Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press In vitro polyadenylation is stimulated by the presence of an upstream intron Maho Niwa, Scott D. Rose, and Susan M. Berget Marrs McClean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030 USA The majority of vertebrate pre-mRNAs are both spliced and polyadenylated. To investigate the mechanism whereby processing factors recognize last exons containing both splicing and polyadenylation consensus elements, chimeric precursor RNAs containing a single intron and a poly(A) site were constructed and assayed for in vitro splicing and polyadenylation. Chimeric RNAs underwent splicing and polyadenylation. Both reactions occurred in a single RNA. The presence of an intron enhanced the rate of polyadenylation at a downstream poly(A) site. The extent of stimulation varied from two- to fivefold, depending on the magnesium concentration. Maximal stimulation of polyadenylation by an upstream intron required a 3' splice site but not a 5' splice site, suggesting that the structure of the terminal exon was more important than the presence of a complete upstream intron. We suggest that splicing and polyadenylation factors interact to recognize terminal, poly(A) site-containing exons. Such interaction may explain why all known intron-containing eukaryotic pre- mRNAs generate their 3' ends by polyadenylation. [Key Words: pre-mRNA; polyadenylation; splicing] Received June 1, 1990; revised version accepted July 13, 1990. The production of mRNA in higher eukaryotes entails ysis of the influence of one type of signal on the other considerable RNA processing. Most vertebrate genes processing reaction requires investigation of the nuclear contain introns. Most, but not all, pre-mRNAs are poly- phenotype of the mutation. Few experiments of this adenylated. Although many intronless pre-mRNAs are type have been reported. Those that have are suggestive polyadenylated, at least one major class, that coding for of some link between the two processing steps. Villar- histone proteins, is not. In contrast, no mechanism real and White (1983) reported that deletion of splicing other than polyadenylation for 3'-end generation of signals depressed the level of polyadenylation of nuclear spliced pre-mRNAs has ever been reported. This restric- RNA at the downstream poly(A) site, resulting in tion hints at the existence of interaction between normal levels of nuclear RNA with dispersed 3' termini. splicing and polyadenylation. 3'-Terminal exons begin Furthermore, placing an intron upstream, but not down- with a 3' splice site and terminate with a poly(A) site. stream, of a poly(A) site increases expression from trans- They are longer, on average, than internal exons. A re- fected genes (Buchman and Berg 1988) and raises the cent study of vertebrate exon size indicated an average level of nuclear poly(A) + RNA (Huang and Gorman length of 3'-terminal exons of 632 nucleotides versus a 1990), suggesting that maximal polyadenylation and 137-nucleotide average for internal exons (Hawkins transport to the cytoplasm require both the presence of 1988). Some terminal exons are quite large. This differ- splicing signals and the correct positioning of these ence also suggests that the processing machinery might signals upstream of the poly(A) site. recognize internal and 3'-terminal exons by different Several experiments have indicated that vertebrate mechanisms. poly(A) sites work only within the appropriate context. Experiments investigating in vitro splicing and polya- Placing a functional polyadenylation cassette in the denylation normally uncouple the two reactions. middle of an intron results in no apparent usage of the Poly(A) site-containing precursor RNAs lacking splicing poly(A) site and normal levels of splicing, suggesting signals polyadenylate, and vice versa. Furthermore, frac- that poly(A) sites cannot be recognized when located be- tionation efforts indicate that the splicing and polyaden- tween 5' and 3' splice sites (Adami and Nevins 1988; ylation cleavage activities are distinct (Christofori and Brady and Wold 1988; Levitt et al. 1989). The same cas- Keller 1988; Gilmartin et al. 1988; Takagaki et al. 1988). sette placed within an exon, however, directs efficient At first glance, these results would seem to contradict polyadenylation. When placed in an intron, mutation of the possibility that splicing and polyadenylation com- the flanking splicing signals permits recognition of the municate. poly(A) site (Adami and Nevins 1988; Levitt et al. 1989). Mutation of either splicing or polyadenylation signals These results suggest that either splicing occurs so inhibits production of mature cytoplasmic RNA. Anal- quickly that polyadenylation cannot compete or that 1552 GENES & DEVELOPMENT 4:1552-1559 91990 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/90 $1.00 Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Polyadenylation stimulated by presence oI upstream intron there is a required polarity for the arrangement of pro- A cessing signals in a pre-mRNA for both splicing and 100 200 300 400 polyadenylation. I II t I II We chose to address the question of interaction be- BamHI SfaNI Hpal Dral tween these two steps in processing by constructing chi- A meric splicing/polyadenylation precursor RNAs and as- saying their activity in in vitro systems competent for 120 -]1,,-55 MXSVL both reactions. We find that the presence of an intron increases the rate of polyadenylation at a downstream D25~5' 3' MINX poly(A) site. Maximal activity was dependent on the 120 ~ A presence of a 3' splice site but not a 5' splice site, sug- gesting that poly(A) sites are recognized as parts of ter- ;D"~ 141~_]~55 lj~" SVL minal exons. Results B EcoRI c.~Taeaec~ ~TTe~c.e~ 50 To examine the interaction of polyadenylation and S' Spl/ee Site Hind HI splicing factors during pre-mRNA processing, we con- [~c~ T~GAGCeTA G~TGTAG~ eTGGTTAeeT G~GCe~G 100 structed precursor RNAs that contain both splicing and polyadenylation signals (Fig. 1). The chimeric DNA con- structed for this study (MXSVL) consists of 217 nucleo- CTTGCTGCAC GTCTAGGGCG CAGTAGTCCA GGGTTTCCTT GATGATGTCA 150 Branch Point 3' Splice Site tides from the major late transcription unit of human t adenovirus containing two exons and a single intron TACTTATCCT GTCCCTTTTT TTTCCACAG~::=:r162 200 Bum tlI (MX) fused within exon 2 to a standard polyadenylation t cassette of 237 nucleotides from the late transcription GATC~GACA T~Ta~7~ATA CATTGATGAG ] 250 unit of SV40 (SVL). Precursor RNAs made from only the splicing or polyadenylation portion of the chimeric gene I TTTGGACAAA CCACAACTAG AATGCAGTGA AAAAAATGCT TTATTTGTGA [ 300 splice or polyadenylate well, respectively, in in vitro HeLa cell extracts (Sperry and Berget 1986; Zillmann et AATTTGTGAT GCTATTGCTT TATTTGTAAC CATTATAAGC TGCAATAAAC [ 350 al. 1987). MX DNA contains no known polyadenylation Hpa I Polyadenylatlon Site signals, and the SVL cassette contains no splicing t I AAGTTAACAA CAACAATTGC ATTCATTTTA TGTTTCAGGT TCAGGGGGAG [ 400 signals. Fusion creates a DNA that transcribes into a l~ra I 416-nucleotide precursor RNA with a second exon of t 237 nucleotides, with a cleavage and poly(A) addition I GTGTGGGAGG TTTTTTAAAG I 420 site located 182 nucleotides downstream of the 3' splice Figure 1. Chimeric splicing/polyadenylation precursor RNAs. site. (A) Large boxes indicate exon sequences; lines represent introns Processing reactions were performed in HeLa nuclear and sequences downstream of the poly(A) addition site; wavy extracts capable of both splicing and polyadenylation lines indicate vector sequences. Solid and lined boxes represent under conditions that represented a compromise be- exon sequences from the adenovirus 2 major late and SV40 late transcription units, respectively. Splice sites (5' and 3'), poly(A) tween standard splicing and polyadenylation assays (1.5 addition sites, and restriction enzyme sites used to truncate in rnM MgC12; see Materials and methods). Reactions con- vitro transcription templates are indicated. (B) Sequence of tained cordycepin (3' dATP) to permit addition of only a MXSVL chimeric precursor RNA. Shaded boxes demarcate single A residue following cleavage. The chimeric exon sequences from the adenovirus major late transcription MXSVL substrate underwent both splicing and polya- unit; open boxes indicate sequences from the SV40 late tran- denylation (Fig. 2). Final spliced and polyadenylation scription unit. MXSVL5' was a deletion of this sequence, re- product RNA (referred to as S+A + RNA) appeared by 20 moving the sequences from the indicated EcoRI-HindlII sites. min of reaction. Two other nonlariat RNAs, corre- MXSVL3' was a point mutant in which the 3' splice site was sponding in molecular weight to polyadenylated, but not altered from an AG to an AA. spliced, RNA (A+S - RNA) and spliced, but not polyade- nylated, RNA (S+A - RNA), were also observed. The re- action products designated as polyadenylated (A +) in that the site of cleavage was the correct in vivo-utilized Figure 2 disappeared when reactions were performed in site (data not shown). the presence of ATP instead of cordycepin; instead, pro- Production of S+A + RNA species indicated that both cessed material appeared above precursor RNA, indi- splicing and polyadenylation reactions occurred on the cating the addition of a poly(A) tail and confirming the same precursor molecule. A+S - RNA consistently ap- identification of the A + species. S+A - RNA was present peared before S+A + RNA. More complete time courses in equal amounts in reactions performed in the presence detected A+S - RNA as early as 3 min into the reaction of cordycepin or ATP, indicating that this species was (data not shown, but see Fig. 3D), indicating that the not polyadenylated. Mapping of the polyadenylated chimeric substrates were extremely active for polyaden- product RNA with complementary riboprobes indicated ylation. S+A - RNA and S+A + RNA, however, were not GENES & DEVELOPMENT 1553 Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Niwa et al.