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Efficiency of Cleavage at the Polyadenylation Site MOSHE SADOFSKY and JAMES C

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Efficiency of Cleavage at the Polyadenylation Site MOSHE SADOFSKY and JAMES C MOLECULAR AND CELLULAR BIOLOGY, Aug. 1984, p. 1460-1468 Vol. 4. No. 8 0270-7306/84/081460-09$02.00/0 Copyright ©D 1984, American Society for Microbiology Sequences on the 3' Side of Hexanucleotide AAUAAA Affect Efficiency of Cleavage at the Polyadenylation Site MOSHE SADOFSKY AND JAMES C. ALWINE* Department of Microbiology, School of Medicine/G2, UnivSersity of Pennsylvania, Phtiladelphia, Pennsylvania /9104 Received 3 April 1984/Accepted 8 May 1984 The hexanucleotide AAUAAA has been demonstrated to be part of the signal for cleavage and polyadenyla- tion at appropriate sites on eucaryotic mRNA precursors. Since this sequence is not unique to polyadenylation sites, it cannot be the entire signal for the cleavage event. We have extended the definition of the polyadenylation cleavage signal by examining the cleavage event at the site of polyadenylation for the simian virus 40 late mRNAs. Using viable mutants, we have determined that deletion of sequences between 3 and 60 nucleotides on the 3' side of the AAUAAA decreases the efficiency of utilization of the normal polyadenylation site. These data strongly indicate a second major element of the polyadenylation signal. The phenotype of these deletion mutants is an enrichment of viral late transcripts longer than the normally polyadenylated RNA in infected cells. These extended transcripts appear to have an increased half-life due to the less efficient cleavage at the normal polyadenylation site. The enriched levels of extended transcripts in cells infected with the deletion mutants allowed us to examine regions of the late transcript which normally are difficult to study. The extended transcripts have several discrete 3' ends which we have analyzed in relation to polyadenylation and other RNA processing events. Two of these ends map to nucleotides 2794 and 2848, which lie within a region of extensive secondary structure which marks the putative processing signal for the formation of the simian virus 40- associated small RNA. A third specific 3' end reveals a cryptic polyadenylation site at approximately nucleotides 2980 to 2985, more than 300 nucleotides beyond the normal polyadenylation site. This site appears to be utilized only in mutants with debilitated normal sites. The significance of sequences on the 3' side of an AAUAAA for efficient polyadenylation at a specific site is discussed. The 3' ends of polyadenylated messages in higher eucary- type and mutants the cleavage at the normal polyadenylation otes appear to be generated by cleavage from much larger site is relatively efficient. However, cells infected with primary transcripts. The specificity of cleavage and poly- certain mutants, which contain deletions on the 3' side of the adenylation is partly determined by the sequence AAUAAA AAUAAA, generated enriched levels of late RNAs which (17, 37), or close homologs, found 11 to 30 bases 5' to the extend beyond the normal polyadenylation cleavage site. actual cleavage site. This mechanism has been demonstrated The enrichment of the population of primary, extended in viral (1, 3, 11, 18, 19, 28, 29, 34) and cellular (22, 26) genes. transcripts in the mutants suggests that the normal process- Although the necessity for the AAUAAA sequence has been ing of these RNAs has been made less efficient by the clearly demonstrated (17, 24, 33), this sequence alone cannot specific deletions. These observations imply that specific be sufficient to signal the processing, because it also occurs sequences downstream from the AAUAAA are part of the within coding regions of messages, such as within the simian processing recognition signal. virus 40 (SV40) early coding region (43), the adenovirus type The enriched levels of extended transcripts have allowed 12 ElA transcription unit (36), and the chicken ovalbumin us to examine regions of the primary late transcript which gene (31). are normally difficult to detect. By mapping distinct 3' ends To understand better the mechanism underlying the utili- of the extended transcripts, several important observations zation of specific AAUAAA signals as polyadenylation sites, have been made. (i) Through alteration of the efficient we chose to study the polyadenylation of the late messages polyadenylation at the normal site, we have been able to of SV40. The 3' ends of SV40 late mRNAs (see Fig. 1) are detect an alternative polyadenylation site within the extend- formed specifically at nucleotide 2674 (SV numbering) (43) ed region at approximately nucleotide 2980. The resulting by cleavage of much larger nuclear transcripts (3, 28, 29). polyadenylated RNA is transported to the cytoplasm. (ii) We The region of the viral genome surrounding the polyadenyla- have located the processing site for the 5' end of the SV40- tion site is untranslated in both the early and the late senses; associated small RNA (SAS RNA; 4, 5, 6, 30). This site lies for this reason it is amenable to extensive mutation and within a region of extensive secondary structure indicative deletion analysis without loss of viral viability. This feature of a processing signal. allowed Fitzgerald and Shenk (17) to demonstrate that deletions surrounding the AAUAAA of the late polyadenyla- MATERIALS AND METHODS tion site permitted polyadenylation to occur, but deletion of the hexanucleotide prevented polyadenylation at this. site. Cells and infection conditions. All experiments were per- Since these studies analyzed only polyadenylated RNAs, the formed with the established line CV-1P of African green effect of the deletions on the efficiency of polyadenylation at monkey kidney cells grown in Dulbecco modified Eagle the normal site could not be determined. Using the mutants medium supplemented with glutamine (2 mM), penicillin of Fitzgerald and Shenk (17), we have found that in both wild (100 U/ml), streptomycin (100 FLg/ml), and 10% fetal bovine serum for propagation or 2% serum for maintenance and viral infection. * Corresponding author. T75 flasks with a confluent monolayer of cells were 1460 VOL. 4, 1984 EFFICIENCY OF POLYADENYLATION 1461 infected with a specific virus at 10 PFU per cell for 2 h in 3 ml used directly or further treated with a specific restriction of medium (2% fetal bovine serum) at room temperature with enzyme to generate a molecule labeled on only one strand. rocking. Cells were then fed and incubated at 37°C. The probes are purified by gel electrophoresis to eliminate Viruses. The wild-type SV40 strain 776 was used. Deletion unincorporated material and to isolate specific restriction mutant strains d1882 (41) and d11455, d11457, dl1458, d11453, fragments. and dl1465 (17) were generously provided by T. Shenk. Nuclease S1 analysis. Si analysis followed the procedure of Deletion strains dl1263 and d11265 (13, 14) were gifts of C. Berk and Sharp (10), with hybridization temperatures opti- Cole. The double deletion strain d11465-1263 was construct- mized for each probe. The probe generated from the EcoRI ed by ligating the BelI-to-BglI B fragment (nucleotides 2771 site was hybridized at 53°C. The BamHI probe was hybrid- to 5235) of dl1263 to the corresponding A fragment (nucleo- ized at 46°C. tides 5236 to 2770) of dl1465. Viral stocks were prepared Computer analysis of DNA sequences. RNA secondary after plaque purification. structure was deduced from a dot-matrix analysis of the RNA and DNA extraction. RNA was extracted from cells corresponding DNA sequences by using the programs of between 40 and 48 h after infection either as total cellular Fristensky et al. (20), as modified for an Apple II Plus RNA or as separate nuclear and cytoplasmic pools by the computer with Epson MX-80 printer. method of Villareal (47). The RNA preparations were freed of DNA by treatment with RNase-free DNase prepared by RESULTS the method of Tullis and Rubin (45). Viral DNA was Experimental design. To establish the effect of specific extracted by the method of Hirt (25), followed by CsCl deletions on the efficiency of polyadenylation processing at equilibrium gradient centrifugation in the presence of ethi- the SV40 late RNA polyadenylation site, we analyzed the dium bromide (38). normally polyadenylated RNA as well as the extended Selection of polyadenylated RNA. For somne experiments, primary transcripts by quantitative nuclease S1 hybridiza- polyadenylated RNA was selected by column chromatogra- tion analysis (10). However, we found that this analysis was phy on oligodeoxythymidylate cellulose (8). The RNA prep- not straightforward, due to the marked change in the AT aration was passed through the column three times in loading content of the genomic region surrounding the late polyaden- buffer (0.5 M NaCl, 10 mM Tris-hydrochloride [pH 7.5]), and ylation site (71% AT within the 160 nucleotides preceding the flowthrough was designated the nonpolyadenylated pool. the AAUAAA signal and 53% AT within the 150 nucleotides The retentate was eluted in 10 mM Tris-hydrochloride (pH following it). The unique BamHI site at base no. 2533 (Fig. 1 7.5), and the whole process was repeated for a total of three and 2) is only 141 bases upstream from the polyadenylation selections. The final elution was designated the polyadeny- site in the wild-type virus and less in the case of many of the lated pool. Between samples, the column was cleared with deletions. The richness of AT content on the 5' side of the 0.1 M NaOH and then reequilibrated with loading buffer. polyadenylation signal caused hybrid instability in this re- DNA hybridization probe preparation with T4 DNA poly- gion. Thus, a DNA probe extending from the BamHI site merase. The T4 polymerase replacement synthesis technique was effective in quantitating and mapping the extended of O'Farrell et al. (35) was adapted to label DNA for Si transcripts but formed short, unstable hybrids with the probes. By this technique a segment of DNA is labeled to the normally polyadenylated RNAs. Therefore, an alternative same extent as by nick-translation methods without leaving a probe extending from the EcoRI site, containing more se- residual nick that would interfere with nuclease Si analysis.
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