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A Cap for All Occasions Gabriele Varani View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Minireview 855 A cap for all occasions Gabriele Varani The 5¢ end of each polymerase II transcript is capped by a are modified and recognized is therefore central to methylated guanosine triphosphate. The cap earmarks the understanding how gene expression is regulated post- mRNA for subsequent processing and nucleocytoplasmic transcriptionally. transport, protects the mRNA from degradation and promotes efficient initiation of protein synthesis. The Capping enzymes recently solved structures of capping enzymes and The modification of the mature 5′ end of an mRNA cap–protein complexes shed light on how the 5¢ ends of occurs very early during transcription; in Drosophila, the mRNAs are modified, and reveals the mechanisms by majority of mRNA strands that have reached 30 nucleo- which the cap is recognized and how it functions in a tides in length are already capped [1]. Capping occurs in diverse range of processes. three steps: removal of the first phosphate by RNA tri- phosphatase to generate diphosphate-terminated pre- Address: MRC Laboratory of Molecular Biology, Hills Road, mRNA; capping with a GMP nucleoside mediated by Cambridge CB2 2QH, UK. RNA guanylyltransferase — a reaction which occurs E-mail: [email protected] through an enzyme–GMP adduct intermediate; and N7- Structure 15 July 1997, 5:855–858 methylation by RNA (guanine-7)methyltransferase [2]. http://biomednet.com/elecref/096921260050855 Although all mono- or diphosphate terminated RNAs are good substrates for guanylyl transfer, only polymerase II © Current Biology Ltd ISSN 0969-2126 transcripts are capped in vivo. This specificity is probably due to interactions between the capping and the tran- Post-transcriptional regulation of gene expression in- scription machineries. volves the recognition by protein factors of specific sig- nals located throughout messenger RNAs. These signals The crystal structure of the Chlorella virus guanylyltrans- include distinctive terminal features, namely the N7- ferase enzyme, PBCV-1, has remarkable similarities with methylated guanosine triphosphate cap at the 5′ end and DNA and RNA ligases. Sequence motifs that are con- the polyadenosine tail at the 3′ end. The cap, which has served in cellular and viral capping enzymes and in DNA the same structure in all transcripts generated by poly- and RNA ligases cluster around the nucleotide-binding merase II, directs pre-mRNAs to the processing and site in the PBCV-1 and T7 DNA ligase structures [3,4]. transport pathways in the cell nucleus and regulates both The GTP–PBCV-1 and ATP–ligase interactions involve mRNA turnover and the initiation of translation (Fig- similar contacts between the nucleotide and residues ure 1). Understanding how the 5′ ends of mature mRNAs from these conserved motifs, whereas non-conserved Figure 1 (a) Co-transcriptional (b) Pre-mRNA (c) Nucleocytoplasmic (d) mRNA (e) Translational capping splicing transport turnover initiation ? Capping α CBC Importin Pab1p Pab1p enzymes CBC AAAAA AAAAA Pre-mRNA Pre-mRNA ? mRNA mRNA mRNP ? eIF-4G Pol II SnRNPs eIF-4E ne Nuclear membra Decapping enzymes Importin β 5′–3′ exonucleases DNA CBC Ribosome mRNA Multiple roles of the 5′ mRNA cap (red sphere) during gene expression in eukaryotes. CBC is the cap-binding complex made up of two subunits; Pab1p is the cytoplasmic poly(A)-binding protein; eIF-4E and eIF-4G are translation initiation factors. For details of each process (a–e) see text. 856 Structure 1997, Vol 5 No 7 amino acids in the nucleotide-binding site define the Cap-modifying enzymes specificity of each enzyme [4]. Residues of the conserved Specific classes of capped RNAs are subjected to further motifs line a groove between two structural domains modifications. For example, the first nucleotide of (Figure 2). This cleft, which is probably the site of sub- certain viral mRNAs is methylated at the ribose 2′-OH, strate binding, is much narrower in the capping enzyme and the base N2 of the cap guanine is hypermethylated than in the ligase; this probably reflects their different in spliceosomal U snRNAs. A cap-specific 2′-O-methyl- specificities for single-stranded RNA versus double- transferase, VP39, from vaccinia virus recognizes the stranded DNA substrates. canonical N7-methyl guanine cap structure and methy- lates the ribose hydroxyl group of the first transcribed The crystal structure of PBCV-1 guanylyltransferase re- nucleotide. The VP39 structure is homologous to other vealed that it has two distinct conformations [4]. In the methyltransferases [5], and is superficially similar to crystal structure of guanylyltransferase, the deep cleft sepa- RNA-binding proteins [6]. The fold of VP39 is distinct rating the two domains is ‘closed’ in one molecule in the from typical αβ RNA-binding proteins, however, reveal- unit cell by a rigid displacement of domain 2 (Figure 2). ing a novel architecture for RNA recognition. The β Upon soaking the crystals with divalent cations, a GMP– sheet of VP39 is not exposed to solvent as in RNA- lysine adduct is formed, in which GMP binds within the binding proteins but buried in the protein core, whereas signature KXDG sequence [2] of the ‘closed’ but not of the the exposed surface is formed by loops and α helices ‘open’ conformation. A strong electron density for GTP is (Figure 3) [5]. observed only for the ‘closed’ conformation, suggesting that the catalytically active ‘closed’ structure is induced by In the VP39–cap complex, the N7-methylated guanosine GTP binding [4]. As the ‘closed’ form is not wide enough occupies a deep aromatic cleft and is sandwiched be- to accommodate the RNA substrate, both conformations tween the sidechains of Phe180 and Tyr22 (Figure 3) [7]. are probably important for catalysis. These stacking interactions are likely to contribute to the specific recognition of the methylated base through en- hanced π–π interactions, as observed for small aromatic Figure 2 molecules. Thus, the unique electronic structure of the methylated base contributes to specific recognition of the mRNA cap and (probably) of alkylated bases by DNA excision repair enzymes. Figure 3 Superposition of ‘closed’ (yellow) and ‘open’ (green) conformations of Structure of the vaccinia virus VP39 methyltransferase–cap complex, the chlorella virus guanylyltransferase, with GTP (purple) bound to the highlighting the stacking interactions between the modified guanine ‘closed’ state of the enzyme [4]. (purple) and essential aromatic amino acids (yellow) [7]. Minireview A cap for all occasions Varani 857 Pre-mRNA splicing default pathway for mRNA degradation, decapping occurs The role of the cap in mRNA splicing is mediated by a only after de-adenylation. Yeast strains lacking Pab1p (the nuclear cap-binding complex (CBC; Figure 1b), which is cytoplasmic poly(A)-binding protein) are decapped prior composed of two tightly associated proteins, CBP20 and to de-adenylation [12], suggesting that Pab1p protects the CBP80 [8,9]. CBP20 is a member of the RNP superfamily cap from decapping enzymes (Figure 1d). of RNA-binding proteins [6], but CBP80 has no clear homology with other proteins. CBC (but not CBP80 or Translational initiation CBP20 in isolation) interacts specifically with the mono- The rate-limiting step in the initiation of protein synthesis methylated guanosine cap, both in vitro and in vivo [8,9], requires engagement between the 40S subunit of the ribo- and promotes an efficient interaction between U1 snRNP some and the mRNA [14]. The cap plays a critical role in and the 5′ splice site. This interaction between U1 snRNP this process, through the cytoplasmic cap-binding com- and the 5′ splice site is one of the earliest steps in spliceo- plex eIF-4F, again in synergy with the poly(A) tail (Figure some assembly [8] and is necessary to define the 5′ splice 1e) [15]. The cytoplasmic cap-binding protein, eIF-4E, site of the very first intron. This essential function of the specifically recognizes the cap and another component of CBC–cap complex and (probably) the mechanism by which eIF-4F, the ‘adapter’ protein eIF-4G [14]. In yeast, eIF- it promotes mRNA splicing are conserved between yeast 4G also interacts with Pab1p [15,16], suggesting that stim- and mammals, but the identity of the factor(s) that ulation of recruitment of the 40S subunit by the cap and mediate the interaction between CBC and the splicing the poly(A) tail are mediated by the same mechanism, as machinery and the structural basis for cap–CBC recogni- illustrated in Figure 1e. tion remain to be established. The requirement for the cap in translation is not Nucleocytoplasmic export and mRNA turnover absolute. For example, internal ribosome entry sites of The CBC associates with nascent transcripts and remains some cellular messenger and picornaviral RNAs bypass bound to the cap throughout the splicing cycle, even the cap requirement. Those mRNAs that have a low when the mature mRNA leaves the spliceosome [8]. At affinity for eIF-4E, such as heat-shock gene transcripts, this stage, mRNA has to be packaged into a ribonucleo- and mRNAs that are expressed during early develop- protein particle (mRNP) for export to the cytoplasm ment, are likely to rely on the poly(A) tail rather than the through the nucleopore complex [10]. The cap–CBC com- cap for translational initiation [16]. On the other hand, plex is critical for the export of spliceosomal components the translation of mRNAs in which very stable secondary (U snRNAs). The complex also plays a significant but structures are present between the cap and the initiation nonessential role in mRNA export. Presumably, mRNPs codon is strongly dependent on the eIF-4E–cap inter- contain multiple, redundant, export signals [9,10]. In yeast action. The activity of eIF-4E is down-regulated by cells and Xenopus oocytes, the CBC forms an abundant protein inhibitors (4E-BPs) that block the eIF-4E–eIF- cap-dependent complex with a component of the nuclear 4G interaction.
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