Mille viae in eukaryotic mRNA decapping

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An in vivo control map for the eukaryotic mRNA translation machinery

Rate control analysis defines the in vivo control map governing yeast protein synthesis and generates an extensively parameterized digital model of the translation pathway. Among other non‐intuitive outcomes, translation demonstrates a high degree of functional modularity and comprises a non‐stoichiometric combination of proteins manifesting functional convergence on a shared maximal translation rate. In exponentially growing cells, polypeptide elongation (eEF1A, eEF2, and eEF3) exerts the strongest control. The two other strong control points are recruitment of mRNA and tRNA_i to the 40S ribosomal subunit (eIF4F and eIF2) and termination (eRF1; Dbp5). In contrast, factors that are found to promote mRNA scanning efficiency on a longer than‐average 5′untranslated region (eIF1, eIF1A, Ded1, eIF2B, eIF3, and eIF5) exceed the levels required for maximal control. This is expected to allow the cell to minimize scanning transition times, particularly for longer 5′UTRs. The analysis reveals these and other collective adaptations of control shared across the factors, as well as features that reflect functional modularity and system robustness. Remarkably, gene duplication is implicated in the fine control of cellular protein synthesis.     

New insights into the control of mRNA decapping

mRNA decapping irreversibly targets mRNAs for fast decay. Cap removal is catalyzed by decapping protein Dcp2 but also requires Dcp1. Recently, two groups have provided a first glimpse of the regulation mechanism of this crucial step in gene expression. Resolution of the yeast Dcp2 structure has enabled identification of the residues that are important for its interaction with Dcp1. However, the human decapping machinery seems to be more complex because a third component, Hedls, is required for a functional Dcp1-Dcp2 interaction.     

Crystal structure of Dcp1p and its functional implications in mRNA decapping

A major pathway of eukaryotic mRNA turnover begins with deadenylation, followed by decapping and 5'-->3' exonucleolytic degradation. A critical step in this pathway is decapping, which is carried out by an enzyme composed of Dcp1p and Dcp2p. The crystal structure of Dcp1p shows that it markedly resembles the EVH1 family of protein domains. Comparison of the proline-rich sequence (PRS)-binding sites in this family of proteins with Dcp1p indicates that it belongs to a novel class of EVH1 domains. Mapping of the sequence conservation on the molecular surface of Dcp1p reveals two prominent sites. One of these is required for the function of the Dcp1p-Dcp2p complex, and the other, corresponding to the PRS-binding site of EVH1 domains, is probably a binding site for decapping regulatory proteins. Moreover, a conserved hydrophobic patch is shown to be critical for decapping.     

Structural analyses of the pre‐mRNA splicing machinery

Pre‐mRNA splicing is a critical event in the gene expression pathway of all eukaryotes. The splicing reaction is catalyzed by the spliceosome, a huge protein‐RNA complex that contains five snRNAs and hundreds of different protein factors. Understanding the structure of this large molecular machinery is critical for understanding its function. Although the highly dynamic nature of the spliceosome, in both composition and conformation, posed daunting challenges to structural studies, there has been significant recent progress on structural analyses of the splicing machinery, using electron microscopy, crystallography, and nuclear magnetic resonance. This review discusses key recent findings in the structural analyses of the spliceosome and its components and how these findings advance our understanding of the function of the splicing machinery.     

Structural/functional homology between the bacterial and eukaryotic cytoskeletons

Structural proteins are now known to be as necessary for controlling cell division and cell shape in prokaryotes as they are in eukaryotes. Bacterial ParM and MreB not only have atomic structures that resemble eukaryotic actin and form similar filaments, but they are also equivalent in function: the assembly of ParM drives intracellular motility and MreB maintains the shape of the cell. FtsZ resembles tubulin in structure and in its dynamic assembly, and is similarly controlled by accessory proteins. Bacterial MinD and eukaryotic dynamin appear to have similar functions in membrane control. In dividing eukaryotic organelles of bacterial origin, bacterial and eukaryotic proteins work together.     

Structural similarities and functional diversity of eukaryotic discoidin-like domains

The discoidin domain is a approximately 150 amino acid motif common in both eukaryotic and prokaryotic proteins. It is found in a variety of extracellular, intracellular and transmembrane multidomain proteins characterized by a considerable functional diversity, mostly involved in developmental processes. The biological role of the domain depends on its interactions with different molecules, including growth factors, phospholipids and lipids, galactose or its derivatives, and collagen. The conservation of the motif, as well as the serious physiological consequences of discoidin domain disorders underscore the importance of the fold, while the ability to accommodate such an extraordinarily broad range of ligand molecules makes it a fascinating research target. In present review we characterize the distinctive features of discoidin domains and briefly outline the biological role of this module in various eukaryotic proteins.     

mRNA decapping activities and their biological roles

The 5′ cap structure of eukaryotic mRNAs is significant for a variety of cellular events and also serves to protect mRNAs from premature degradation. Analysis of mRNA decay in Saccharomyces cerevisiae has shown that removal of the 5′ cap structure is a key step in the turnover of many yeast mRNAs, and that this decapping is carried out by Dcplp. In addition to the yeast decapping enzyme, other activities that can cleave the 5′ cap structure have been described. These include two mammalian enzymes and two viral activities that cleave cellular mRNA cap structures as part of their life cycle. Here we review these various decapping activities and discuss their biological roles.     

Identification of functional domains in Arabidopsis thaliana mRNA decapping enzyme (AtDcp2)

The Arabidopsis thaliana decapping enzyme (AtDcp2) was characterized by bioinformatics analysis and by biochemical studies of the enzyme and mutants produced by recombinant expression. Three functionally significant regions were detected: (i) a highly disordered C-terminal region with a putative PSD-95, Discs-large, ZO-1 (PDZ) domain-binding motif, (ii) a conserved Nudix box constituting the putative active site and (iii) a putative RNA binding domain consisting of the conserved Box B and a preceding loop region. Mutation of the putative PDZ domain-binding motif improved the stability of recombinant AtDcp2 and secondary mutants expressed in Escherichia coli. Such recombinant AtDcp2 specifically hydrolysed capped mRNA to produce 7-methyl GDP and decapped RNA. AtDcp2 activity was Mn²⁺- or Mg²⁺-dependent and was inhibited by the product 7-methyl GDP. Mutation of the conserved glutamate-154 and glutamate-158 in the Nudix box reduced AtDcp2 activity up to 400-fold and showed that AtDcp2 employs the catalytic mechanism conserved amongst Nudix hydrolases. Unlike many Nudix hydrolases, AtDcp2 is refractory to inhibition by fluoride ions. Decapping was dependent on binding to the mRNA moiety rather than to the 7-methyl diguanosine triphosphate cap of the substrate. Mutational analysis of the putative RNA-binding domain confirmed the functional significance of an 11-residue loop region and the conserved Box B.     

The eukaryotic mRNA decapping protein Dcp1 interacts physically and functionally with the eIF4F translation initiation complex

Dcp1 plays a key role in the mRNA decay process in Saccharomyces cerevisiae, cleaving off the 5' cap to leave an end susceptible to exonucleolytic degradation. The eukaryotic initiation factor complex eIF4F, which in yeast contains the core components eIF4E and eIF4G, uses the cap as a binding site, serving as an initial point of assembly for the translation apparatus, and also binds the poly(A) binding protein Pab1. We show that Dcp1 binds to eIF4G and Pab1 as free proteins, as well as to the complex eIF4E-eIF4G-Pab1. Dcp1 interacts with the N-terminal region of eIF4G but does not compete significantly with eIF4E or Pab1 for binding to eIF4G. Most importantly, eIF4G acts as a function-enhancing recruitment factor for Dcp1. However, eIF4E blocks this effect as a component of the high affinity cap-binding complex eIF4E-eIF4G. Indeed, cooperative enhancement of the eIF4E-cap interaction stabilizes yeast mRNAs in vivo. These data on interactions at the interface between translation and mRNA decay suggest how events at the 5' cap and 3' poly(A) tail might be coupled.     

Functional analysis of mRNA scavenger decapping enzymes

Eukaryotic cells primarily utilize exoribonucleases and decapping enzymes to degrade their mRNA. Two major decapping enzymes have been identified. The hDcp2 protein catalyzes hydrolysis of the 5' cap linked to an RNA moiety, whereas the scavenger decapping enzyme, DcpS, functions on a cap structure lacking the RNA moiety. DcpS is a member of the histidine triad (HIT) family of hydrolases and catalyzes the cleavage of m7GpppN. HIT proteins are homodimeric and contain two conserved 100-amino-acid HIT fold domains with independent active sites that are each sufficient to bind and hydrolyze cognate substrates. We carried out a functional characterization of the DcpS enzyme and demonstrate that unlike previously described HIT proteins, DcpS is a modular protein that requires both the core HIT fold at the carboxyl-terminus and sequences at the amino-terminus of the protein for cap binding and hydrolysis. Interestingly, DcpS can efficiently compete for and hydrolyze the cap structure even in the presence of excess eIF4E, implying that DcpS could function to alleviate the accumulation of complexes between eIF4E and cap structure that would otherwise accumulate following mRNA decay. Using immunofluorescence microscopy, we demonstrate that DcpS is predominantly a nuclear protein, with low levels of detected protein in the cytoplasm. Furthermore, analysis of the endogenous hDcp2 protein reveals that in addition to the cytoplasmic foci, it is also present in the nucleus. These data reveal that both decapping enzymes are contained in the nuclear compartment, indicating that they may fulfill a greater function in the nucleus than previously appreciated.     

Structural and functional properties of calmodulin from the eukaryotic microorganism Dictyostelium discoideum

Calmodulin was purified from the eukaryotic microorganism Dictyostelium discoideum and characterized in terms of its nearly complete primary structure and quantitative activator activity. The strategy for amino acid sequence analysis took advantage of the highly conserved structure of calmodulin and employed a new procedure for limited cleavage of calmodulin that uses a protease from mouse submaxillary gland. Fourteen amino acid sequence differences between Dictyostelium and bovine calmodulin were identified unequivocally, as well as an unmethylated lysine at residue 115 instead of N epsilon, N epsilon, N epsilon-trimethyllysine. Seven of the amino acid substitutions in Dictyostelium calmodulin are novel in that the residues at these positions are invariant in all calmodulin sequences previously examined, most notably an additional residue at the carboxy terminus. Comparison of the Dictyostelium calmodulin sequence with other calmodulin sequences shows that the region with the greatest extended sequence identity includes parts of the first and second structural domains and the interdomain region between domains 1 and 2. Dictyostelium calmodulin activated bovine brain cyclic nucleotide phosphodiesterase in a manner indistinguishable from that of bovine brain calmodulin. However, Dictyostelium calmodulin activated pea NAD kinase to a maximal level 4.6-fold greater than that produced by bovine brain calmodulin. This functional difference demonstrates the potential biological importance of the limited number of amino acid sequence differences between Dictyostelium calmodulin and other calmodulins and provides further insight into the structure, function, and evolution of the calmodulin family of proteins.