Vera P. Pisareva

The Role of ABCE1 in Eukaryotic Posttermination Ribosomal Recycling

After termination, eukaryotic 80S ribosomes remain associated with mRNA, P-site deacylated tRNA, and release factor eRF1 and must be recycled by dissociating these ligands and separating ribosomes into subunits. Although recycling of eukaryotic posttermination complexes (post-TCs) can be mediated by initiation factors eIF3, eIF1, and eIF1A (Pisarev et al., 2007), this energy-free mechanism can function only in a narrow range of low Mg(2+) concentrations. Here, we report that ABCE1, a conserved and essential member of the ATP-binding cassette (ABC) family of proteins, promotes eukaryotic ribosomal recycling over a wide range of Mg(2+) concentrations. ABCE1 dissociates post-TCs into free 60S subunits and mRNA- and tRNA-bound 40S subunits. It can hydrolyze ATP, GTP, UTP, and CTP. NTP hydrolysis by ABCE1 is stimulated by post-TCs and is required for its recycling activity. Importantly, ABCE1 dissociates only post-TCs obtained with eRF1/eRF3 (or eRF1 alone), but not post-TCs obtained with puromycin in eRF1s absence.

Dissociation by Pelota, Hbs1 and ABCE1 of mammalian vacant 80S ribosomes and stalled elongation complexes.

No‐go decay (NGD) and non‐stop decay (NSD) are eukaryotic surveillance mechanisms that target mRNAs on which elongation complexes (ECs) are stalled by, for example, stable secondary structures (NGD) or due to the absence of a stop codon (NSD). Two interacting proteins Dom34(yeast)/Pelota(mammals) and Hbs1, which are paralogues of eRF1 and eRF3, are implicated in these processes. Dom34/Hbs1 were shown to promote dissociation of stalled ECs and release of intact peptidyl‐tRNA. Using an in vitro reconstitution approach, we investigated the activities of mammalian Pelota/Hbs1 and report that Pelota/Hbs1 also induced dissociation of ECs and release of peptidyl‐tRNA, but only in the presence of ABCE1. Whereas Pelota and ABCE1 were essential, Hbs1 had a stimulatory effect. Importantly, ABCE1/Pelota/Hbs1 dissociated ECs containing only a limited number of mRNA nucleotides downstream of the P‐site, which suggests that ABCE1/Pelota/Hbs1 would disassemble NSD complexes stalled at the 3′‐end, but not pre‐cleavage NGD complexes stalled in the middle of mRNA. ABCE1/Pelota/Hbs1 also dissociated vacant 80S ribosomes, which stimulated 48S complex formation, suggesting that Pelota/Hbs1 have an additional role outside of NGD.

Structural insights into eRF3 and stop codon recognition by eRF1.

Eukaryotic translation termination is mediated by two interacting release factors, eRF1 and eRF3, which act cooperatively to ensure efficient stop codon recognition and fast polypeptide release. The crystal structures of human and Schizosaccharomyces pombe full-length eRF1 in complex with eRF3 lacking the GTPase domain revealed details of the interaction between these two factors and marked conformational changes in eRF1 that occur upon binding to eRF3, leading eRF1 to resemble a tRNA molecule. Small-angle X-ray scattering analysis of the eRF1/eRF3/GTP complex suggested that eRF1s M domain contacts eRF3s GTPase domain. Consistently, mutation of Arg192, which is predicted to come in close contact with the switch regions of eRF3, revealed its important role for eRF1s stimulatory effect on eRF3s GTPase activity. An ATP molecule used as a crystallization additive was bound in eRF1s putative decoding area. Mutational analysis of the ATP-binding site shed light on the mechanism of stop codon recognition by eRF1.

Kinetic Analysis of Interaction of Eukaryotic Release Factor 3 with Guanine Nucleotides

Eukaryotic translation termination is mediated by two release factors: eRF1 recognizes stop codons and triggers peptidyl-tRNA hydrolysis, whereas eRF3 accelerates this process in a GTP-dependent manner. Here we report kinetic analysis of guanine nucleotide binding to eRF3 performed by fluorescence stopped-flow technique using GTP/GDP derivatives carrying the fluorescent methylanthraniloyl (mant-) group, as well as thermodynamic analysis of eRF3 binding to unlabeled guanine nucleotides. Whereas the kinetics of eRF3 binding to mant-GDP is consistent with a one-step binding model, the double-exponential transients of eRF3 binding to mant-GTP indicate a two-step binding mechanism, in which the initial eRF3·mant-GTP complex undergoes subsequent conformational change. The affinity of eRF3 for GTP (Kd, ∼70 μm) is about 70-fold lower than for GDP (Kd, ∼ 1 μm) and both nucleotides dissociate rapidly from eRF3 (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(k_{-1}^{\mathrm{mant-GDP}}\) \end{document} ∼ 2.4 s-1; \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(k_{-2}^{\mathrm{mant-GTP}}\) \end{document} ∼ 3.3 s-1). Whereas not influencing eRF3 binding to GDP, association of eRF3 with eRF1 at physiological Mg2+ concentrations specifically changes the kinetics of eRF3/mant-GTP interaction and stabilizes eRF3·GTP binding by two orders of magnitude (Kd ∼ 0.7 μm) due to lowering of the dissociation rate constant ∼24-fold (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(k_{-1}^{\mathrm{mant-GTP}}{\sim}0.14\mathrm{s}^{-1}\) \end{document} ∼ 0.14 s-1). Thus, eRF1 acts as a GTP dissociation inhibitor (TDI) for eRF3, promoting efficient ribosomal recruitment of its GTP-bound form. 80 S ribosomes did not influence guanine nucleotide binding/exchange on the eRF1·eRF3 complex. Guanine nucleotide binding and exchange on eRF3, which therefore depends on stimulation by eRF1, is entirely different from that on prokaryotic RF3 and unusual among GTPases.

Bypassing of stems versus linear base-by-base inspection of mammalian mRNAs during ribosomal scanning

Initiation codon selection in eukaryotes involves base‐by‐base inspection of the 5′‐untranslated region of mRNA by scanning ribosomal 43S preinitiation complexes. We employed in vitro reconstitution to investigate factor requirements for this process and report that in the absence of eIF1 and DHX29, eIFs 4A, 4B and 4G promote efficient bypassing of stable stems by scanning 43S complexes and formation of 48S initiation complexes on AUG codons immediately upstream and downstream of such stems, without their unwinding. However, intact stems are not threaded through the entire mRNA Exit channel of the 40S subunit, resulting in incorrect positioning of mRNA upstream of the ribosomal P site in 48S complexes formed on AUG codons following intact stems, which renders them susceptible to dissociation by eIF1. In 48S complexes formed on AUG codons preceding intact stems, the stems are accommodated in the A site. Such aberrant complexes are destabilized by DHX29, which also ensures that mRNA enters the mRNA‐binding cleft in a single‐stranded form and therefore undergoes base‐by‐base inspection during scanning.

eIF5 and eIF5B together stimulate 48S initiation complex formation during ribosomal scanning

48S initiation complex (48S IC) formation is the first stage in the eukaryotic translation process. According to the canonical mechanism, 40S ribosomal subunit binds to the 5′-end of messenger RNA (mRNA) and scans its 5′-untranslated region (5′-UTR) to the initiation codon where it forms the 48S IC. Entire process is mediated by initiation factors. Here we show that eIF5 and eIF5B together stimulate 48S IC formation influencing initiation codon selection during ribosomal scanning. Initiation on non-optimal start codons—following structured 5′-UTRs, in bad AUG context, within few nucleotides from 5′-end of mRNA and CUG start codon—is the most affected. eIF5-induced hydrolysis of eIF2-bound GTP is essential for stimulation. GTP hydrolysis increases the probability that scanning ribosomal complexes will recognize and arrest scanning at a non-optimal initiation codon. Such 48S ICs are less stable owing to dissociation of eIF2*GDP from initiator tRNA, and eIF5B is then required to stabilize the initiator tRNA in the P site of 40S subunit. Alternative model that eIF5 and eIF5B cause 43S pre-initiation complex rearrangement favoring more efficient initiation codon recognition during ribosomal scanning is equally possible. Mutational analysis of eIF1A and eIF5B revealed distinct functions of eIF5B in 48S IC formation and subunit joining.

Characterization of Novel Ribosome-Associated Endoribonuclease SLFN14 from Rabbit Reticulocytes

Turnover of mRNA is a critical step that allows cells to control gene expression. Endoribonucleases, enzymes cleaving RNA molecules internally, are some of the key components of the degradation process. Here we provide a detailed characterization of novel endoribonuclease SLFN14 purified from rabbit reticulocyte lysate. Schlafen genes encode a family of proteins limited to mammals. Their cellular function is unknown or incompletely understood. In reticulocytes, SLFN14 is strongly overexpressed, represented exclusively by the short form, all tethered to ribosomes, and appears to be one of the major ribosome-associated proteins. SLFN14 binds to ribosomes and ribosomal subunits in the low part of the body and cleaves RNA but preferentially rRNA and ribosome-associated mRNA. This results in the degradation of ribosomal subunits. This process is strictly Mg2+- and Mn2+-dependent, NTP-independent, and sequence nonspecific. However, in other cell types, SLFN14 is a full-length solely nuclear protein, which lacks ribosomal binding and nuclease activities. Mutational analysis revealed the ribosomal binding site and the aspartate essential for the endonucleolytic activity of protein. Only few endoribonucleases participating in ribosome-mediated processes have been characterized to date. Moreover, none of them are shown to be directly associated with the ribosome. Therefore, our findings expand the general knowledge of endoribonucleases involved in mammalian translation control.

DHX29 reduces leaky scanning through an upstream AUG codon regardless of its nucleotide context

During eukaryotic translation initiation, the 43S preinitiation complex (43S PIC), consisting of the 40S ribosomal subunit, eukaryotic initiation factors (eIFs) and initiator tRNA scans mRNA to find an appropriate start codon. Key roles in the accuracy of initiation codon selection belong to eIF1 and eIF1A, whereas the mammalian-specific DHX29 helicase substantially contributes to ribosomal scanning of structured mRNAs. Here, we show that DHX29 stimulates the recognition of the AUG codon but not the near-cognate CUG codon regardless of its nucleotide context during ribosomal scanning. The stimulatory effect depends on the contact between DHX29 and eIF1A. The unique DHX29 N-terminal domain binds to the ribosomal site near the mRNA entrance, where it contacts the eIF1A OB domain. UV crosslinking assays revealed that DHX29 may rearrange eIF1A and eIF2α in key nucleotide context positions of ribosomal complexes. Interestingly, DHX29 impedes the 48S initiation complex formation in the absence of eIF1A perhaps due to forming a physical barrier that prevents the 43S PIC from loading onto mRNA. Mutational analysis allowed us to split the mRNA unwinding and codon selection activities of DHX29. Thus, DHX29 is another example of an initiation factor contributing to start codon selection.