A major pathway of mRNA turnover in eukaryotic cells initiates with deadenylation, leading to mRNA decapping and subsequent 5′ to 3′ exonuclease digestion. We show that a highly conserved member of the DEAD box family of helicases, Dhh1p, stimulates mRNA decapping in yeast. In dhh1Δ mutants, mRNAs accumulate as deadenylated, capped species. Dhh1p's effects on decapping only occur on normal messages as nonsense-mediated decay still occurs in dhh1Δ mutants. The role of Dhh1p in decapping appears to be direct, as Dhh1p physically interacts with several proteins involved in mRNA decapping including the decapping enzyme Dcp1p, as well as Lsm1p and Pat1p/Mrt1p, which function to enhance the decapping rate. Additional observations suggest Dhh1p functions to coordinate distinct steps in mRNA function and decay. Dhh1p also associates with Pop2p, a subunit of the mRNA deadenylase. In addition, genetic phenotypes suggest that Dhh1p also has a second biological function. Interestingly, Dhh1p homologs in others species function in maternal mRNA storage. This provides a novel link between the mechanisms of decapping and maternal mRNA translational repression.

The Upf1 protein in yeast has been implicated in the modulation of efficient translation termination as well as in the accelerated turnover of mRNAs containing premature stop codons, a phenomenon called nonsense-mediated mRNA decay (NMD). A human homolog of the yeast UPF1, termed HUpf1/RENT1, has also been identified. The HUpf1 has also been shown to play a role in NMD in mammalian cells. Comparison of the yeast and human UPF1 proteins demonstrated that the amino terminal cysteine/histidine-rich region and the region comprising the domains that define this protein as a superfamily group I helicase have been conserved. The yeast Upf1p demonstrates RNA-dependent ATPase and 5′ → 3′ helicase activities. In this paper, we report the expression, purification, and characterization of the activities of the human Upf1 protein. We demonstrate that human Upf1 protein displays a nucleic-acid-dependent ATPase activity and a 5′ → 3′ helicase activity. Furthermore, human Upf1 is an RNA-binding protein whose RNA-binding activity is modulated by ATP. Taken together, these results indicate that the activities of the Upf1 protein are conserved across species, reflecting the conservation of function of this protein throughout evolution.

The human coronavirus 229E replicase gene encodes a protein, p66HEL, that contains a putative zinc finger structure linked to a putative superfamily (SF) 1 helicase. A histidine-tagged form of this protein, HEL, was expressed using baculovirus vectors in insect cells. The purified recombinant protein had in vitro ATPase activity that was strongly stimulated by poly(U), poly(dT), poly(C), and poly(dA), but not by poly(G). The recombinant protein also had both RNA and DNA duplex-unwinding activities with 5′-to-3′ polarity. The DNA helicase activity of the enzyme preferentially unwound 5′-oligopyrimidine-tailed, partial-duplex substrates and required a tail length of at least 10 nucleotides for effective unwinding. The combined data suggest that the coronaviral SF1 helicase functionally differs from the previously characterized RNA virus SF2 helicases.

Translation termination is the final step that completes the synthesis of a polypeptide. Premature translation termination by introduction of a nonsense mutation leads to the synthesis of a truncated protein. We report the identification and characterization of the product of the MTT1 gene, a helicase belonging to the Upf1-like family of helicases that is involved in modulating translation termination. MTT1 is homologous to UPF1, a factor previously shown to function in both mRNA turnover and translation termination. Overexpression of MTT1 induced a nonsense suppression phenotype in a wild-type yeast strain. Nonsense suppression is apparently not due to induction of [PSI+], even though cooverexpression of HSP104 alleviated the nonsense suppression phenotype observed in cells overexpressing MTT1, suggesting a more direct role of Hsp104p in the translation termination process. The MTT1 gene product was shown to interact with translation termination factors and is localized to polysomes. Taken together, these results indicate that at least two members of a family of RNA helicases modulate translation termination efficiency in cells.

Eukaryotic translation initiation factor 4A (eIF4A) has been proposed to use the energy of ATP hydrolysis to remove RNA structure in the 5′ untranslated region (UTR) of mRNAs, helping the 43S ribosomal complex bind to an mRNA and scan to find the 5′-most AUG initiator codon. We have examined the effect of changing the atomic composition and length of single-stranded oligonucleotides on binding to eIF4A and on stimulation of its ATPase activity once bound. Substitution of 2′-OH groups with 2′-H or 2′-OCH3 groups reduces ATPase stimulation at least 100-fold, to background levels, without significantly affecting oligonucleotide affinity. These effects suggest that 2′-OH groups participate in an eIF4A conformational change that occurs subsequent to oligonucleotide binding and is required for ATPase stimulation. Replacing nonbridging oxygen atoms in phosphodiester linkages with sulfur atoms to make phosphorothioate linkages has no significant effect on stimulation, while substantially increasing affinity. Extending the length of an RNA oligonucleotide from 4 to ∼15 nt gradually increases oligonucleotide affinity and ATPase stimulation. Consistent with this observation, the increase in affinity and stimulation provided by phosphorothioate linkages and 2′-OH groups is proportional to the number of these groups present within larger oligonucleotides. Further, changing the position of blocks of phosphorothioate linkages or 2′-OH groups within a larger oligonucleotide does not affect affinity and has only a small effect on stimulation. These observations suggest that numerous interactions between the oligonucleotide and eIF4A contribute individually to binding and ATPase stimulation. Nevertheless, significant stimulation is observed with as few as four RNA residues. These properties may allow eIF4A to operate within regions of 5′ UTRs containing only short stretches of exposed single-stranded RNA. As stimulation increases when longer stretches of single-stranded RNA are available, it is possible that the accessibility of single-stranded RNA in a 5′ UTR influences translation efficiency.

Hepatitis C virus (HCV) nonstructural protein 3 (NS3) has been shown to possess protease and helicase activities and has also been demonstrated to spontaneously associate with nonstructural protein NS4A (NS4A) to form a stable complex. Previous attempts to produce the NS3/NS4A complex in recombinant baculovirus resulted in a protein complex that aggregated and precipitated in the absence of nonionic detergent and high salt. A single-chain form of the NS3/NS4A complex (His-NS4A21–32–GSGS-NS33–631) was constructed in which the NS4A core peptide is fused to the N-terminus of the NS3 protease domain as previously described (Taremi et al., 1998). This protein contains a histidine tagged NS4A peptide (a.a. 21–32) fused to the full-length NS3 (a.a. 3–631) through a flexible tetra amino acid linker. The recombinant protein was expressed to high levels in Escherichia coli, purified to homogeneity, and examined for NTPase, nucleic acid unwinding, and proteolytic activities. The single-chain recombinant NS3-NS4A protein possesses physiological properties equivalent to those of the NS3/NS4A complex except that this novel construct is stable, soluble and sixfold to sevenfold more active in unwinding duplex RNA. Comparison of the helicase activity of the single-chain recombinant NS3-NS4A with that of the full-length NS3 (without NS4A) and that of the helicase domain alone suggested that the presence of the protease domain and at least the NS4A core peptide are required for optimal unwinding activity.

The nonsense-mediated mRNA decay pathway decreases the abundance of mRNAs that contain premature termination codons and prevents suppression of nonsense alleles. The UPF1 gene in the yeast Saccharomyces cerevisiae was shown to be a trans-acting factor in this decay pathway. The Upf1p demonstrates RNA-dependent ATPase, RNA helicase, and RNA binding activities. The results presented here investigate the binding affinity of the Upf1p for ATP and the consequences of ATP binding on its affinity for RNA. The results demonstrate that the Upf1p binds ATP in the absence of RNA. Consistent with this result, the TR800AA mutant form of the Upf1p still bound ATP, although it does not bind RNA. ATP binding also modulates the affinity of Upf1p for RNA. The RNA binding activity of the DE572AA mutant form of the Upf1p, which lacks ATPase activity, still bound ATP as efficiently as the wild-type Upf1p and destabilized the Upf1p–RNA complex. Similarly, ATPγS, a nonhydrolyzable analogue of ATP, interacted with Upf1p and promoted disassociation of the Upf1p–RNA complex. The conserved lysine residue (K436) in the helicase motif Ia in the Upf1p was shown to be critical for ATP binding. Taken together, these findings formally prove that ATP can bind Upf1p in the absence of RNA and that this interaction has consequences on the formation of the Upf1p–RNA complex. Further, the results support the genetic evidence indicating that ATP binding is important for the Upf1p to increase the translation termination efficiency at a nonsense codon. Based on these findings, a model describing how the Upf1p functions in modulating translation and turnover and the potential insights into the mechanism of the Upf1p helicase will be discussed.