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Four Factors Are Required for 3'-End Cleavage of Pre-Mrnas

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Four Factors Are Required for 3'-End Cleavage of Pre-Mrnas Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Four factors are required for 3'-end cleavage of pre-mRNAs Yoshio Takagaki, Lisa C. Ryner/ and James L. Manley Columbia University, Department of Biological Sciences, New York, New York 10027 USA We reported previously that authentic polyadenylation of pre-mRNAs in vitro requires at least two factors: a cleavage/specificity factor (CSF) and a fraction containing nonspecific poly(A) polymerase activity. To study the molecular mechanisms underlying 3' cleavage of pre-mRNAs, we fractionated CSF further and show that it consists of four separable subunits. One of these, called specificity factor (SF; M„ —290,000), is required for both specific cleavage and for specific polyadenylation and thus appears responsible for the specificity of the reaction. Although SF has not been purified to homogeneity, several lines of evidence suggest that it may not contain an essential RNA component. Two other factors, designated cleavage factors I (CFI; M„ -130,000) and II (CFII; M„ —110,000), are sufficient to reconstitute accurate cleavage when mixed with SF. A fourth factor, termed cleavage stimulation factor (CstF; M„ —200,000), enhances cleavage efficiency significantly when added to a mixture of the three other factors. CFI, CFII, and CstF do not contain RNA components, nor do they affect specific polyadenylation in the absence of cleavage. Although these four factors are necessary and sufficient to reconstitute efficient cleavage of one pre-RNA tested, poly(A) polymerase is also required to cleave several others. A model suggesting how these factors interact with the pre-mRNA and with each other is discussed. [Key Words: Cleavage/specificity factor; poly(A); pre-RNA] Received June 21, 1989; accepted August 8, 1989. Polyadenylation of eukaryotic pre-mRNAs is one of the cleavage reaction is also influenced by sequences that lie most important steps in maturation of RNA polymerase just downstream of the cleavage site (for review, see II transcripts. This process plays an important role in Manley 1988). Several studies have indicated that large gene expression, as mutations that block 3'-end forma­ complexes form on pre-mRNAs, which presumably con­ tion prevent accumulation of mature mRNA (lor re­ tain the factors that catalyze the 3'-end formation reac­ views, see Bimstiel et al. 1985; Manley 1988). In addi- tion (Humphrey et al. 1987; Skolnik-David et al. 1987; tioU; polyadenylation can play a role in the regulation of Zarkower and Wickens 1987; Zhang and Cole 1987; gene expression, because selection of alternative poly(A) McLauchlan et al. 1988; Moore et al. 1988b; Stefano and sites in a single gene can lead to the synthesis of dif­ Adams 1988). Based on UV cross-linking experiments, it ferent mRNAs (for review, see Leff et al. 1986). has been proposed that two proteins (M^, 64,000-68,000 HeLa cell nuclear extracts are able to accurately pro­ and 155,000) interact specifically with the sequences cess exogenously added pre-RNA molecules that contain surrounding the poly(A) signal AAUAAA (Moore et al. the signals required for 3'-end formation (Moore and 1988a; Wilusz and Shenk 1988). Sharp 1985). Using such extracts, it has been shown that Recently, several groups have begun fractionating the two steps of the 3'-end processing reaction, i.e., en- HeLa cell nuclear extracts to identify the factors in­ donucleolytic cleavage and polyadenylation, can be un­ volved in the cleavage and polyadenylation reactions (for coupled and assayed separately (Moore and Sharp 1985; review, see Humphrey and Proudfoot 1988). We reported Moore et al. 1986; Zarkower et al. 1986; Sheets et al. previously that a cleavage/specificity factor (CSF) that 1987). In the absence of divalent cation, pre-RNAs are efficiently cleaves SV40 late pre-RNA at its poly(A) ad­ accurately cleaved but not polyadenylated, generating dition site can be separated chromatographically from a upstream and downstream cleavage products. In the poly(A) polymerase (PAP; Takagaki et al. 1988). Al­ presence of Mg^^, pre-RNAs can be polyadenylated at though this PAP activity functions only nonspecifically the 3' ends of either the pre-RNAs, themselves, or up­ by itself, addition of CSF causes it to function in a stream cleavage products (Manley 1983; Manley et al. poly(A) signal (AAUAAA)-dependent manner. On the 1985; Moore and Sharp 1985; Zarkower et al. 1986). Both other hand, the PAP activity is also required to cleave all reactions absolutely require the conserved AAUAAA other pre-RNAs tested so far. Using different fraction­ signal sequence (Proudfoot and Brownlee 1976), and the ation methods, it has been shown that multiple factors are required for both cleavage and polyadenylation reac­ 'Cuitent address: Department of Biological Sciences, Stanford Univer­ tions (Gilmartin et al. 1988; McDevitt et al. 1988). sity, Stanford, California 94305 USA. Christofori and Keller (1988) have recently demon- GENES & DEVELOPMENT 3:1711-1724 © 1989 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/89 $1.00 1711 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Takagaki et al. strated that three factors are required for cleavage of To elucidate the molecular mechanisms underlying both SV40 late and Ad2 L3 pre-RNAs. Although two of 3'-end processing of pre-mRNA, it is essential to iden­ these factors—cleavage polyadenylation factor and PAP tify and characterize all the factors that are involved in —are also required for polyadenylation, a third factor— this process. In this study we have isolated from CSF cleavage factor—is necessary only for the cleavage reac­ four different factors that are necessary and sufficient to tion. reconstitute accurate and efficient cleavage of SV40 late Analogous to other pre-mRNA processing reactions, pre-RNA and characterized some of their functions and e.g., splicing (for review, see Green 1986; Padgett et al. physical properties. 1986; Maniatis and Reed 1987) and histone mRNA 3'- end formation (for review, see Bimstiel et al. 1985; Results Mowry and Steitz 1988), the involvement of small nu­ clear ribonucleoproteins (snRNPs) in 3'-end formation of CSF can be fractionated into three factors polyadenylated mRNAs has been suggested (Moore and We showed previously that a CSF that can efficiently Sharp 1984, 1985; Hashimoto and Steitz 1986; Sperry cleave SV40 late pre-RNA at its poly (A) addition site can and Berget 1986; Christofori and Keller 1988; Gilmartin be separated from a nonspecific PAP. In addition, CSF is et al. 1988). However, it is unlikely that a major species also required, along with PAP, to catalyze AAUAAA-de- of snRNA (e.g., Ul, U2, U4, U5, and U6) is required for pendent polyadenylation (Takagaki et al. 1988). To study this process, because degradation of these snRNAs does CSF further, we subjected the factor to additional frac­ not affect 3'-end processing reactions (Berget and Rob- tionation steps, as indicated in Figure 1. berson 1986; Ryner and Manley 1987). Recently, Chris­ As a first step, CSF obtained by Superose 6 chromatog­ tofori and Keller (1988) reported that a fraction of Ull raphy (Materials and methods; Takagaki et al. 1988) was snRNP (Kramer 1987; Montzka and Steitz 1988) cofrac- applied to a Mono Q anion exchange column. Fractions tionated with a factor required for both cleavage and po­ were assayed by incubating aliquots with a ^^P-labeled lyadenylation reactions. The activity of this factor, how­ 233-nucleotide SV40 late pre-RNA, either in the pres­ ever, was not strictly correlated with the amount of Ul 1 ence of Mg^"^ and crude, Superose 6 PAP to assay specific snRNA. In addition, in contrast to snRNAs involved in polyadenylation, or in the absence of Mg^"^ and other splicing and 3'-end processing of histone pre-RNAs, the fractions to assay cleavage. Reaction products were ana­ sequence of Ul 1 snRNA displays no complementarity to lyzed by denaturing polyacrylamide gel electrophoresis, sequences known to be required for polyadenylation (e.g. and the results of the experiment in which PAP was AAUAAA; Montzka and Steitz 1988). The question of added are shown in Figure 2. A single, strong peak of snRNP involvement in pre-mRNA 3'-end processing specific polyadenylation activity was detected in frac­ thus remains an enigma. tions 19-21 (Fig. 2B). In contrast, when each of the HeLa Cell Nuclear Extract I Ammonium Sulfate Fractionation (20-40% Saturation) ; Superose 6 Poly(A) Polymerase (PAP) Cleavage/Specificity Factor (CSF) Mono Q 1 Flow Through Low Salt High Salt Cleavage Stimulation Cleavage Factor Specificity Factor Factor (CstF) (CF) (SF) Heparin Agarose Mono S Phenyl Superose 1 ' 1 Low Salt High Salt Figure 1. Fractionation of factors required for in CFI CFII vitro 3'-end processing of pre-mRNAs. The frac­ tionation methods used to separate and charac­ • J' T terize the factors involved in 3'-end processing of Glycerol Density 1 Gradient Centrifugation CsCI Buoyant Density pre-mRNAs are shown schematically. (Bottom) Centnfugation Sedimentation coefficients obtained by glycerol density gradient centrifugation, estimated molec­ CstF CFI CFII SF ular mass, and density (d) for SF. 9.0S. 200ltd 6.5S, 130kd 5.8S, 110kd I2.5S, 290kd, d:>1.28g/ml 1712 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press 3' End cleavage of pre-mRNAs B 5^« ;: r.floo>0-j:in5t«5^«»0jjcvi n PolylA) 30S Nl 18C m F.T. Low salt High salt (CstF) (CF) TSF) 110 % • iMiM MqCl^ Dral PG3SVL-A AATAAA -^ lsP6 RNA Polymcrat* 10 15 Fraction Number GpppG- — AAUAAA—I 233 nt Figure 2. Purification of CSF by Mono Q anion exchange chromatography. [A] The elution profile of proteins was monitored by UV absorbance at 280 nm.
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