Ribosomal collision is not a prerequisite for ZNF598-mediated ribosome ubiquitination and disassembly of ribosomal complexes by ASCC.

Ribosomal stalling induces the ribosome-associated quality control (RQC) pathway targeting aberrant polypeptides. RQC is initiated by K63-polyubiquitination of ribosomal protein uS10 located at the mRNA entrance of stalled ribosomes by the E3 ubiquitin ligase ZNF598 (Hel2 in yeast). Ubiquitinated ribosomes are dissociated by the ASC-1 complex (ASCC) (RQC-Trigger (RQT) complex in yeast). A cryo-EM structure of the ribosome-bound RQT complex suggested the dissociation mechanism, in which the RNA helicase Slh1 subunit of RQT (ASCC3 in mammals) applies a pulling force on the mRNA, inducing destabilizing conformational changes in the 40S subunit, whereas the collided ribosome acts as a wedge, promoting subunit dissociation. Here, using an in vitro reconstitution approach, we found that ribosomal collision is not a strict prerequisite for ribosomal ubiquitination by ZNF598 or for ASCC-mediated ribosome release. Following ubiquitination by ZNF598, ASCC efficiently dissociated all polysomal ribosomes in a stalled queue, monosomes assembled in RRL, in vitro reconstituted 80S elongation complexes in pre- and post-translocated states, and 48S initiation complexes, as long as such complexes contained ≥ 30-35 3'-terminal mRNA nt. downstream from the P site and sufficiently long ubiquitin chains. Dissociation of polysomes and monosomes both involved ribosomal splitting, enabling Listerin-mediated ubiquitination of 60S-associated nascent chains.

Extraction of mRNA from Stalled Ribosomes by the Ski Complex.

The evolutionarily conserved Ski2-Ski3-Ski8 (Ski) complex containing the 3'→5' RNA helicase Ski2 binds to 80S ribosomes near the mRNA entrance and facilitates 3'→5' exosomal degradation of mRNA during ribosome-associated mRNA surveillance pathways. Here, we assayed Ski's activity using an in vitro reconstituted translation system and report that this complex efficiently extracts mRNA from 80S ribosomes in the 3'→5' direction in a nucleotide-by-nucleotide manner. The process is ATP dependent and can occur on pre- and post-translocation ribosomal complexes. The Ski complex can engage productively with mRNA and extract it from 80S complexes containing as few as 19 (but not 13) 3'-terminal mRNA nucleotides starting from the P site. The mRNA-extracting activity of the Ski complex suggests that its role in mRNA quality control pathways is not limited to acceleration of exosomal degradation and could include clearance of stalled ribosomes from mRNA, poising mRNA for degradation and rendering stalled ribosomes recyclable by Pelota/Hbs1/ABCE1.

In Vitro Characterization of the Activity of the Mammalian RNA Exosome on mRNAs in Ribosomal Translation Complexes.

The RNA exosome is a multisubunit protein complex that exhibits a 3' to 5' exoribonuclease activity, endoribonuclease activity, and participates in a variety of RNA processing and degradation pathways in both the nucleus and the cytoplasm. Exosomes interact with various cofactors which target them to specific RNA substrates and processes. Investigation of the mechanisms by which mammalian RNA exosomes are targeted to specific RNA substrates requires the development of in vitro approaches for purification of exosomes and their co-factors, assembly of substrates and monitoring of the exosomal activity. Here, we describe protocols for in vitro reconstitution of ribosomal 80S elongation complexes on cap-labeled mRNAs and for assaying exosomal degradation of mRNAs in such complexes depending on the presence of GTPBP1, which has previously been implicated in directing the exosome to mRNA targets.

Two classes of EF1-family translational GTPases encoded by giant viruses.

Giant viruses have extraordinarily large dsDNA genomes, and exceptionally, they encode various components of the translation apparatus, including tRNAs, aminoacyl-tRNA synthetases and translation factors. Here, we focused on the elongation factor 1 (EF1) family of viral translational GTPases (trGTPases), using computational and functional approaches to shed light on their functions. Multiple sequence alignment indicated that these trGTPases clustered into two groups epitomized by members of Mimiviridae and Marseilleviridae, respectively. trGTPases in the first group were more closely related to GTP-binding protein 1 (GTPBP1), whereas trGTPases in the second group were closer to eEF1A, eRF3 and Hbs1. Functional characterization of representative GTPBP1-like trGTPases (encoded by Hirudovirus, Catovirus and Moumouvirus) using in vitro reconstitution revealed that they possess eEF1A-like activity and can deliver cognate aa-tRNAs to the ribosomal A site during translation elongation. By contrast, representative eEF1A/eRF3/Hbs1-like viral trGTPases, encoded by Marseillevirus and Lausannevirus, have eRF3-like termination activity and stimulate peptide release by eRF1. Our analysis identified specific aspects of the functioning of these viral trGTPases with eRF1 of human, amoebal and Marseillevirus origin.

Release of Ubiquitinated and Non-ubiquitinated Nascent Chains from Stalled Mammalian Ribosomal Complexes by ANKZF1 and Ptrh1.

The ribosome-associated quality control (RQC) pathway degrades nascent chains (NCs) arising from interrupted translation. First, recycling factors split stalled ribosomes, yielding NC-tRNA/60S ribosome-nascent chain complexes (60S RNCs). 60S RNCs associate with NEMF, which recruits the E3 ubiquitin ligase Listerin that ubiquitinates NCs. The mechanism of subsequent ribosomal release of Ub-NCs remains obscure. We found that, in non-ubiquitinated 60S RNCs and 80S RNCs formed on non-stop mRNAs, tRNA is not firmly fixed in the P site, which allows peptidyl-tRNA hydrolase Ptrh1 to cleave NC-tRNA, suggesting the existence of a pathway involving release of non-ubiquitinated NCs. Association with NEMF and Listerin and ubiquitination of NCs results in accommodation of NC-tRNA, rendering 60S RNCs resistant to Ptrh1 but susceptible to ANKZF1, which induces specific cleavage in the tRNA acceptor arm, releasing proteasome-degradable Ub-NCs linked to four 3'-terminal tRNA nucleotides. We also found that TCF25, a poorly characterized RQC component, ensures preferential formation of the K48-ubiquitin linkage.

Functions of unconventional mammalian translational GTPases GTPBP1 and GTPBP2.

GTP-binding protein 1 (GTPBP1) and GTPBP2 comprise a divergent group of translational GTPases with obscure functions, which are most closely related to eEF1A, eRF3, and Hbs1. Although recent reports implicated GTPBPs in mRNA surveillance and ribosome-associated quality control, how they perform these functions remains unknown. Here, we demonstrate that GTPBP1 possesses eEF1A-like elongation activity, delivering cognate aminoacyl-transfer RNA (aa-tRNA) to the ribosomal A site in a GTP-dependent manner. It also stimulates exosomal degradation of mRNAs in elongation complexes. The kinetics of GTPBP1-mediated elongation argues against its functioning in elongation per se but supports involvement in mRNA surveillance. Thus, GTP hydrolysis by GTPBP1 is not followed by rapid peptide bond formation, suggesting that after hydrolysis, GTPBP1 retains aa-tRNA, delaying its accommodation in the A site. In physiological settings, this would cause ribosome stalling, enabling GTPBP1 to elicit quality control programs; e.g., by recruiting the exosome. GTPBP1 can also deliver deacylated tRNA to the A site, indicating that it might function via interaction with deacylated tRNA, which accumulates during stresses. Although GTPBP2's binding to GTP was stimulated by Phe-tRNAPhe, suggesting that its function might also involve interaction with aa-tRNA, GTPBP2 lacked elongation activity and did not stimulate exosomal degradation, indicating that GTPBP1 and GTPBP2 have different functions.

Structural basis for LeishIF4E-1 modulation by an interacting protein in the human parasite Leishmania major.

Leishmania parasites are unicellular pathogens that are transmitted to humans through the bite of infected sandflies. Most of the regulation of their gene expression occurs post-transcriptionally, and the different patterns of gene expression required throughout the parasites' life cycle are regulated at the level of translation. Here, we report the X-ray crystal structure of the Leishmania cap-binding isoform 1, LeishIF4E-1, bound to a protein fragment of previously unknown function, Leish4E-IP1, that binds tightly to LeishIF4E-1. The molecular structure, coupled to NMR spectroscopy experiments and in vitro cap-binding assays, reveal that Leish4E-IP1 allosterically destabilizes the binding of LeishIF4E-1 to the 5' mRNA cap. We propose mechanisms through which Leish4E-IP1-mediated LeishIF4E-1 inhibition could regulate translation initiation in the human parasite.

5' UTR m(6)A Promotes Cap-Independent Translation.

Protein translation typically begins with the recruitment of the 43S ribosomal complex to the 5' cap of mRNAs by a cap-binding complex. However, some transcripts are translated in a cap-independent manner through poorly understood mechanisms. Here, we show that mRNAs containing N(6)-methyladenosine (m(6)A) in their 5' UTR can be translated in a cap-independent manner. A single 5' UTR m(6)A directly binds eukaryotic initiation factor 3 (eIF3), which is sufficient to recruit the 43S complex to initiate translation in the absence of the cap-binding factor eIF4E. Inhibition of adenosine methylation selectively reduces translation of mRNAs containing 5'UTR m(6)A. Additionally, increased m(6)A levels in the Hsp70 mRNA regulate its cap-independent translation following heat shock. Notably, we find that diverse cellular stresses induce a transcriptome-wide redistribution of m(6)A, resulting in increased numbers of mRNAs with 5' UTR m(6)A. These data show that 5' UTR m(6)A bypasses 5' cap-binding proteins to promote translation under stresses.

The eIF3 complex of Leishmania-subunit composition and mode of recruitment to different cap-binding complexes.

Eukaryotic initiation factor 3 (eIF3) is a multi-protein complex and a key participant in the assembly of the translation initiation machinery. In mammals, eIF3 comprises 13 subunits, most of which are characterized by conserved structural domains. The trypanosomatid eIF3 subunits are poorly conserved. Here, we identify 12 subunits that comprise the Leishmania eIF3 complex (LeishIF3a-l) by combining bioinformatics with affinity purification and mass spectrometry analyses. These results highlight the strong association of LeishIF3 with LeishIF1, LeishIF2 and LeishIF5, suggesting the existence of a multi-factor complex. In trypanosomatids, the translation machinery is tightly regulated in the different life stages of these organisms as part of their adaptation and survival in changing environments. We, therefore, addressed the mechanism by which LeishIF3 is recruited to different mRNA cap-binding complexes. A direct interaction was observed in vitro between the fully assembled LeishIF3 complex and recombinant LeishIF4G3, the canonical scaffolding protein of the cap-binding complex in Leishmania promastigotes. We further highlight a novel interaction between the C-terminus of LeishIF3a and LeishIF4E1, the only cap-binding protein that efficiently binds the cap structure under heat shock conditions, anchoring a complex that is deficient of any MIF4G-based scaffolding subunit.

Multiple mechanisms of reinitiation on bicistronic calicivirus mRNAs.

Reinitiation is a strategy used by viruses to express several cistrons from one mRNA. Although extremely weak after translation of long open reading frames (ORFs) on cellular mRNAs, reinitiation occurs efficiently on subgenomic bicistronic calicivirus mRNAs, enabling synthesis of minor capsid proteins. The process is governed by a short element upstream of the restart AUG, designated "termination upstream ribosomal binding site" (TURBS). It contains the conserved Motif 1 complementary to h26 of 18S rRNA, displayed in the loop of a hairpin formed by species-specific Motifs 2/2(∗). To determine the advantages conferred on reinitiation by TURBS, we reconstituted this process in vitro on two model bicistronic calicivirus mRNAs. We found that post-termination ribosomal tethering of mRNA by TURBS allows reinitiation by post-termination 80S ribosomes and diminishes dependence on eukaryotic initiation factor 3 (eIF3) of reinitiation by recycled 40S subunits, which can be mediated either by eIFs 2/1/1A or by Ligatin following ABCE1-dependent or -independent splitting of post-termination complexes.

Nutritional stress affects an atypical cap-binding protein in Leishmania.

Many eukaryotes encode multiple isoforms of the cap-binding translation initiation factor (eIF4E). Leishmanias and other trypanosomatids encode four paralogs of this protein, but none can complement the eIF4E function in a yeast mutant. A low conservation is observed between the four paralogs, suggesting they assist these organisms survive a multitude of conditions encountered throughout the life cycle. Earlier attempts to decipher their function led to identification of LeishIF4E-4 as the canonical translation initiation factor. LeishIF4E-1 appears to function during thermal stress, via a mechanism not yet understood. LeishIF4E-3 hardly binds cap-4 and is, therefore, less likely to serve as a typical initiation factor. Although it interacts with an eIF4G homolog, LeishIF4G-4, the two polypeptides do not co-migrate on sucrose gradients. While LeishIF4E-3 enters large particles that increase in size during nutritional stress, LeishIF4G-4 is found only in the top fractions. Confocal microscopy localized LeishIF4E-3 (but not LeishIF4G-4) within nutritional stress-induced granules. Accordingly, interaction between the two proteins reduced upon starvation. We therefore propose that under normal conditions, LeishIF4G-4 sequesters LeishIF4E-3 in the cytoplasm. During a nutritional stress, LeishIF4E-3 is modified and released from LeishIF4G-4 to enter stress granules, where inactive mRNAs are stored. Binding of LeishIF4G-4 to LeishIF4E-3 requires a short peptide within the LeishIF4G-4 N-terminus, which bears no similarity to the consensus 4E-binding peptide, YXXXXLΦ. Mutational analysis combined with structure prediction indicates that this interaction is based on an obligatory, conserved α helix in LeishIF4G-4. These features further highlight the uniqueness of LeishIF4E-3 and how it interacts with its binding partners.

Gene duplication in trypanosomatids - two DED1 paralogs are functionally redundant and differentially expressed during the life cycle.

DED1/VAS belong to the DEAD-box family of RNA helicases that are associated with translation initiation in higher eukaryotes. Here we report on two DED1/VAS homologs that were identified in the genome of Leishmania. The two paralogs include all the domains that are typical of DEAD-box proteins and a phylogenetic analysis suggests that their duplication predates the branching of DED1 and VAS, which took place along with the appearance of early metazoans. The two Leishmania DED1 paralogs complement a yeast strain that fails to express the endogenous DED1, suggesting that they are responsible for a similar function. This is also supported by RNAi-mediated silencing experiments performed in Trypanosoma brucei. The two proteins are functionally redundant, since defects in protein synthesis and cell growth arrest were observed only when both paralogs were eliminated. A partial stage-specific specialization is observed, as LeishDED1-2 is more abundant in promastigotes, whereas expression of LeishDED1-1 increases in amastigotes. Duplication of an essential gene usually offers a safety net against mutations but in this case it also generated two proteins with stage specific expression.

Evolutionary conservation and diversification of the translation initiation apparatus in trypanosomatids.

Trypanosomatids are ancient eukaryotic parasites that migrate between insect vectors and mammalian hosts, causing a range of diseases in humans and domestic animals. Trypanosomatids feature a multitude of unusual molecular features, including polycistronic transcription and subsequent processing by trans-splicing and polyadenylation. Regulation of protein coding genes is posttranscriptional and thus, translation regulation is fundamental for activating the developmental program of gene expression. The spliced-leader RNA is attached to all mRNAs. It contains an unusual hypermethylated cap-4 structure in its 5' end. The cap-binding complex, eIF4F, has gone through evolutionary changes in accordance with the requirement to bind cap-4. The eIF4F components in trypanosomatids are highly diverged from their orthologs in higher eukaryotes, and their potential functions are discussed. The cap-binding activity in all eukaryotes is a target for regulation and plays a similar role in trypanosomatids. Recent studies revealed a novel eIF4E-interacting protein, involved in directing stage-specific and stress-induced translation pathways. Translation regulation during stress also follows unusual regulatory cues, as the increased translation of Hsp83 following heat stress is driven by a defined element in the 3' UTR, unlike higher eukaryotes. Overall, the environmental switches experienced by trypanosomatids during their life cycle seem to affect their translational machinery in unique ways.

A novel 4E-interacting protein in Leishmania is involved in stage-specific translation pathways.

In eukaryotes, exposure to stress conditions causes a shift from cap-dependent to cap-independent translation. In trypanosomatids, environmental switches are the driving force of a developmental program of gene expression, but it is yet unclear how their translation machinery copes with their constantly changing environment. Trypanosomatids have a unique cap structure (cap-4) and encode four highly diverged paralogs of the cap-binding protein, eIF4E; none were found to genetically complement a yeast mutant failing to express eIF4E. Here we show that in promastigotes, a typical cap-binding complex is anchored through LeishIF4E-4, which associates with components of the cap-binding pre-initiation complex. In axenic amastigotes, expression of LeishIF4E-4 decreases and the protein does not bind the cap, whereas LeishIF4E-1 maintains its expression level and associates with the cap structure and with translation initiation factors. However, LeishIF4E-1 does not interact with eIF4G-like proteins in both life stages, excluding its involvement in cap-dependent translation. Using pull-down assays and mass-spectrometry, we identified a novel, non-conserved 4E-Interacting Protein (Leish4E-IP), which binds to LeishIF4E-1 in promastigotes, but not in amastigotes. Yeast two-hybrid and NMR spectroscopy confirmed the specificity of this interaction. We propose that Leish4E-IP is a translation regulator that is involved in switching between cap-dependent and alternative translation pathways.

Leishmania parasites act as a Trojan horse that paralyzes the translation system of host macrophages.

GP63 is an abundant GPI-anchored surface metalloprotease of Leishmania. Jaramillo et al. (2011) show that GP63 manipulates the translation system of host macrophages by cleaving mTOR, which leads to 4E-BP1 dephosphorylation. This study pioneers the observation that Leishmania parasites metabolically paralyze their host cells using an elegant translation shutoff mechanism.

Evolutionary changes in the Leishmania eIF4F complex involve variations in the eIF4E-eIF4G interactions.

Translation initiation in eukaryotes is mediated by assembly of the eIF4F complex over the m(7)GTP cap structure at the 5'-end of mRNAs. This requires an interaction between eIF4E and eIF4G, two eIF4F subunits. The Leishmania orthologs of eIF4E are structurally diverged from their higher eukaryote counterparts, since they have evolved to bind the unique trypanosomatid cap-4 structure. Here, we characterize a key eIF4G candidate from Leishmania parasites (LeishIF4G-3) that contains a conserved MIF4G domain. LeishIF4G-3 was found to coelute with the parasite eIF4F subunits from an m(7)GTP-Sepharose column and to bind directly to LeishIF4E. In higher eukaryotes the eIF4E-eIF4G interaction is based on a conserved peptide signature [Y(X(4))Lphi], where X is any amino acid and Phi is a hydrophobic residue. A parallel eIF4E-binding peptide was identified in LeishIF4G-3 (20-YPGFSLDE-27). However, the binding motif varies extensively: in addition to Y20 and L25, binding strictly requires the presence of F23, whereas the hydrophobic amino acid (Phi) is dispensable. The LeishIF4E-LeishIF4G-3 interaction was also confirmed by nuclear magnetic resonance (NMR) studies. In view of these diversities, the characterization of the parasite eIF4E-eIF4G interaction may not only serve as a novel target for inhibiting Leishmaniasis but also provide important insight for future drug discovery.