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Polyadenylation: a Tail of Two Dispatch Complexes View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Current Biology, Vol. 12, R855–R857, December 23, 2002, ©2002 Elsevier Science Ltd. All rights reserved. PII S0960-9822(02)01353-2 Polyadenylation: A Tail of Two Dispatch Complexes Nick Proudfoot and Justin O’Sullivan been shown to bind these U-rich poly(A) signals [5–7]. Interestingly, Yhh1p is homologous to CPSF160, which has been known for some time to directly bind the Recent studies have uncovered new connections AAUAAA sequence. Indeed the originally erroneous between the enzymes of mRNA 3′ end processing view that the highly conserved AAUAAA must be rec- and RNA polymerase II. These connections improve ognized by interaction with a small nuclear (sn)RNA the efficiency of polyadenylation and signal to the was only laid to rest when it was finally proven that polymerase to terminate transcription; their discov- CPSF160 binds directly to mammalian AAUAAA [8]. For ery reveals another level of gene regulation. the rest of this article, the names of cleavage-poly(A) and transcription factors will be preceded by sc, sp or hs to indicate species: S. cerevisiae, S. pombe and In eukaryotes, formation of the mature 3′ end of a mes- Homo sapiens, respectively. senger (m)RNA involves cleavage of the nascent tran- As with the other molecular mechanisms of gene script and polyadenylation. The mechanisms involved expression, polyadenylation can no longer be consid- in this process have been under scrutiny for over two ered in isolation. Connecting the polyadenylation of decades. The mRNA sequence itself provides the pre-mRNA with the actual transcription process has signals that determine the site of polyadenylation: now become a major research focus in many of the these are the ubiquitous AAUAAA element, 20–30 labs that study these phenomena. This story started nucleotides upstream of the cleavage site where the two decades ago, when it was realized that mutating poly(A) is added, and immediately downstream of this poly(A) signals not only block mRNA polyadenylation a GU-rich sequence. These sequences are recognized — and consequently mRNA accumulation — but also by two key proteins: the ‘cleavage and polyadenylation disrupt the normal site of transcriptional termination stimulation factor’ CPSF, and the ‘cleavage stimulation by RNA polymerase II, causing nascent transcription factor’ CstF, which associate with the additional cleav- to run on into the gene’s 3′ flanking region [9]. age factors CF1 and CF2. The pre-mRNA is cleaved The significance of these results became clearer after these factors bind to it, and the 3′ end so formed with work on mammalian gene systems, in particular, is then immediately polyadenylated to A200 by poly(A) analysis of the key molecular anchor of the RNA polymerase [1] (Figure 1A). polymerase II elongation complex, the large subunit Genetic studies in yeast — both budding yeast carboxy-terminal domain (CTD), which consists of an Saccharomyces cerevisiae and fission yeast Schizosac- extended 52 unit heptad repeat sequence. Each repeat charomyces pombe — have identified a set of essential has two serines — in positions 2 and 5 of the heptad — gene products associated with cleavage and poly- which are susceptible to phosphorylation, at least in adenylation. These factors have also been biochem- part mediated by the hsTFIIH-associated kinase ically characterised. These studies have initially hsCdk7 [9,10]. This phosphorylation process, initially ascribed a seemingly more complex set of proteins to largely on serines at the 5 positions on the CTD, is the cleavage and polyadenylation mechanism in yeast associated with the switch from initiation complexes to than in mammalian cells (Figure 1B), but it seems likely the more streamlined elongation complexes. Additional that most components of the yeast apparatus will turn phosphates are then added, largely to serines in the 2 out to have clear mammalian counterparts [2,3]. As positions, by several other kinases, including hsCdk9 shown in Figure 1B, yeast CF1A contains subunits which in association with hscyclinT comprises the tran- homologous to those of mammalian CstF and CFIIm [4], scription elongation factor hsPTEFb [9–11]. while the yeast cleavage and polyadenylation factor Biochemical analysis then revealed that hsCPSF CPF, previously regarded as two separate sub-com- and hsCstF both associate with the CTD — hsCPSF plexes, CFII and PFI, contains homologs of CPSF as showing no preference for phosphorylation state — well as of other mammalian cleavage and polyadenyla- during transcription elongation, and in the case of tion factors. hsCPSF also during initiation, so that they are avail- Confusingly, the cis elements for yeast polyadeny- able to cleave and polyadenylate the nascent tran- lation are poorly defined, though three redundant and script as it emerges from polymerase II’s RNA exit degenerate sequences are generally identifiable — an channel [9,10]. Indeed, it appears that the phospho- efficiency element, a positioning element and the CTD is an active component of the cleavage appara- poly(A) site — spread over about 100 nucleotides of tus, as it significantly stimulates in vitro 3′ end pre-mRNA sequence [2]. The latest twist on poly(A) processing [12]. site signals in yeast is that many have U-rich regions These experiments set the stage for recent studies flanking their cleavage sites [5]. Ydh1p, Yth1p and in yeast which have further elucidated the connection Yhh1p, components of yeast CPF (Figure 1B), have all between cleavage–polyadenylation and the phospho- CTD. The yeast polymerase II CTD has just 24 heptad Sir William Dunn School of Pathology, University of Oxford, repeats, so there would seem to be less room on this South Parks Road, Oxford OX1 3RE,. UK. protein domain for the multiple molecular interactions Dispatch R856 ABFigure 1. Comparison of cleavage- CPF polyadenylation factors associated with poly(A) signals in mammals and budding Pta1 Pfs1 Pfs2 yeast. Pol II Ysh1 CstF In mammals (A), the five factors involved CTD mRNA Ydh1 Fip1 are indicated, showing the subunit struc- CPSF 50 64 Yth1 Yhh1 tures and molecular weights of CPSF and Pap1 160 77 CstF. The polymerase II CTD (grey) is (G/U) UUE Py(A)n DUE PAP also shown. In yeast (B), three multi- 100 73 mRNA subunit factors are involved with the 30 PE CFIm subunit components indicated. The posi- AAUAAA CFIIm RNA15 ? tions of the different factors are intended EE Hrp1 RNA14 to indicate (where known) the interac- tions between different factors and their CFIB subunits. The sequence elements that Pcf11 CFIA comprise the poly(A) signals are also indi- Clp1 cated by black rectangles, and the site of cleavage (and subsequent polyadenyla- Current Biology tion) is shown by a red arrow bolt. Homologous factors between yeast and mammals are color matched. Known homologous subunits between yeast and human include: Pta1p = symplekin; Pfs2p = CstF50; Ysh1p = CPSF73; Ydh1p = CPSF100; Yth1p = CPSF30; Yhh1p = CPSF160; Pap1p = PAP; Rna14p = CstF77; Rna15p = CstF64 [3]. Clp1p and Pcf11 are components of CFIIm [4]. ascribed to the longer mammalian CTD. So while scCPF also directly contacts the phospho-CTD the CTD is a key player in mammalian pre-mRNA through its scYhh1p subunit [7], which recruits the rest processing, the process in yeast might be simpler and of this large complex, including scPta1p, which had less demanding. previously been implicated in CTD interaction [16]. As An elegant experiment has now shown that the scYhh1p also directly interacts with the mRNA poly(A) polymerase II CTD is not obligatory for cleavage-poly- signal [7], this protein provides a direct contact adenylation in yeast. A yeast strain was engineered to between the nascent RNA and the elongating poly- express T7 bacteriophage RNA polymerase, which merase II complex. Indeed there appear to be multiple lacks a CTD, so that it can transcribe a T7 promoter- molecular contacts between the cleavage-polyadeny- driven gene encoding pre-RNA that has an intron and lation apparatus and the polymerase II CTD (Figure 2). a poly(A) signal [13]. The mRNA transcribed from this How these large protein complexes jostle for space on gene was not subject to the usual 5′ ‘capping’, but the relatively short yeast polymerase II CTD domain is cleavage-polyadenylation was readily detected. Some hard to envisage. But in mammals, the longer 52 reduction in the specificity of poly(A) site recognition heptad repeat may allow position specialisation, with was noticed, but the usual cleavage-polyadenylation the more carboxy-terminal repeats involved in 3′ factors — at least scRna15p and scPap1p — were processing function — and directly interacting with required for the processing reaction. hsCstF50 — leaving the rest of the CTD to cope with Although this experiment indicates that the RNA capping and splicing functions [17]. polymerase does not require a CTD for 3′ end pro- Transcription factors that directly contact the cessing to take place, there are contrary indications cleavage-polyadenylation apparatus have been from work on yeast where the endogenous enzyme identified in several independent studies. Firstly, the has been engineered to lack a CTD. In this case, the carboxy-terminal region of hsCstF64 — Rna15p in S. mutant yeast exhibited a reduction in 3′ end process- cerevisiae and Ctf1 in S. pombe — was shown to be ing of endogenous pre-mRNA transcripts, with a ten- critical for promoting transcriptional termination, even dency to use cryptic poly(A) sites downstream of the though it is dispensable for mRNA 3′ end cleavage normal poly(A) signals [14]. These data indicate that [18]. Clues to the function of this termination domain the CTD at least influences cleavage-polyadenylation come from the observation that it interacts with PC4 in yeast. — Sub1p in S. cerevisiae [19] — which in turn inter- Regardless of whether or not the CTD is required to acts with the general transcription factor hsTFIIB.
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