Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press A multisubunit factor, CstF, is required for polyadenylation of mammalian pre-mRNAs Yoshio Takagaki/ James L. Manley,* Clinton C. MacDonald,^ Jeffrey Wilusz,^ and Thomas Shenk^ ^Department of Biological Sciences, Columbia University, New York, New York 10027 USA; ^^Howard Hughes Medical Institute, Department of Biology, Princeton University, Princeton, New Jersey 08544 USA; ^Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, New Jersey 07103 USA We have purified and characterized a factor required for accurate polyadenylation of mammalian pre-mRNAs in vitro. This factor, called cleavage-stimulation factor (CstF), is composed of three distinct polypeptide subunits of 77, 64, and 50 kD. Using monoclonal antibodies directed against the 64- and 50-kD subunits, we show that CstF is required for efficient cleavage of polyadenylation substrates. Furthermore, CstF present in unfractionated nuclear extracts interacts with pre-mRNAs containing the signal sequence AAUAAA, but not AAGAAA, in such a manner that the 64-kD subunit can be cross-linked to the RNA by UV light. This polypeptide is thus identical to the previously described 64-kD nuclear protein that binds to AAUAAA- containing RNAs. Finally, indirect immunofluorescence of fixed cells indicates that CstF is distributed diffusely throughout the nucleus in a granular pattern distinct from the "speckled" pattern displayed by factors involved in pre-mRNA splicing, but similar to that of heterogeneous nuclear ribonucleoproteins. A model is presented in which CstF binds specifically to nascent RNA polymerase II transcripts and, by interacting with other factors, results in a rapid initiation of 3'-end processing of pre-mRNAs. {Key V^ords: Multisubunit factor CstF; polyadenylation; mRNA processing] Received August 10, 1990; revised version accepted September 18, 1990. Nearly all mammalian mRNAs are post-transcription- 200-290 kD, is required for both reactions (Christofori ally modified by the addition of a 3' poly(A) tail. This and Keller 1988; Gilmartin and Nevins 1989; Takagaki occurs by a two-step reaction in which an RNA poly­ et al. 1989). Poly(A) polymerase (PAP; 40-60 kD), merase II primary transcript is first endonucleolytically which, by itself, synthesizes poly(A) tracts onto any cleaved at the site of polyadenylation, and a poly(A) RNA primer, is the only other factor necessary for stretch of 200-300 nucleotides is then added to the AAUAAA-dependent poly(A) addition (Christofori and 3'end of the upstream cleavage product (Nevins and Keller 1989; Gilmartin and Nevins 1989; Ryner et al. Darnell 1978; Manley et al. 1982; Moore et al. 1986; 1989; Bardwell et al. 1990). Three additional factors, des­ Sheets et al. 1987; for reviews, see Humphrey and ignated cleavage factors I (CFI; 130 kD) and II (CFII; 110 Proudfoot 1988; Manley 1988). Although these two steps kD) and cleavage-stimulation factor (CstF; 200 kD), to­ are tightly coupled in vivo, they can be uncoupled and gether with SF, are necessary and sufficient to reconsti­ studied separately in vitro (Manley 1983; Moore and tute efficient cleavage activity with an SV40 late pre- Sharp 1985; Zarkower et al. 1986). Using such assays, it mRNA (Takagaki et al. 1989). To cleave other pre- has been shown that the nearly ubiquitous AAUAAA mRNAs, however, PAP is also necessary (Christofori consensus sequence located 10-30 nucleotides up­ and Keller 1988, 1989; Takagaki et al. 1988, 1989; Gil­ stream of the polyadenylation site (Proudfoot and martin and Nevins 1989; Ryner et al. 1989; Terns and Brownlee 1976; Fitzgerald and Shenk 1981; Higgs et al. Jacob 1989; Bardwell et al. 1990). To date, none of the 1983) is necessary for both cleavage and polyadenylation factors required for polyadenylation have been purified (Manley et al. 1985; Zarkower et al. 1986; Skolnik- to homogeneity. Using crude nuclear extracts, however, David et al. 1987) and that a complex set of traiis-acting it was found that 64-kD (Moore et al. 1988b; Wilusz and factors is required to catalyze accurate polyadenylation. Shenk 1988) and 155-kD (Moore et al. 1988b) polypep­ Biochemical fractionation of nuclear extracts has tides can be specifically UV cross-linked to pre-mRNAs demonstrated that multiple factors are required for both that contain the AAUAAA sequence. Recently, it was cleavage and polyadenylation reactions. One of these shown that specific cross-linking of the 64-kD protein factors, designated cleavage-polyadenylation or speci­ can be reconstituted with partially purified fractions ficity factor (SF)^ with an estimated molecular mass of (Wilusz et al. 1990). Only SF and CstF fractions are nec- 2112 GENES & DEVELOPMENT 4:2112-2120 © 1990 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/90 $1.00 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press mRNA polyadenylation factor essary, suggesting that the 64-kD protein is a component CstF of one of these two factors. B Apart from the problem of mechanism, an important 200kd 150 question is when and where in the mRNA biosynthetic \ \ a. o E r- r- T- pathway polyadenylation occurs. Early experiments ex­ N 00 O) O ^ 5! amining 3'-end formation in the adenovirus late tran­ scription unit suggested that polyadenylation could 309- occur very rapidly following passage of RNA polymerase 238- ll're past the poly(A) addition site, perhaps while the RNA is 180- i still a nascent chain (Nevins and Darnell 1978; Chen- 77kd • 64 • Kiang et al. 1982; Manley et al. 1982). In keeping with the idea that polyadenylation and transcription might be linked, it has been shown in some cases that 3'-end for­ mation precedes splicing (Weber et al. 1980). However, this situation is not universal (Zeevi et al. 1981), and models that assume a later position for 3'-end processing in the mRNA biosynthetic pathway are in some ways attractive. For example, polyadenylation might be linked to transport of mRNA from nucleus to cyto­ 1 23456739 10 11 plasm. Also, based on experiments showing that certain 12 3 4 5 6 factors involved in pre-mRNA splicing appear to be lo­ calized in discrete "speckles" within the nucleus (Lerner pG3SVL-A-7- et al. 1981; Nyman et al. 1986; Fu and Maniatis 1990), it 1 = has been suggested that splicing might occur only in spe­ -AAUAAA-L- - 233nl cific parts of the nucleus (Fu and Maniatis 1990). Might Figure 1. Cofractionation of three polypeptides with CstF ac­ 3' processing occur in similar (or identical) nuclear sub­ tivity. {A] SDS-polyacrylamide gel profile of glycerol gradient structures? fractions. Fraction numbers and the positions of the glycerol gradient molecular weight markers (200 kD, p-amylasc; 150 To address these issues, we have purified one of the kD, alcohol dehydrogenase) are shown at top. Estimated molec­ factors required for pre-mRNA 3'-end cleavage and ular weights of the three polypeptides are indicated at left. Note studied its biochemical and cell biological character­ that three protein bands are detected in the 64-kD region. [B] istics using monoclonal antibodies. This factor, CstF, is Assay of glycerol gradient fractions for CstF activity. One mi­ composed of three distinct subunits, one of which can be croliter of each of the glycerol gradient fractions (lanes 6-11), specifically cross-linked to AAUAAA-containing RNAs. CstF-containing fraction obtained by Mono S chromatography Indirect immunofluorescence microscopy reveals a dif­ (lane 5), or buffer C containing 50 mM (NH4)2S04 (lane 4) was fuse distribution of CstF throughout the nucleus, sug­ added to reaction mixtures containing SF, CFI, and CFII (see gesting that CstF may bind to nascent RNA polymerase Materials and methods), together with an SV40 late pre-mRNA. II transcripts. The roles of CstF in the 3'-end cleavage of Three microliters of CSF (Takagaki et al. 1988) (lane 3] was also pre-mRNAs are discussed. used as a positive control. The sizes of the Hpall-digested pBR322 DNA fragments (lane 1 ] sue indicated at left. The posi­ tions of the pre-mRNA (lane 2, Pre), and the upstream (solid Results and Discussion arrow) and downstream (open arrow) cleavage products are also shown at right. The structure of the SV40 late pre-mRNA tran­ Purification and characterization of CstF scribed from pG3SVL-A DNA (Takagaki et al. 1988) by SP6 As mentioned above, RNA 3' cleavage and polyadenyla­ RNA polymerase is shown at bottom. The 5' cap structure tion can be uncoupled in vitro and assayed separately. (m''GpppG), polyadenylation signal sequence (AAUAAA), polyadenylation site (arrowhead), and size of the pre-mRNA are For example, a pre-mRNA can be cleaved, but not poly- indicated. adenylated, by a multicomponent complex called cleavage/specificity factor (CSF; Fig. IB, lane 3) (Taka- gaki et al. 1988). We reported previously that CSF can be fractionated into four distinct subunits (Takagaki et al. ular weights of 77, 64, and 50 kD (Fig. lA, fractions 1989). CstF activity can be assayed by adding appropriate 8-10, lanes 2-4) exactly correlated with CstF activity fractions to a mixture of SF- and CFI + CFII-containing (Fig. IB, lanes 7-9). We hypothesized that CstF is com­ fractions (cf. Fig. IB, lanes 4 with 5), using a ^^P-labeled posed of these three polypeptides based on two lines of SV40 late pre-mRNA substrate (for structure, see Fig. IB, evidence. First, the total molecular mass of these three bottom). polypeptides, 191 kD, is close to the native size of CstF, We have purified CstF to near homogeneity by a com­ estimated to be 200 kD by glycerol gradient centrifuga­ bination of chromatography methods and glycerol den­ tion (Fig. lA). Second, the molar ratio of these polypep­ sity gradient centrifugation (see Materials and methods).