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Eukaryotic mRNA 3' processing: a common means to different ends
http://www.100md.com 基因进展 2005年第21期
     University of Vermont, Burlington, Vermont 05405, USA

    In principle, the formation of the 3' end of a eukaryotic mRNA is a simple process. At a minimum, it involves the hydrolysis of a single phosphodiester bond in the nascent transcript. For one unique class of mRNAs, transcripts of the metazoan replication-dependent histone genes, this is indeed the only requirement. All other eukaryotic mRNAs require both cleavage and subsequent poly(A) addition to the newly generated 3' hydroxyl. Despite this apparent simplicity, and more than two decades of work, basic questions concerning the mechanisms of 3'-end formation for both classes of transcripts persist. The accumulated evidence indicates that the mechanisms by which the ends of histone and polyadenylated mRNA are formed are fundamentally distinct, both in the factors responsible for processing and the RNA sequence elements that direct their action (for review, see Marzluff 2005; Zhao et al. 1999a). The 3' processing of polyadenylated mRNAs requires a complex of over a dozen proteins, nearly all of which are conserved from yeast to humans. In contrast, the formation of the 3' ends of the metazoan replication-dependent histone transcripts not only requires a unique set of proteins, but an essential snRNA component as well. In both cases, the identity of the endonuclease has remained largely a matter of speculation.

    Two reports—one from Kolev and Steitz (2005) in this issue of Genes & Development, and the second from Dominski et al. (2005a) have revealed that the mechanisms of poly(A) site processing and histone 3' processing are not quite as unique as they appear. Surprisingly, the two processing machines share a common core of proteins that almost certainly includes the endonuclease. The most exciting aspect of these reports, however, is that they provide a fresh insight into the evolution of what had seemed to be needlessly redundant mechanisms for generating mRNA 3' ends. In essence, these reports indicate that the enzymatic machinery responsible for cleaving all nascent pre-mRNAs is likely to be identical; the differences lie in the elaborate mechanisms that have evolved to recruit this machinery to the two classes of transcripts. The driving forces responsible for the evolution of two distinct 3' processing complexes are likely to involve the conflicting pressures of post-transcriptional regulation: coordinate cell cycle control of the replication-dependent histone mRNAs versus the developmental and cell-type-specific regulation of alternative poly(A) site selection.

    Similarities among contrasts

    The RNA sequence elements that direct the 3' processing of histone and polyadenylated mRNAs could hardly be more different (Fig. 1A). Three elements contribute to mammalian poly(A) site recognition, each of which is recognized by a distinct protein complex (Zhao et al. 1999a). The most well conserved element is the AAUAAA hexamer that generally resides between 10 and 30 nucleotides (nt) upstream of the cleavage site. This element is recognized by a five-subunit complex termed cleavage and polyadenylation specificity factor (CPSF). Downstream of the cleavage site is an amorphous U-rich element that is recognized by the heterotrimeric cleavage stimulatory factor (CstF). A third element of the form UGUA is often present in one or more copies at a variable distance upstream of the cleavage site (Hu et al. 2005), and is recognized by the heterodimeric cleavage factor Im (CFIm) (Brown and Gilmartin 2003; Venkataraman et al. 2005). No conserved RNA structural elements appear to be shared among poly(A) sites; on the contrary, studies have indicated that poly(A) site recognition is facilitated by an "open" or unstructured conformation (Gimmi et al. 1989; Graveley et al. 1996).

    In contrast to polyadenylated transcripts, the 3' processing of histone pre-mRNAs requires both conserved sequence and structural elements (Marzluff 2005). The 3' end of the mature mammalian histone mRNA possesses a highly conserved 26-nt sequence, encompassing a 16-nt stem-loop, located 24-70 nt downstream of the stop codon (Fig. 1B). This element is bound by the stem-loop-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 cleavage occurs 5 nt downstream of the stem-loop. Nine to 12 nt downstream of the cleavage site is the histone downstream element (HDE) that functions in 3' processing through base-pairing to a complementary sequence within the U7 snRNA.

    Figure 1. Mammalian pre-mRNA 3' processing sites. (A) Mammalian poly(A) site. The conserved elements of the poly(A) site are highlighted in bold. (B) Mammalian replication-dependent histone pre-mRNA 3' processing site. The invariant positions within the histone pre-mRNA are highlighted in bold. Sequences downstream of the cleavage site correspond to those of the mouse histone H2A pre-mRNA and are shown base-paired to the mouse U7 snRNA.

    The only common sequence feature of all mammalian pre-mRNA 3' ends appears to be the fact that the cleavage site is often preceded by a CA dinucleotide and is sandwiched between two sets of recognition elements. In each case, factors bound to the flanking regions are bridged by protein:protein interactions across the cleavage site. Unlike the 3' processing of snRNAs, which requires specific promoter sequences (Hernandez and Weiner 1986), the sequences that reside near the 3' end of pre-mRNAs are both necessary and sufficient for 3'-end formation. Several features of the chemistry of the 3' processing of histone and polyadenylated mRNAs also appear to be shared (Dominski and Marzluff 1999). Identical cleavage products, a 3' hydroxyl and a 5' phosphate, are generated by a mechanism that does not require ATP hydrolysis. In addition, in vitro 3' processing of both classes of transcripts yields a similar array of 3' products that have been progressively shortened by a tightly associated 5'-to-3' exonuclease activity. In each case, both the endonuclease and exonuclease activities are resistant to high levels of EDTA, suggesting a role for zinc-dependent catalysis (Ryan et al. 2004; Dominski et al. 2005b).

    As noted above, each mRNA class is bound by a distinct set of factors that function in the recognition of the pre-mRNA. In contrast to the clear similarities between the factors that process polyadenylated mRNAs in mammals and those of yeast, the histone 3' processing factors, both proteins and U7 snRNA, are considerably less conserved among metazoans. This observation, coupled with the apparent processing of both classes of mRNAs by the same endonuclease, and the restriction of non-polyadenylated mRNAs to metazoans, suggests that the histone 3' processing machinery may simply represent a variation on a theme. Rather than U7 snRNA representing 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 3' processing might be a more recent development. As discussed below, the development of a distinct histone 3' processing machine may have been driven by the function of histone mRNA 3' ends in both pre-mRNA 3' processing and the post-transcriptional regulation of the mature message.

    A need for precision and uniformity

    A key to understanding the development of a unique histone 3' processing complex in metazoans may be found in an observation made 20 years ago by Birnstiel et al. (1985). They stated that: "What sets other mRNA genes clearly apart from histone genes is that the distances between the conserved sequences flanking the mature mRNA 3' ends are not tightly conserved." Two decades of work has subsequently revealed the basis for the spatial constraints exhibited by histone 3' processing sites (Marzluff 2005) and suggests a basis for the spatial flexibility of poly(A) sites.

    The replication-dependent histone proteins function with a defined stoichiometry with respect to each other and to DNA. Thus the defining aspect of the post-transcriptional regulation of replication-dependent histone genes is that the entire class of mRNAs responds in a coordinate manner to cell cycle and DNA replication signals. The determinants of this coordinate regulation reside primarily at the 3' end of the message (Harris et al. 1991). Histone mRNA levels increase 35-fold as cells progress from G1 to S phase, of which transcription accounts for a three- to fivefold increase. Upon completion of DNA replication, histone mRNAs are rapidly and specifically degraded. DNA replication inhibitors also induce the rapid disappearance of this entire class of transcripts. The key player in these events is SLBP (Zeng et al. 2003). SLBP initially binds the histone pre-mRNA in the nucleus where it acts to stabilize the binding of U7 snRNA to the HDE, an interaction bridged by the zinc finger protein ZFP100 (Pillai et al. 2003). As a component of the mature histone mRNP, SLBP is exported to the cytoplasm with the mRNA, where it acts to stimulate translation initiation (Gorgoni et al. 2005). Xenopus possesses an additional, oocyte-specific form of SLBP that participates in the silencing of stored histone mRNAs during early development (Wang et al. 1999). Most importantly, SLBP serves as the key modulator of the rapid, coordinate degradation of histone mRNA in response to both the end of S phase and the inhibition of DNA synthesis. In addition to SLBP, this rapid turnover requires active translation and the nonsense-mediated decay factor Upf1 (Kaygun and Marzluff 2005a). The function of SLBP in regulated mRNA decay requires that it be positioned within a narrow window with respect to both the stop codon and the 3' end of the transcript (Kaygun and Marzluff 2005b). The primary candidate for the nuclease responsible for regulated histone mRNA decay is a 3'-to-5' exonuclease, termed 3' hExo (Dominski et al. 2003). High-affinity binding of 3' hExo to the histone mRNA requires both the stem-loop and the highly conserved 3'-terminal ACCCA sequence. Extension of the RNA beyond the ACCCA terminus reduced the binding of 3' hExo by two orders of magnitude. 3' hExo binds the stem-loop independently as well as simultaneously with SLBP. When bound together with SLBP, 3' hExo degradation of the histone mRNA is blocked.

    The common set of 3' ends shared by the replication-dependent histone mRNAs is essential for their regulation. Precise pre-mRNA processing is required to generate a class of transcripts in which the RNA-binding proteins required for post-transcriptional regulation are strictly positioned with respect to the 3' end. Cleavage site precision is achieved by the base-pairing of U7 snRNA with the histone pre-mRNA HDE within a very narrowly defined window downstream of the cleavage site (Scharl and Steitz 1994, 1996; Cho et al. 1995). Scharl and Steitz (1994) proposed that the HDE:U7 snRNP interaction functions as a "molecular ruler" to precisely determine the site of cleavage. The binding of U7 snRNP to the pre-mRNA is necessary, and in some cases sufficient, for the recruitment of a functional 3' processing complex, as well as for cleavage site precision. The use of RNA:RNA base-pairing in trans to confer catalytic specificity can be seen in a wide variety of processes, including pre-mRNA splicing, translation, snoRNA-directed rRNA processing and modification, and the action of miRNA and siRNAs.

    A comparable set of rigid constraints is not observed in the 3' processing of polyadenylated mRNAs. Poly(A) sites exhibit a relatively wide range of both sequence and spatial variability. As depicted in Figure 1A, the AAUAAA hexamer resides within a window of 20 nt upstream of the cleavage site. Although the AAUAAA hexamer is the most conserved element among mammalian poly(A) sites, only 53% of human poly(A) sites contain this sequence. An AUUAAA hexamer is present in 17% of human poly(A) sites, and the remaining 30% contain other hexamer variants (Tian et al. 2005). The position of the downstream element, which lacks an identified consensus, varies within an 30-nt window with respect to the cleavage site. Although flexible, the spatial arrangement of these sequences appears to be more constrained than those of the UGUA elements, which vary not only in their position relative to the cleavage site, but in copy number as well (Brown and Gilmartin 2003; Hu et al. 2005; Venkataraman et al. 2005). The variability in poly(A) site organization is reflected in the variability of cleavage site choice. Bioinformatic studies indicate that cleavage site choice within a poly(A) site is inherently imprecise (Pauws et al. 2001; Tian et al. 2005). In addition to cleavage site heterogeneity, alternative selection among different poly(A) sites (that range over distances in the kilobase range) is observed within the majority of human pre-mRNAs (Iseli et al. 2002; Tian et al. 2005). The consequences of alternative poly(A) site choice may impact the protein coding capacity of the message, as well as its localization, translation efficiency, and stability (Edwalds-Gilbert et al. 1997). Metazoan 3' UTRs have been found to function as repositories for a wide variety of post-transcriptional regulatory elements (recognized by both proteins and miRNAs) that may function in a developmental or tissue-specific manner (Kuersten and Goodwin 2003; Xie et al. 2005). While there are cases of alternative 3' processing of histone genes, the only choice observed is between the nonadenylated processing site and a downstream poly(A) site (Dominski and Marzluff 1999).

    An intrinsic difference between the 3' processing of replication-dependent histone mRNAs and polyadenylated mRNAs, as observed by Birnstiel et al. (1985), is therefore the precision and uniformity of the former class and the diversity and flexibility of the latter. The metazoan replication-dependent histone mRNA 3' processing machinery appears to have evolved to support the coordinate post-transcriptional regulation of an entire class of transcripts through its ability to generate a common set of 3' ends. In contrast, the polyadenylation machinery appears to have evolved to support a diverse array of developmental and tissue-specific post-transcriptional regulatory responses. Thus while the evidence indicates that the 3' processing complexes of these two classes of transcripts share a common catalytic core, the mechanisms that have been developed to specify the site of catalysis are distinct. The mechanisms for the recruitment of the catalytic core to the pre-mRNA are not restricted to the factors bound at the 3' ends, but also involve both splicing and transcription. Whereas poly(A) site processing is clearly coupled to splicing (Minviella-Sebastia and Keller 1999), the 3' processing of metazoan replication-dependent histone pre-mRNAs (which uniformly lack introns) appears to be inherently incompatible with splicing (Pandey et al. 1990). Furthermore, whereas poly(A) site processing is functionally coupled to transcription (Adamson et al. 2005), this does not appear to be the case for histone mRNA 3' processing (Adamson and Price 2003).

    A common catalytic core

    Kolev and Steitz (2005) have identified all five subunits of CPSF, two subunits of CstF, and symplekin as constituents of a factor termed HLF (heat labile factor) that is required for histone 3' processing in vitro. CPSF and symplekin not only function in poly(A) site processing in the nucleus (Zhao et al. 1999a,b; Xing et al. 2004), they are also required for developmentally regulated poly(A) addition in the cytoplasm (Barnard et al. 2004). Dominski et al. (2005a) have captured the 73-kDa subunit of CPSF at the site of cleavage of a mouse H2A pre-mRNA by UV cross-linking. Their evidence suggests that CPSF-73 functions as both the endonuclease that cleaves the nascent RNA and as the 5'-to-3' exonuclease that initiates the degradation of the downstream cleavage product. The presence of CPSF-73 at the histone pre-mRNA 3' cleavage site provides new support for the previously proposed role of this protein as the poly(A) site endonuclease. An endonucleolytic function for CPSF-73 was initially suggested by the identification of a domain shared with a group of predicted or proven nucleases, termed the -CASP domain (Callebaut et al. 2002). The work of Ryan et al. (2004) provided additional support for CPSF-73 as the poly(A) site endonuclease, but conclusive evidence has remained elusive.

    Several key questions remain to be addressed. The histone mRNA 3' processing and polyadenylation complexes clearly share a core set of proteins that likely encompasses the endonuclease, but the composition of this core remains to be defined. Symplekin, a proposed assembly/scaffolding factor (Takagaki and Manley 2000), is clearly an essential component, and CPSF-73 is very likely to be as well, but the additional CPSF and CstF subunits identified as constituents of HLF are as yet guilty by association. Thus the complete complement of factors required for 3' processing has yet to be conclusively identified for either metazoan histone mRNAs or for polyadenylated mRNAs. Furthermore, the absence of nonpolyadenylated histone mRNAs in all organisms other than metazoans prompts the question of what mechanisms these organisms have evolved to regulate histone gene expression. The apparent conservation of a 5'-to-3' exonuclease activity along with an endonuclease activity in the processing of these two classes of transcripts raises the question as to whether it may have a role in transcription termination. Exonucleases have been proposed to contribute to RNA polymerase II transcription termination in both yeast and humans (Kim et al. 2004; West et al. 2004), and transcription termination has been shown to be coupled to the 3' processing of both histone and polyadenylated mRNAs (Chodchoy et al. 1991; Proudfoot et al. 2002).

    Taken together, the reports of Kolev and Steitz (2005) and Dominski et al. (2005a) demonstrate that while the 3' processing of metazoan replication-dependent histone genes is unique in its outcome, its mechanism is not. These findings indicate that the 3' processing machinery of all eukaryotic mRNA likely share a common evolutionary origin. Furthermore, the divergence of histone and poly(A) site processing may simply reflect a selection for precision versus flexibility. The essential difference between the two classes of mRNAs appears to be that they have evolved distinct mechanisms for the recruitment and activation of a shared catalytic activity. Which just goes to show, it's how you get there that makes all the difference in the end.

    Acknowledgments

    I thank Zbigniew Dominski, Xiao-cui Yang, and Bill Marzluff for sharing their work prior to publication.

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