The putative RNA helicase DDX1 associates with the nuclear RNA exosome and modulates RNA/DNA hybrids (R-loops).

The RNA exosome is a ribonuclease complex that mediates both RNA processing and degradation. This complex is evolutionarily conserved, ubiquitously expressed, and required for fundamental cellular functions, including rRNA processing. The RNA exosome plays roles in regulating gene expression and protecting the genome, including modulating the accumulation of RNA-DNA hybrids (R-loops). The function of the RNA exosome is facilitated by cofactors, such as the RNA helicase MTR4, which binds/remodels RNAs. Recently, missense mutations in RNA exosome subunit genes have been linked to neurological diseases. One possibility to explain why missense mutations in genes encoding RNA exosome subunits lead to neurological diseases is that the complex may interact with cell- or tissue-specific cofactors that are impacted by these changes. To begin addressing this question, we performed immunoprecipitation of the RNA exosome subunit, EXOSC3, in a neuronal cell line (N2A), followed by proteomic analyses to identify novel interactors. We identified the putative RNA helicase, DDX1, as an interactor. DDX1 plays roles in double-strand break repair, rRNA processing, and R-loop modulation. To explore the functional connections between EXOSC3 and DDX1, we examined the interaction following double-strand breaks and analyzed changes in R-loops in N2A cells depleted for EXOSC3 or DDX1 by DNA/RNA immunoprecipitation followed by sequencing. We find that EXOSC3 interaction with DDX1 is decreased in the presence of DNA damage and that loss of EXOSC3 or DDX1 alters R-loops. These results suggest EXOSC3 and DDX1 interact during events of cellular homeostasis and potentially suppress unscrupulous expression of genes promoting neuronal projection.

A Biallelic Variant of the RNA Exosome Gene EXOSC4 Causes Translational Defects Associated with a Neurodevelopmental Disorder.

The RNA exosome is an evolutionarily conserved complex required for both precise RNA processing and decay. Mutations in EXOSC genes encoding structural subunits of the complex are linked to several autosomal recessive disorders. Here, we describe a missense allele of the EXOSC4 gene, which causes a collection of clinical features in two affected siblings. This missense mutation (NM_019037.3: exon3:c.560T>C), changes a leucine residue within a highly conserved region of EXOSC4 to proline (p.Leu187Pro). The two affected individuals presented with prenatal growth restriction, failure to thrive, global developmental delay, intracerebral and basal ganglia calcifications, and kidney failure. Homozygosity for the damaging variant was identified through exome sequencing and Sanger sequencing confirmed segregation. To explore the functional consequences of this amino acid change, we modeled EXOSC4-L187P in the corresponding budding yeast protein, Rrp41 (Rrp41-L187P). Cells that express Rrp41-L187P as the sole copy of the essential Rrp41 protein show significant growth defects. The steady-state level of both the Rrp41-L187P and the EXOSC4-L187P proteins is significantly decreased compared to control Rrp41/EXOSC4. Consistent with this observation, targets of the RNA exosome accumulate in rrp41-L187P cells, including the 7S precursor of 5.8S rRNA. Polysome profiles show a significant decrease in translation in rrp41-L187P cells as compared to control cells with apparent incorporation of 7S pre-rRNA into polysomes. Taken together, this work adds the EXOSC4 subunit of the RNA exosome to the structural subunits of this complex that have been linked to human disease and defines foundational molecular defects that could contribute to the adverse growth phenotypes caused by this novel EXOSC4 pathogenic variant.

Sinefungin, a natural nucleoside analog of S-adenosyl methionine, impairs the pathogenicity of Candida albicans.

Candida albicans, an opportunistic fungal human pathogen, is a major threat to the healthcare system due to both infections in immunocompromised individuals and the emergence of antifungal resistance. Fungal infection caused by C. albicans, candidiasis, is a life-threatening condition in immunocompromised patients and the current treatments are mostly restricted to polyenes, azoles, and echinocandins. Use of these antifungals is limited by toxicity, drug-drug interactions, and the emergence of resistance, underscoring the importance of identifying novel therapeutic targets and the need for new treatment approaches. C. albicans can undergo a morphological transition from yeast to hyphae and this transition is central to C. albicans virulence. Here, we determine the impact of sinefungin, a natural nucleoside analog of S-adenosyl methionine, on the virulence of C. albicans strain SC5314 by evaluating treatment effects on the morphological transition, human epithelial cell adhesion, and biofilm formation. Our data indicate that sinefungin impairs pathogenic traits of C. albicans including hyphal lengthening, biofilm formation and the adhesion to the human epithelial cell lines, without adversely affecting human cells, therefore highlighting sinefungin as a potential avenue for therapeutic intervention. We determine that the formation of N6-methyladenosine (m6A) is particularly disturbed by sinefungin. More broadly, this study underscores the importance of considering the post-transcriptional control mechanisms of pathogenicity when designing therapeutic solutions to fungal infection.

Comparative analyses of disease-linked missense mutations in the RNA exosome modeled in budding yeast reveal distinct functional consequences in translation.

The RNA exosome is an evolutionarily conserved exoribonuclease complex that consists of a 3-subunit cap, a 6-subunit barrel-shaped core, and a catalytic base subunit. Missense mutations in genes encoding structural subunits of the RNA exosome cause a growing family of diseases with diverse pathologies, collectively termed RNA exosomopathies. The disease symptoms vary and can manifest as neurological defects or developmental disorders. The diversity of the RNA exosomopathy pathologies suggests that the different missense mutations in structural genes result in distinct in vivo consequences. To investigate these functional consequences and distinguish whether they are unique to each RNA exosomopathy mutation, we generated a collection of in vivo models using budding yeast by introducing pathogenic missense mutations in orthologous S. cerevisiae genes. We then performed a comparative RNA-seq analysis to assess broad transcriptomic changes in each mutant model. Three of the mutant models rrp4-G226D, rrp40-W195R and rrp46-L191H, which model mutations in the genes encoding structural subunits of the RNA exosome, EXOSC2, EXOSC3 and EXOSC5 showed the largest transcriptomic differences. Further analyses revealed shared increased transcripts enriched in translation or ribosomal RNA modification/processing pathways across the three mutant models. Studies of the impact of the mutations on translation revealed shared defects in ribosome biogenesis but distinct impacts on translation. Collectively, our results provide the first comparative analysis of several RNA exosomopathy mutant models and suggest that different RNA exosomopathy mutations result in in vivo consequences that are both unique and shared across each variant, providing more insight into the biology underlying each distinct pathology.

RNA structural probing of guanine and uracil nucleotides in yeast.

RNA structure can be essential for its cellular function. Therefore, methods to investigate the structure of RNA in vivo are of great importance for understanding the role of cellular RNAs. RNA structure probing is an indirect method to asess the three-dimensional structure of RNA by analyzing the reactivity of different nucleotides to chemical modifications. Dimethyl sulfate (DMS) is a well-established compound that reports on base pairing context of adenine (A) and cytidine (C) in-vitro and in-vivo, but is not reactive to guanine (G) or uracil (U). Recently, new compounds were used to modify Gs and Us in plant, bacteria, and human cells. To complement the scope of RNA structural probing by chemical modifications in the model organism yeast, we analyze the effectiveness of guanine modification by the glyoxal family in Saccharomyces cerevisiae and Candida albicans. We show that within glyoxal family of compounds, phenylglyoxal (PGO) is the best guanine probe for structural probing in S. cerevisiae and C. albicans. Further, we show that PGO treatment does not affect the processing of different RNA species in the cell and is not toxic for the cells under the conditions we have established for RNA structural probing. We also explore the effectiveness of uracil modification by Cyclohexyl-3-(2-Morpholinoethyl) Carbodiimide metho-p-Toluenesulfonate (CMCT) in vivo and demonstrate that uracils can be modified by CMCT in S. cerevisiae in vivo. Our results provide the conditions for in vivo probing the reactivity of guanine and uracil nucleotides in RNA structures in yeast and offer a valuable tool for studying RNA structure and function in two widely used yeast model systems.

The RNA helicase DDX1 associates with the nuclear RNA exosome and modulates R-loops.

The RNA exosome is a ribonuclease complex that mediates both RNA processing and degradation. This complex is evolutionarily conserved, ubiquitously expressed, and required for fundamental cellular functions, including rRNA processing. The RNA exosome plays roles in regulating gene expression and protecting the genome, including modulating the accumulation of RNA-DNA hybrids (R-loops). The function of the RNA exosome is facilitated by cofactors, such as the RNA helicase MTR4, which binds/remodels RNAs. Recently, missense mutations in RNA exosome subunit genes have been linked to neurological diseases. One possibility to explain why missense mutations in genes encoding RNA exosome subunits lead to neurological diseases is that the complex may interact with cell- or tissue-specific cofactors that are impacted by these changes. To begin addressing this question, we performed immunoprecipitation of the RNA exosome subunit, EXOSC3, in a neuronal cell line (N2A) followed by proteomic analyses to identify novel interactors. We identified the putative RNA helicase, DDX1, as an interactor. DDX1 plays roles in double-strand break repair, rRNA processing, and R-loop modulation. To explore the functional connections between EXOSC3 and DDX1, we examined the interaction following double-strand breaks, and analyzed changes in R-loops in N2A cells depleted for EXOSC3 or DDX1 by DNA/RNA immunoprecipitation followed by sequencing (DRIP-Seq). We find that EXOSC3 interaction with DDX1 is decreased in the presence of DNA damage and that loss of EXOSC3 or DDX1 alters R-loops. These results suggest EXOSC3 and DDX1 interact during events of cellular homeostasis and potentially suppress unscrupulous expression of genes promoting neuronal projection.

Studies of mutations of assembly factor Hit1 in budding yeast suggest translation defects as the molecular basis for PEHO syndrome.

Regulation of protein synthesis is critical for control of gene expression in all cells. Ribosomes are ribonucleoprotein machines responsible for translating cellular proteins. Defects in ribosome production, function, or regulation are detrimental to the cell and cause human diseases, such as progressive encephalopathy with edema, hypsarrhythmia, and optic atrophy (PEHO) syndrome. PEHO syndrome is a devastating neurodevelopmental disorder caused by mutations in the ZNHIT3 gene, which encodes an evolutionarily conserved nuclear protein. The precise mechanisms by which ZNHIT3 mutations lead to PEHO syndrome are currently unclear. Studies of the human zinc finger HIT-type containing protein 3 homolog in budding yeast (Hit1) revealed that this protein is critical for formation of small nucleolar ribonucleoprotein complexes that are required for rRNA processing and 2'-O-methylation. Here, we use budding yeast as a model system to reveal the basis for the molecular pathogenesis of PEHO syndrome. We show that missense mutations modeling those found in PEHO syndrome patients cause a decrease in steady-state Hit1 protein levels, a significant reduction of box C/D snoRNA levels, and subsequent defects in rRNA processing and altered cellular translation. Using RiboMethSeq analysis of rRNAs isolated from actively translating ribosomes, we reveal site-specific changes in the rRNA modification pattern of PEHO syndrome mutant yeast cells. Our data suggest that PEHO syndrome is a ribosomopathy and reveal potential new aspects of the molecular basis of this disease in translation dysregulation.

Ribosomal RNA 2'-O-methylations regulate translation by impacting ribosome dynamics.

SignificanceThe presence of RNA chemical modifications has long been known, but their precise molecular consequences remain unknown. 2'-O-methylation is an abundant modification that exists in RNA in all domains of life. Ribosomal RNA (rRNA) represents a functionally important RNA that is heavily modified by 2'-O-methylations. Although abundant at functionally important regions of the rRNA, the contribution of 2'-O-methylations to ribosome activities is unknown. By establishing a method to disturb rRNA 2'-O-methylation patterns, we show that rRNA 2'-O-methylations affect the function and fidelity of the ribosome and change the balance between different ribosome conformational states. Our work links 2'-O-methylation to ribosome dynamics and defines a set of critical rRNA 2'-O-methylations required for ribosome biogenesis and others that are dispensable.

Phosphorylation regulates arginine-rich RNA-binding protein solubility and oligomerization.

Posttranslational modifications (PTMs) such as phosphorylation of RNA-binding proteins (RBPs) regulate several critical steps in RNA metabolism, including spliceosome assembly, alternative splicing, and mRNA export. Notably, serine-/arginine- (SR)-rich RBPs are densely phosphorylated compared with the remainder of the proteome. Previously, we showed that dephosphorylation of the splicing factor SRSF2 regulated increased interactions with similar arginine-rich RBPs U1-70K and LUC7L3. However, the large-scale functional and structural impact of these modifications on RBPs remains unclear. In this work, we dephosphorylated nuclear extracts using phosphatase in vitro and analyzed equal amounts of detergent-soluble and -insoluble fractions by mass-spectrometry-based proteomics. Correlation network analysis resolved 27 distinct modules of differentially soluble nucleoplasm proteins. We found classes of arginine-rich RBPs that decrease in solubility following dephosphorylation and enrich the insoluble pelleted fraction, including the SR protein family and the SR-like LUC7L RBP family. Importantly, increased insolubility was not observed across broad classes of RBPs. We determined that phosphorylation regulated SRSF2 structure, as dephosphorylated SRSF2 formed high-molecular-weight oligomeric species in vitro. Reciprocally, phosphorylation of SRSF2 by serine/arginine protein kinase 2 (SRPK2) in vitro decreased high-molecular-weight SRSF2 species formation. Furthermore, upon pharmacological inhibition of SRPKs in mammalian cells, we observed SRSF2 cytoplasmic mislocalization and increased formation of cytoplasmic granules as well as cytoplasmic tubular structures that associated with microtubules by immunocytochemical staining. Collectively, these findings demonstrate that phosphorylation may be a critical modification that prevents arginine-rich RBP insolubility and oligomerization.

A conserved Bcd1 interaction essential for box C/D snoRNP biogenesis.

Precise modification and processing of rRNAs are required for the production of ribosomes and accurate translation of proteins. Small nucleolar ribonucleoproteins (snoRNPs) guide the folding, modification, and processing of rRNAs and are thus critical for all eukaryotic cells. Bcd1, an essential zinc finger HIT protein functionally conserved in eukaryotes, has been implicated as an early regulator for biogenesis of box C/D snoRNPs and controls steady-state levels of box C/D snoRNAs through an unknown mechanism. Using a combination of genetic and biochemical approaches, here we found a conserved N-terminal motif in Bcd1 from Saccharomyces cerevisiae that is required for interactions with box C/D snoRNAs and the core snoRNP protein, Snu13. We show that both the Bcd1-snoRNA and Bcd1-Snu13 interactions are critical for snoRNP assembly and ribosome biogenesis. Our results provide mechanistic insight into Bcd1 interactions that likely control the early steps of snoRNP maturation and contribute to the essential role of this protein in maintaining the steady-state levels of snoRNAs in the cell.

Rrp5 establishes a checkpoint for 60S assembly during 40S maturation.

Even though the RNAs contained in the small (40S) and large (60S) ribosomal subunits are cotranscribed, their assembly proceeds largely separately, involving entirely distinct machineries. Nevertheless, separation of the two subunits, an event that is critical for assembly of the small subunit, is delayed until domain I of the large subunit is transcribed, indicating crosstalk between the two assembly pathways. Here we show that this crosstalk is mediated by the assembly factor Rrp5, one of only three proteins required for assembly of both ribosomal subunits. Quantitative RNA binding and cleavage data demonstrate that early on, Rrp5 blocks separation of the two subunits, and thus 40S maturation by inhibiting the access of Rcl1 to promote cleavage of the nascent rRNA. Upon transcription of domain I of 25S rRNA, the 60S assembly factors Noc1/Noc2 bind both this RNA and Rrp5 to change the Rrp5 RNA binding mode to enable pre-40S rRNA processing. Mutants in the HEAT-repeat domain of Noc1 are deficient in the separation of the subunits, which is rescued by overexpression of wild-type but not inactive Rcl1 in vivo. Thus, Rrp5 establishes a checkpoint for 60S assembly during 40S maturation to ensure balanced levels of the two subunits.

The DEAD-box Protein Rok1 Orchestrates 40S and 60S Ribosome Assembly by Promoting the Release of Rrp5 from Pre-40S Ribosomes to Allow for 60S Maturation.

DEAD-box proteins are ubiquitous regulators of RNA biology. While commonly dubbed "helicases," their activities also include duplex annealing, adenosine triphosphate (ATP)-dependent RNA binding, and RNA-protein complex remodeling. Rok1, an essential DEAD-box protein, and its cofactor Rrp5 are required for ribosome assembly. Here, we use in vivo and in vitro biochemical analyses to demonstrate that ATP-bound Rok1, but not adenosine diphosphate (ADP)-bound Rok1, stabilizes Rrp5 binding to 40S ribosomes. Interconversion between these two forms by ATP hydrolysis is required for release of Rrp5 from pre-40S ribosomes in vivo, thereby allowing Rrp5 to carry out its role in 60S subunit assembly. Furthermore, our data also strongly suggest that the previously described accumulation of snR30 upon Rok1 inactivation arises because Rrp5 release is blocked and implicate a previously undescribed interaction between Rrp5 and the DEAD-box protein Has1 in mediating snR30 accumulation when Rrp5 release from pre-40S subunits is blocked.

Structural integrity of the PCI domain of eIF3a/TIF32 is required for mRNA recruitment to the 43S pre-initiation complexes.

Transfer of genetic information from genes into proteins is mediated by messenger RNA (mRNA) that must be first recruited to ribosomal pre-initiation complexes (PICs) by a mechanism that is still poorly understood. Recent studies showed that besides eIF4F and poly(A)-binding protein, eIF3 also plays a critical role in this process, yet the molecular mechanism of its action is unknown. We showed previously that the PCI domain of the eIF3c/NIP1 subunit of yeast eIF3 is involved in RNA binding. To assess the role of the second PCI domain of eIF3 present in eIF3a/TIF32, we performed its mutational analysis and identified a 10-Ala-substitution (Box37) that severely reduces amounts of model mRNA in the 43-48S PICs in vivo as the major, if not the only, detectable defect. Crystal structure analysis of the a/TIF32-PCI domain at 2.65-Å resolution showed that it is required for integrity of the eIF3 core and, similarly to the c/NIP1-PCI, is capable of RNA binding. The putative RNA-binding surface defined by positively charged areas contains two Box37 residues, R363 and K364. Their substitutions with alanines severely impair the mRNA recruitment step in vivo suggesting that a/TIF32-PCI represents one of the key domains ensuring stable and efficient mRNA delivery to the PICs.

Cofactor-dependent specificity of a DEAD-box protein.

DEAD-box proteins, a large class of RNA-dependent ATPases, regulate all aspects of gene expression and RNA metabolism. They can facilitate dissociation of RNA duplexes and remodeling of RNA-protein complexes, serve as ATP-dependent RNA-binding proteins, or even anneal duplexes. These proteins have highly conserved sequence elements that are contained within two RecA-like domains; consequently, their structures are nearly identical. Furthermore, crystal structures of DEAD-box proteins with bound RNA reveal interactions exclusively between the protein and the RNA backbone. Together, these findings suggest that DEAD-box proteins interact with their substrates in a nonspecific manner, which is confirmed in biochemical experiments. Nevertheless, this contrasts with the need to target these enzymes to specific substrates in vivo. Using the DEAD-box protein Rok1 and its cofactor Rrp5, which both function during maturation of the small ribosomal subunit, we show here that Rrp5 provides specificity to the otherwise nonspecific biochemical activities of the Rok1 DEAD-domain. This finding could reconcile the need for specific substrate binding of some DEAD-box proteins with their nonspecific binding surface and expands the potential roles of cofactors to specificity factors. Identification of helicase cofactors and their RNA substrates could therefore help define the undescribed roles of the 19 DEAD-box proteins that function in ribosome assembly.

Crystal structure analysis of a fatty acid double-bond hydratase from Lactobacillus acidophilus.

Bacteria have evolved mechanisms for the hydrogenation of unsaturated fatty acids. Hydroxy fatty acid formation may be the first step in such a process; however, knowledge of the structural and mechanistic aspects of this reaction is scarce. Recently, myosin cross-reactive antigen was shown to be a bacterial FAD-containing hydratase which acts on the 9Z and 12Z double bonds of C16 and C18 non-esterified fatty acids, with the formation of 10-hydroxy and 10,13-dihydroxy fatty acids. These fatty acid hydratases form a large protein family which is conserved across Gram-positive and Gram-negative bacteria with no sequence similarity to any known protein apart from the FAD-binding motif. In order to shed light on the substrate recognition and the mechanism of the hydratase reaction, the crystal structure of the hydratase from Lactobacillus acidophilus (LAH) was determined by single-wavelength anomalous dispersion. Crystal structures of apo LAH and of LAH with bound linoleic acid were refined at resolutions of 2.3 and 1.8 Å, respectively. LAH is a homodimer; each protomer consists of four intricately connected domains. Three of them form the FAD-binding and substrate-binding sites and reveal structural similarity to three domains of several flavin-dependent enzymes, including amine oxidoreductases. The additional fourth domain of LAH is located at the C-terminus and consists of three α-helices. It covers the entrance to the hydrophobic substrate channel leading from the protein surface to the active site. In the presence of linoleic acid, the fourth domain of one protomer undergoes conformational changes and opens the entrance to the substrate-binding channel of the other protomer of the LAH homodimer. The linoleic acid molecule is bound at the entrance to the substrate channel, suggesting movement of the lid domain triggered by substrate recognition.

Novel insights into the architecture and protein interaction network of yeast eIF3.

Translation initiation in eukaryotes is a multistep process requiring the orchestrated interaction of several eukaryotic initiation factors (eIFs). The largest of these factors, eIF3, forms the scaffold for other initiation factors, promoting their binding to the 40S ribosomal subunit. Biochemical and structural studies on eIF3 need highly pure eIF3. However, natively purified eIF3 comprise complexes containing other proteins such as eIF5. Therefore we have established in vitro reconstitution protocols for Saccharomyces cerevisiae eIF3 using its five recombinantly expressed and purified subunits. This reconstituted eIF3 complex (eIF3(rec)) exhibits the same size and activity as the natively purified eIF3 (eIF3(nat)). The homogeneity and stoichiometry of eIF3(rec) and eIF3(nat) were confirmed by analytical size exclusion chromatography, mass spectrometry, and multi-angle light scattering, demonstrating the presence of one copy of each subunit in the eIF3 complex. The reconstituted and native eIF3 complexes were compared by single-particle electron microscopy showing a high degree of structural conservation. The interaction network between eIF3 proteins was studied by means of limited proteolysis, analytical size exclusion chromatography, in vitro binding assays, and isothermal titration calorimetry, unveiling distinct protein domains and subcomplexes that are critical for the integrity of the protein network in yeast eIF3. Taken together, the data presented here provide a novel procedure to obtain highly pure yeast eIF3, suitable for biochemical and structural analysis, in addition to a detailed picture of the network of protein interactions within this complex.

Crystal structure of the RNA recognition motif of yeast translation initiation factor eIF3b reveals differences to human eIF3b.

BACKGROUND: The multi-subunit eukaryotic initiation factor3 (eIF3) plays a central role in the initiation step of protein synthesis in eukaryotes. One of its large subunits, eIF3b, serves as a scaffold within eIF3 as it interacts with several other subunits. It harbors an RNA Recognition Motif (RRM), which is shown to be a non-canonical RRM in human as it is not capable to interact with oligonucleotides, but rather interacts with eIF3j, a sub-stoichiometric subunit of eIF3. PRINCIPAL FINDING: We have analyzed the high-resolution crystal structure of the eIF3b RRM domain from yeast. It exhibits the same fold as its human ortholog, with similar charge distribution on the surface interacting with the eIF3j in human. Thermodynamic analysis of the interaction between yeast eIF3b-RRM and eIF3j revealed the same range of enthalpy change and dissociation constant as for the human proteins, providing another line of evidence for the same mode of interaction between eIF3b and eIF3j in both organisms. However, analysis of the surface charge distribution of the putative RNA-binding ß-sheet suggested that in contrast to its human ortholog, it potentially could bind oligonucleotides. Three-dimensional positioning of the so called "RNP1" motif in this domain is similar to other canonical RRMs, suggesting that this domain might indeed be a canonical RRM, conferring oligonucleotide binding capability to eIF3 in yeast. Interaction studies with yeast total RNA extract confirmed the proposed RNA binding activity of yeast eIF3b-RRM. CONCLUSION: We showed that yeast eIF3b-RRM interacts with eIF3j in a manner similar to its human ortholog. However, it shows similarities in the oligonucleotide binding surface to canonical RRMs and interacts with yeast total RNA. The proposed RNA binding activity of eIF3b-RRM may help eIF3 to either bind to the ribosome or recruit the mRNA to the 43S pre-initiation complex.

The iron-sulphur protein RNase L inhibitor functions in translation termination.

The iron-sulphur (Fe-S)-containing RNase L inhibitor (Rli1) is involved in ribosomal subunit maturation, transport of both ribosomal subunits to the cytoplasm, and translation initiation through interaction with the eukaryotic initiation factor 3 (eIF3) complex. Here, we present a new function for Rli1 in translation termination. Through co-immunoprecipitation experiments, we show that Rli1 interacts physically with the translation termination factors eukaryotic release factor 1 (eRF1)/Sup45 and eRF3/Sup35 in Saccharomyces cerevisiae. Genetic interactions were uncovered between a strain depleted for Rli1 and sup35-21 or sup45-2. Furthermore, we show that downregulation of RLI1 expression leads to defects in the recognition of a stop codon, as seen in mutants of other termination factors. By contrast, RLI1 overexpression partly suppresses the read-through defects in sup45-2. Interestingly, we find that although the Fe-S cluster is not required for the interaction of Rli1 with eRF1 or its other interacting partner, Hcr1, from the initiation complex eIF3, it is required for its activity in translation termination; an Fe-S cluster mutant of RLI1 cannot suppress the read-through defects of sup45-2.