suggestions that signals at both ends of an exon are required for effective splicing of an upstream intron. [Key Words: Polyadenylation signal; splicing; vertebrate RNAs] Received July 31, 1991; revised version accepted September 10, 1991. The majority of vertebrate mRNAs undergo both splic- Using chimeric RNAs competent for both in vitro ing and polyadenylation during maturation. The rela- splicing and polyadenylation, we recently reported that tionship between the two processing steps has been un- in vitro polyadenylation is stimulated when an intron is clear. Polyadenylation has been reported to occur shortly placed upstream of a poly(A) site (Niwa et al. 1990). after RNA polymerase passes the poly{A) cleavage site Stimulation required the presence of splicing signals at and prior to splicing (Ford and Hsu 1978; Nevins and the 3' end of the intron but not at the 5'end. This obser- Damell 1978; Weber et al. 1980). Other experiments, vation suggests that the presence of the poly(A) site however, demonstrate splicing of introns distal to the within a terminal exon (and thereby placement down- poly(A) site, both before polyadenylation and during stream of sequences at the 3' end of the intron) is im- transcription (Berget and Sharp 1978; Keohavong et al. portant for stimulation. It suggests further that the splic- 1982; Green et al. 1983; Mariman et al. 1983; Beyer and ing and polyadenylation machineries interact. Osheim 1988; LeMaire and Thummel 1990), indicating A number of mutants with altered polyadenylation no obligate ordering of the two processing steps. The signals have been isolated and characterized. Mutation possibility remained, however, that splicing of the last of the conserved AAUAAA element drastically reduces intron might be coupled to polyadenylation. levels of cytoplasmic RNA. Few, if any, studies have Polyadenylation is readily assayed in vitro with pre- investigated the effect of such mutations on the splicing cursor RNAs that lack splicing signals. Likewise, splic- of upstream introns. Here, we report the effect of the ing is readily assayed with precursor RNAs that lack presence of a poly(A) site on in vitro splicing. poly(A) sites. Furthermore, the two activities do not cof- We found that a wild-type poly(A) site was required for ractionate (Christofori and Keller 1988; Gilmartin et al. maximal removal of the upstream intron. Mutation of 1988; Kramer 1988; Takagaki et al. 1988), indicating that the essential AAUAAA polyadenylation element im- the two processing steps are catalyzed by different enti- paired splicing. Using constructs containing multiple, ties. At face value, these results suggest that splicing and identical introns we observed that the effect of the polyadenylation are uncoupled. Several recent reports, poly(A) site was limited to splicing of the last intron; however, indicate that mutation or deletion of splicing upstream introns were unaffected. We conclude that consensus sequences depresses polyadenylation in vivo splicing of 3'-terminal introns is influenced by the pres- (Villereal and White 1983; Huang and Gorman 1990). ence of a poly(A) site. 2086 GENES& DEVELOPMENT 5:2086-2095 9 1991by Cold Spring Harbor Laboratory ISSN 0890-9369/91 $3.00 Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Polyadenylation and splicing Results Wild-type chimeric RNA spliced 5- to 10-fold better than either the mutant RNA or RNA containing only splicing Polyadenylation signals maximize splicing signals. Thus, the presence of a poly{A) site in the chi- of an upstream intron meric RNA significantly enhanced splicing of the up- We have been investigating the relationship between stream intron. This observation strongly suggests a syn- splicing and polyadenylation by examining the in vitro ergistic relationship between splicing and polyadenyla- processing of simple chimeric precursor RNAs contain- tion. ing both splicing and polyadenylation signals (Niwa et Precursor RNAs have a finite lifetime in in vitro pro- al. 1990; Niwa and Berget 1991). We find that these pre- cessing extracts. Analysis of the total amount of RNA in cursor RNAs are readily recognized in in vitro processing the reactions of the three types of precursor indicated extracts and are both polyadenylated and spliced to pro- that these RNAs all had a similar half-life in extract (data duce a product RNA in which both reactions have oc- not shown). Therefore, the difference in activity between curred {referred to here as S + A + RNA). Either polyade- wild-type and mutant substrates was not the result of nylation or splicing can occur first, although only the differential stability of substrate or product RNA. former pathway produces mature doubly processed Splicing of chimeric RNA was also examined under RNA. RNA that splices first behaves as a dead-end in- conditions more standard for measurement of in vitro termediate that cannot be processed further. Choice of splicing (Fig. 2). At 1.5 mM magnesium, greater splicing the pathway of processing is dependent on the magne- activity was observed for both MXSVL WT and MXSVL sium concentration: Polyadenylation is favored at low AAGAAA substrates than at lower magnesium (Fig. 2A). magnesium, and the primary product observed is doubly For wild-type precursor RNA, half of the precursor was processed RNA. Splicing dominates at higher magne- processed by initial polyadenylation and half was pro- sium, producing considerable RNA product that has un- cessed by initial splicing. Quantitation of splicing activ- dergone only splicing. ity indicated that total splicing of the wild-type precur- To investigate the effect of polyadenylation signals on sor RNA was about twofold higher than splicing of the splicing we compared the splicing activity of a wild-type mutant precursor RNA (Fig. 2B). Thus, even at magne- single-intron precursor RNA (MXSVL) to that of a mu- sium concentration favoring splicing, the presence of a tant precursor RNA containing altered polyadenylation wild-type poly(A) site resulted in more splicing than in a signals. The mutant MXSVL contained a single point similar construct containing a mutated poly(A) site. mutation converting the required polyadenylation con- Wild-type chimeric RNA produced two types of sensus sequence AAUAAA to AAGAAA (Fig. 1A). This spliced RNA: RNA that had been spliced but not poly- mutation effectively abolishes polyadenylation of SV40 adenylated (S+ RNA), and RNA that had been spliced late-precursor RNA in vivo and in vitro (Sheets et al. and polyadenylated (S + A RNA). Figure 2C compares the 1990). Figure 1B compares the splicing activities of these rates of production of each of these RNA species to the chimeric substrates, as well as the splicing activity of a rate of production of spliced RNA by using the AA- substrate containing only splicing signals (SB2) under the GAAA mutant precursor RNA. The production of mu- low magnesium conditions that both favor polyadenyla- tant spliced RNA had kinetics of appearance identical to tion and maximize production of doubly processed RNA the spliced but not polyadenylated (S + ) population from from MXSVL (Niwa and Berget 1991). The SB2 splicing wild-type RNA. The difference in total splicing between template was spliced very poorly under these conditions. mutant and wild-type precursor RNAs was therefore re- In contrast, MXSVL was both polyadenylated and spliced flected in the presence of a pool of doubly processed to produce doubly processed product RNA {A + S + RNA S + A + RNA by using wild-type precursor RNA that was in Fig. 1B). MXSVL containing the AAGAAA point mu- not produced from mutant precursor RNA because of the tation was not polyadenylated because of the mutation mutated polyadenylation signal. Production of equiva- and was only marginally spliced. Thus, the presence of a lent amounts of S + RNA by mutant and wild-type chi- poly(A) site in the chimeric RNA permitted splicing in meric RNA, coupled with the inability of the S + popu- conditions under which it would normally not occur, lation to polyadenylate (Niwa and Berget 1991), raises and this splicing was dependent on a wild-type polyade- the possibility that wild-type RNA was processed by two nylation sequence. distinct pathways at moderate magnesium concentra- To quantitate splicing activity, the gel in Figure 1B tions, one that

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