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Eukaryotic Mrna 3 Processing Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press PERSPECTIVE Eukaryotic mRNA 3؅ processing: a common means to different ends Gregory M. Gilmartin1 University of Vermont, Burlington, Vermont 05405, USA In principle, the formation of the 3Ј end of a eukaryotic for the evolution of two distinct 3Ј processing complexes mRNA is a simple process. At a minimum, it involves are likely to involve the conflicting pressures of post- the hydrolysis of a single phosphodiester bond in the transcriptional regulation: coordinate cell cycle control nascent transcript. For one unique class of mRNAs, tran- of the replication-dependent histone mRNAs versus the scripts of the metazoan replication-dependent histone developmental and cell-type-specific regulation of alter- genes, this is indeed the only requirement. All other eu- native poly(A) site selection. karyotic mRNAs require both cleavage and subsequent poly(A) addition to the newly generated 3Ј hydroxyl. De- spite this apparent simplicity, and more than two de- Similarities among contrasts cades of work, basic questions concerning the mecha- Ј nisms of 3Ј-end formation for both classes of transcripts The RNA sequence elements that direct the 3 process- persist. The accumulated evidence indicates that the ing of histone and polyadenylated mRNAs could hardly mechanisms by which the ends of histone and polyade- be more different (Fig. 1A). Three elements contribute to nylated mRNA are formed are fundamentally distinct, mammalian poly(A) site recognition, each of which is both in the factors responsible for processing and the recognized by a distinct protein complex (Zhao et al. RNA sequence elements that direct their action (for re- 1999a). The most well conserved element is the view, see Marzluff 2005; Zhao et al. 1999a). The 3Ј pro- AAUAAA hexamer that generally resides between 10 cessing of polyadenylated mRNAs requires a complex of and 30 nucleotides (nt) upstream of the cleavage site. over a dozen proteins, nearly all of which are conserved This element is recognized by a five-subunit complex from yeast to humans. In contrast, the formation of the termed cleavage and polyadenylation specificity factor 3Ј ends of the metazoan replication-dependent histone (CPSF). Downstream of the cleavage site is an amor- transcripts not only requires a unique set of proteins, but phous U-rich element that is recognized by the heterotri- an essential snRNA component as well. In both cases, meric cleavage stimulatory factor (CstF). A third ele- the identity of the endonuclease has remained largely a ment of the form UGUA is often present in one or more matter of speculation. copies at a variable distance upstream of the cleavage Two reports—one from Kolev and Steitz (2005) in this site (Hu et al. 2005), and is recognized by the heterodi- issue of Genes & Development, and the second from meric cleavage factor Im (CFIm) (Brown and Gilmartin Dominski et al. (2005a) have revealed that the mecha- 2003; Venkataraman et al. 2005). No conserved RNA nisms of poly(A) site processing and histone 3Ј process- structural elements appear to be shared among poly(A) ing are not quite as unique as they appear. Surprisingly, sites; on the contrary, studies have indicated that poly(A) the two processing machines share a common core of site recognition is facilitated by an “open” or unstruc- proteins that almost certainly includes the endonucle- tured conformation (Gimmi et al. 1989; Graveley et al. ase. The most exciting aspect of these reports, however, 1996). Ј is that they provide a fresh insight into the evolution of In contrast to polyadenylated transcripts, the 3 pro- what had seemed to be needlessly redundant mecha- cessing of histone pre-mRNAs requires both conserved Ј nisms for generating mRNA 3Ј ends. In essence, these sequence and structural elements (Marzluff 2005). The 3 reports indicate that the enzymatic machinery respon- end of the mature mammalian histone mRNA possesses sible for cleaving all nascent pre-mRNAs is likely to be a highly conserved 26-nt sequence, encompassing a 16-nt identical; the differences lie in the elaborate mecha- stem-loop, located 24–70 nt downstream of the stop nisms that have evolved to recruit this machinery to the codon (Fig. 1B). This element is bound by the stem-loop- Ј two classes of transcripts. The driving forces responsible binding protein (SLBP) that functions in 3 processing, as well as translation and the coupling of message stability to DNA replication and the cell cycle. Endonucleolytic 1Correspondence. cleavage occurs 5 nt downstream of the stem-loop. Nine E-MAIL [email protected]; FAX (802) 656-8808. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ to 12 nt downstream of the cleavage site is the histone gad.1378105. downstream element (HDE) that functions in 3Ј process- GENES & DEVELOPMENT 19:2517–2521 © 2005 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/05; www.genesdev.org 2517 Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Gilmartin by the same endonuclease, and the restriction of non- polyadenylated mRNAs to metazoans, suggests that the histone 3Ј processing machinery may simply represent a variation on a theme. Rather than U7 snRNA represent- ing a “functional fossil” (Mowry and Steitz 1988) that has been discarded by the polyadenylation machinery in favor of an all-protein complex, the role of U7 snRNA in histone 3Ј processing might be a more recent develop- ment. As discussed below, the development of a distinct histone 3Ј processing machine may have been driven by the function of histone mRNA 3Ј ends in both pre- mRNA 3Ј processing and the post-transcriptional regu- lation of the mature message. A need for precision and uniformity A key to understanding the development of a unique histone 3Ј processing complex in metazoans may be found in an observation made 20 years ago by Birnstiel et Figure 1. Mammalian pre-mRNA 3Ј processing sites. (A) al. (1985). They stated that: “What sets other mRNA Mammalian poly(A) site. The conserved elements of the poly(A) genes clearly apart from histone genes is that the dis- site are highlighted in bold. (B) Mammalian replication-depen- tances between the conserved sequences flanking the dent histone pre-mRNA 3Ј processing site. The invariant posi- mature mRNA 3Ј ends are not tightly conserved.” Two tions within the histone pre-mRNA are highlighted in bold. decades of work has subsequently revealed the basis for Sequences downstream of the cleavage site correspond to those the spatial constraints exhibited by histone 3Ј processing of the mouse histone H2A pre-mRNA and are shown base- sites (Marzluff 2005) and suggests a basis for the spatial paired to the mouse U7 snRNA. flexibility of poly(A) sites. The replication-dependent histone proteins function ing through base-pairing to a complementary sequence with a defined stoichiometry with respect to each other within the U7 snRNA. and to DNA. Thus the defining aspect of the post-tran- The only common sequence feature of all mammalian scriptional regulation of replication-dependent histone pre-mRNA 3Ј ends appears to be the fact that the cleav- genes is that the entire class of mRNAs responds in a age site is often preceded by a CA dinucleotide and is coordinate manner to cell cycle and DNA replication sandwiched between two sets of recognition elements. signals. The determinants of this coordinate regulation In each case, factors bound to the flanking regions are reside primarily at the 3Ј end of the message (Harris et al. bridged by protein:protein interactions across the cleav- 1991). Histone mRNA levels increase 35-fold as cells age site. Unlike the 3Ј processing of snRNAs, which re- progress from G1 to S phase, of which transcription ac- quires specific promoter sequences (Hernandez and counts for a three- to fivefold increase. Upon completion Weiner 1986), the sequences that reside near the 3Ј end of DNA replication, histone mRNAs are rapidly and spe- of pre-mRNAs are both necessary and sufficient for 3Ј- cifically degraded. DNA replication inhibitors also in- end formation. Several features of the chemistry of the 3Ј duce the rapid disappearance of this entire class of tran- processing of histone and polyadenylated mRNAs also scripts. The key player in these events is SLBP (Zeng et appear to be shared (Dominski and Marzluff 1999). Iden- al. 2003). SLBP initially binds the histone pre-mRNA in tical cleavage products, a 3Ј hydroxyl and a 5Ј phosphate, the nucleus where it acts to stabilize the binding of U7 are generated by a mechanism that does not require ATP snRNA to the HDE, an interaction bridged by the zinc hydrolysis. In addition, in vitro 3Ј processing of both finger protein ZFP100 (Pillai et al. 2003). As a component classes of transcripts yields a similar array of 3Ј products of the mature histone mRNP, SLBP is exported to the that have been progressively shortened by a tightly as- cytoplasm with the mRNA, where it acts to stimulate sociated 5Ј-to-3Ј exonuclease activity. In each case, both translation initiation (Gorgoni et al. 2005). Xenopus pos- the endonuclease and exonuclease activities are resistant sesses an additional, oocyte-specific form of SLBP that to high levels of EDTA, suggesting a role for zinc-depen- participates in the silencing of stored histone mRNAs dent catalysis (Ryan et al. 2004; Dominski et al. 2005b). during early development (Wang et al. 1999). Most im- As noted above, each mRNA class is bound by a dis- portantly, SLBP serves as the key modulator of the rapid, tinct set of factors that function in the recognition of the coordinate degradation of histone mRNA in response to pre-mRNA.
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