ABSTRACT
Transfer RNA (tRNA) modifications are critical for protein synthesis. Queuosine (Q), a 7-deaza-guanosine derivative, is present in tRNA anticodons. In vertebrate tRNAs for Tyr and Asp, Q is further glycosylated with galactose and mannose to generate galQ and manQ, respectively. However, biogenesis and physiological relevance of Q-glycosylation remain poorly understood. Here, we biochemically identified two RNA glycosylases, QTGAL and QTMAN, and successfully reconstituted Q-glycosylation of tRNAs using nucleotide diphosphate sugars. Ribosome profiling of knockout cells revealed that Q-glycosylation slowed down elongation at cognate codons, UAC and GAC (GAU), respectively. We also found that galactosylation of Q suppresses stop codon readthrough. Moreover, protein aggregates increased in cells lacking Q-glycosylation, indicating that Q-glycosylation contributes to proteostasis. Cryo-EM of human ribosome-tRNA complex revealed the molecular basis of codon recognition regulated by Q-glycosylations. Furthermore, zebrafish qtgal and qtman knockout lines displayed shortened body length, implying that Q-glycosylation is required for post-embryonic growth in vertebrates.
Subject(s)
RNA, Transfer , Animals , Humans , Rats , Anticodon , Cell Line , Codon , Glycosylation , Nucleoside Q/chemistry , Nucleoside Q/genetics , Nucleoside Q/metabolism , RNA, Transfer/chemistry , RNA, Transfer/metabolism , Swine , Zebrafish/metabolism , Nucleic Acid ConformationABSTRACT
4.5SH RNA is a highly abundant, small rodent-specific noncoding RNA that localizes to nuclear speckles enriched in pre-mRNA-splicing regulators. To investigate the physiological functions of 4.5SH RNA, we have created mutant mice that lack the expression of 4.5SH RNA. The mutant mice exhibited embryonic lethality, suggesting that 4.5SH RNA is an essential species-specific noncoding RNA in mice. RNA-sequencing analyses revealed that 4.5SH RNA protects the transcriptome from abnormal exonizations of the antisense insertions of the retrotransposon SINE B1 (asB1), which would otherwise introduce deleterious premature stop codons or frameshift mutations. Mechanistically, 4.5SH RNA base pairs with complementary asB1-containing exons via the target recognition region and recruits effector proteins including Hnrnpm via its 5' stem loop region. The modular organization of 4.5SH RNA allows us to engineer a programmable splicing regulator to induce the skipping of target exons of interest. Our results also suggest the general existence of splicing regulatory noncoding RNAs.
Subject(s)
RNA Splicing , RNA, Small Untranslated , Mice , Animals , RNA Splicing/genetics , Exons/genetics , Retroelements/genetics , Codon, Nonsense , Alternative SplicingABSTRACT
Translation initiates when the eIF4F complex binds the 5' mRNA cap, followed by 5' untranslated region scanning for the start codon by scanning ribosomes. Here, we demonstrate that the ASC-1 complex (ASCC), which was previously shown to promote the dissociation of colliding 80S ribosomes, associates with scanning ribosomes to regulate translation initiation. Selective translation complex profiling (TCP-seq) analysis revealed that ASCC3, a helicase domain-containing subunit of ASCC, localizes predominantly to the 5' untranslated region of mRNAs. Ribo-seq, TCP-seq, and luciferase reporter analyses showed that ASCC3 knockdown impairs 43S preinitiation complex loading and scanning dynamics, thereby reducing translation efficiency. Whereas eIF4A, an RNA helicase in the eIF4F complex, is important for global translation, ASCC was found to regulate the scanning process for a specific subset of transcripts. Our results have thus revealed that ASCC is required not only for dissociation of colliding 80S ribosomes but also for efficient translation initiation by scanning ribosomes at a subset of transcripts.
Subject(s)
Eukaryotic Initiation Factor-4F , Ribosomes , Eukaryotic Initiation Factor-4F/genetics , Eukaryotic Initiation Factor-4F/metabolism , 5' Untranslated Regions , Ribosomes/genetics , Ribosomes/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Codon, Initiator , Protein Biosynthesis , Peptide Chain Initiation, TranslationalABSTRACT
Diverse ribonucleoprotein complexes control mRNA processing, translation, and decay. Transcripts in these complexes localize to specific regions of the cell and can condense into non-membrane-bound structures such as stress granules. It has proven challenging to map the RNA composition of these large and dynamic structures, however. We therefore developed an RNA proximity labeling technique, APEX-seq, which uses the ascorbate peroxidase APEX2 to probe the spatial organization of the transcriptome. We show that APEX-seq can resolve the localization of RNAs within the cell and determine their enrichment or depletion near key RNA-binding proteins. Matching the spatial transcriptome, as revealed by APEX-seq, with the spatial proteome determined by APEX-mass spectrometry (APEX-MS), obtained precisely in parallel, provides new insights into the organization of translation initiation complexes on active mRNAs and unanticipated complexity in stress granule composition. Our novel technique allows a powerful and general approach to explore the spatial environment of macromolecules.
Subject(s)
Cytoplasmic Granules/metabolism , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Endonucleases/metabolism , Multifunctional Enzymes/metabolism , Peptide Chain Initiation, Translational , RNA/metabolism , Staining and Labeling , Transcriptome , Cytoplasmic Granules/genetics , DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics , Endonucleases/genetics , HEK293 Cells , Humans , Multifunctional Enzymes/genetics , RNA/geneticsABSTRACT
A class of translation inhibitors, exemplified by the natural product rocaglamide A (RocA), isolated from Aglaia genus plants, exhibits antitumor activity by clamping eukaryotic translation initiation factor 4A (eIF4A) onto polypurine sequences in mRNAs. This unusual inhibitory mechanism raises the question of how the drug imposes sequence selectivity onto a general translation factor. Here, we determined the crystal structure of the human eIF4A1â ATP analogâ RocAâ polypurine RNA complex. RocA targets the "bi-molecular cavity" formed characteristically by eIF4A1 and a sharply bent pair of consecutive purines in the RNA. Natural amino acid substitutions found in Aglaia eIF4As changed the cavity shape, leading to RocA resistance. This study provides an example of an RNA-sequence-selective interfacial inhibitor fitting into the space shaped cooperatively by protein and RNA with specific sequences.
Subject(s)
Benzofurans/metabolism , Eukaryotic Initiation Factor-4A/metabolism , Protein Biosynthesis , Protein Synthesis Inhibitors/metabolism , RNA/metabolism , Ribosomes/metabolism , Adenylyl Imidodiphosphate/chemistry , Adenylyl Imidodiphosphate/metabolism , Aglaia/chemistry , Aglaia/genetics , Aglaia/metabolism , Amino Acid Substitution , Benzofurans/chemistry , Benzofurans/isolation & purification , Benzofurans/pharmacology , Binding Sites , Drug Resistance/genetics , Eukaryotic Initiation Factor-4A/chemistry , Eukaryotic Initiation Factor-4A/genetics , HEK293 Cells , Humans , Models, Molecular , Molecular Structure , Mutation , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/metabolism , Protein Binding , Protein Biosynthesis/drug effects , Protein Biosynthesis/genetics , Protein Interaction Domains and Motifs , Protein Synthesis Inhibitors/chemistry , Protein Synthesis Inhibitors/isolation & purification , Protein Synthesis Inhibitors/pharmacology , RNA/chemistry , Ribosomes/chemistry , Ribosomes/drug effects , Ribosomes/genetics , Structure-Activity RelationshipABSTRACT
The control of mRNA stability plays a central role in regulating gene expression patterns. Recent studies have revealed that codon composition in the open reading frame determines mRNA stability in multiple organisms. Based on genome-wide correlation approaches, this previously unrecognized role for the genetic code is attributable to the kinetics of the codon-decoding process by the ribosome. However, complementary experimental analyses are required to clarify the codon effects on mRNA stability and the related cotranslational mRNA decay pathways, for example, those triggered by aberrant ribosome stalling. In the current study, we performed a set of reporter-based analyses to define codon-mediated mRNA decay and ribosome stall-dependent mRNA decay in zebrafish embryos. Our analysis showed that the effect of codons on mRNA stability stems from the decoding process, independent of the ribosome quality control factor Znf598 and stalling-dependent mRNA decay. We propose that codon-mediated mRNA decay is rather triggered by transiently slowed ribosomes engaging in a productive translation cycle in zebrafish embryos.
Subject(s)
Codon/genetics , RNA Stability/genetics , RNA, Messenger/genetics , Ribosomes/genetics , Amino Acid Sequence , Animals , Open Reading Frames/genetics , Protein Biosynthesis/genetics , Quality Control , Zebrafish/geneticsABSTRACT
Neat1 is an architectural RNA that provides the structural basis for nuclear bodies known as paraspeckles. Although the assembly processes by which Neat1 organizes paraspeckle components are well-documented, the physiological functions of Neat1 are not yet fully understood. This is partly because Neat1 knockout (KO) mice, lacking paraspeckles, do not exhibit overt phenotypes under normal laboratory conditions. During our search for conditions that elicit clear phenotypes in Neat1 KO mice, we discovered that the differentiation of beige adipocytes-inducible thermogenic cells that emerge upon cold exposure-is severely impaired in these mutant mice. Neat1_2, the architectural isoform of Neat1, is transiently upregulated during the early stages of beige adipocyte differentiation, coinciding with increased paraspeckle formation. Genes with altered expression during beige adipocyte differentiation typically cluster at specific chromosomal locations, some of which move closer to paraspeckles upon cold exposure. These observations suggest that paraspeckles might coordinate the regulation of these gene clusters by controlling the activity of certain transcriptional condensates that coregulate multiple genes. We propose that our findings highlight a potential role for Neat1 and paraspeckles in modulating chromosomal organization and gene expression, potentially crucial processes for the differentiation of beige adipocytes.
Subject(s)
Adipocytes, Beige , Cell Differentiation , Cold Temperature , Mice, Knockout , RNA, Long Noncoding , Animals , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Mice , Cell Differentiation/genetics , Adipocytes, Beige/metabolism , Adipocytes, Beige/cytology , Thermogenesis/geneticsABSTRACT
In response to environmental changes, cells flexibly and rapidly alter gene expression through translational controls. In plants, the translation of NIP5;1, a boric acid diffusion facilitator, is downregulated in response to an excess amount of boric acid in the environment through upstream open reading frames (uORFs) that consist of only AUG and stop codons. However, the molecular details of how this minimum uORF controls translation of the downstream main ORF in a boric acid-dependent manner have remained unclear. Here, by combining ribosome profiling, translation complex profile sequencing, structural analysis with cryo-electron microscopy and biochemical assays, we show that the 80S ribosome assembled at AUG-stop migrates into the subsequent RNA segment, followed by downstream translation initiation, and that boric acid impedes this process by the stable confinement of eukaryotic release factor 1 on the 80S ribosome on AUG-stop. Our results provide molecular insight into translation regulation by a minimum and environment-responsive uORF.
Subject(s)
Boric Acids , Protein Biosynthesis , Ribosomes , Ribosomes/metabolism , Boric Acids/chemistry , Peptide Termination Factors/metabolism , Peptide Termination Factors/chemistry , Peptide Termination Factors/genetics , Cryoelectron Microscopy , Open Reading Frames , Codon, Terminator , Arabidopsis/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/geneticsABSTRACT
Gene expression is known to vary among individuals, and this variability can impact the phenotypic diversity observed in natural populations. While the transcriptome and proteome have been extensively studied, little is known about the translation process itself. Here, we therefore performed ribosome and transcriptomic profiling on a genetically and ecologically diverse set of natural isolates of the Saccharomyces cerevisiae yeast. Interestingly, we found that the Euclidean distances between each profile and the expression fold changes in each pairwise isolate comparison were higher at the transcriptomic level. This observation clearly indicates that the transcriptional variation observed in the different isolates is buffered through a phenomenon known as post-transcriptional buffering at the translation level. Furthermore, this phenomenon seemed to have a specific signature by preferentially affecting essential genes as well as genes involved in complex-forming proteins, and low transcribed genes. We also explored the translation of the S. cerevisiae pangenome and found that the accessory genes related to introgression events displayed similar transcription and translation levels as the core genome. By contrast, genes acquired through horizontal gene transfer events tended to be less efficiently translated. Together, our results highlight both the extent and signature of the post-transcriptional buffering.
Subject(s)
Saccharomyces cerevisiae , Transcriptome , Humans , Saccharomyces cerevisiae/genetics , Gene Expression Profiling , Ribosomes/genetics , Genetic Background , Genetic VariationABSTRACT
In higher eukaryotes, tRNA methyltransferase 10A (TRMT10A) is responsible for N1-methylguanosine modification at position nine of various cytoplasmic tRNAs. Pathogenic mutations in TRMT10A cause intellectual disability, microcephaly, diabetes, and short stature in humans, and generate cytotoxic tRNA fragments in cultured cells; however, it is not clear how TRMT10A supports codon translation or brain functions. Here, we generated Trmt10a null mice and showed that tRNAGln(CUG) and initiator methionine tRNA levels were universally decreased in various tissues; the same was true in a human cell line lacking TRMT10A. Ribosome profiling of mouse brain revealed that dysfunction of TRMT10A causes ribosome slowdown at the Gln(CAG) codon and increases translation of Atf4 due to higher frequency of leaky scanning of its upstream open reading frames. Broadly speaking, translation of a subset of mRNAs, especially those for neuronal structures, is perturbed in the mutant brain. Despite not showing discernable defects in the pancreas, liver, or kidney, Trmt10a null mice showed lower body weight and smaller hippocampal postsynaptic densities, which is associated with defective synaptic plasticity and memory. Taken together, our study provides mechanistic insight into the roles of TRMT10A in the brain, and exemplifies the importance of universal tRNA modification during translation of specific codons.
Subject(s)
Brain , Glutamine , Protein Biosynthesis , tRNA Methyltransferases , Animals , Humans , Male , Mice , Brain/metabolism , Codon/genetics , Glutamine/metabolism , Mice, Inbred C57BL , Mice, Knockout , Ribosomes/metabolism , Ribosomes/genetics , RNA, Transfer, Met/metabolism , RNA, Transfer, Met/genetics , tRNA Methyltransferases/genetics , tRNA Methyltransferases/metabolismABSTRACT
Continuous translation elongation, irrespective of amino acid sequences, is a prerequisite for living organisms to produce their proteomes. However, nascent polypeptide products bear an inherent risk of elongation abortion. For example, negatively charged sequences with occasional intermittent prolines, termed intrinsic ribosome destabilization (IRD) sequences, weaken the translating ribosomal complex, causing certain nascent chain sequences to prematurely terminate translation. Here, we show that most potential IRD sequences in the middle of open reading frames remain cryptic and do not interrupt translation, due to two features of the nascent polypeptide. Firstly, the nascent polypeptide itself spans the exit tunnel, and secondly, its bulky amino acid residues occupy the tunnel entrance region, thereby serving as a bridge and protecting the large and small ribosomal subunits from dissociation. Thus, nascent polypeptide products have an inbuilt ability to ensure elongation continuity.
Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Open Reading Frames , Peptides/metabolism , Protein Biosynthesis , Ribosomal Proteins/metabolism , Ribosomes/chemistry , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Peptides/genetics , Ribosomal Proteins/genetics , Ribosomes/genetics , Ribosomes/metabolismABSTRACT
Ribosome pauses are associated with various cotranslational events and determine the fate of mRNAs and proteins. Thus, the identification of precise pause sites across the transcriptome is desirable; however, the landscape of ribosome pauses in bacteria remains ambiguous. Here, we harness monosome and disome (or collided ribosome) profiling strategies to survey ribosome pause sites in Escherichia coli Compared to eukaryotes, ribosome collisions in bacteria showed remarkable differences: a low frequency of disomes at stop codons, collisions occurring immediately after 70S assembly on start codons, and shorter queues of ribosomes trailing upstream. The pause sites corresponded with the biochemical validation by integrated nascent chain profiling (iNP) to detect polypeptidyl-tRNA, an elongation intermediate. Moreover, the subset of those sites showed puromycin resistance, presenting slow peptidyl transfer. Among the identified sites, the ribosome pause at Asn586 of ycbZ was validated by biochemical reporter assay, tRNA sequencing (tRNA-seq), and cryo-electron microscopy (cryo-EM) experiments. Our results provide a useful resource for ribosome stalling sites in bacteria.
Subject(s)
Peptide Chain Elongation, Translational , Peptide Chain Termination, Translational , Ribosomes/metabolism , Codon, Terminator , Escherichia coli , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , RNA, Transfer/genetics , RNA, Transfer/metabolismABSTRACT
Mitochondria possess their own genome that encodes components of oxidative phosphorylation (OXPHOS) complexes, and mitochondrial ribosomes within the organelle translate the mRNAs expressed from the mitochondrial genome. Given the differential OXPHOS activity observed in diverse cell types, cell growth conditions, and other circumstances, cellular heterogeneity in mitochondrial translation can be expected. Although individual protein products translated in mitochondria have been monitored, the lack of techniques that address the variation in overall mitochondrial protein synthesis in cell populations poses analytic challenges. Here, we adapted mitochondrial-specific fluorescent noncanonical amino acid tagging (FUNCAT) for use with fluorescence-activated cell sorting (FACS) and developed mito-FUNCAT-FACS. The click chemistry-compatible methionine analog L-homopropargylglycine (HPG) enabled the metabolic labeling of newly synthesized proteins. In the presence of cytosolic translation inhibitors, HPG was selectively incorporated into mitochondrial nascent proteins and conjugated to fluorophores via the click reaction (mito-FUNCAT). The application of in situ mito-FUNCAT to flow cytometry allowed us to separate changes in net mitochondrial translation activity from those of the organelle mass and detect variations in mitochondrial translation in cancer cells. Our approach provides a useful methodology for examining mitochondrial protein synthesis in individual cells.
Subject(s)
Amino Acids , Protein Biosynthesis , Amino Acids/chemistry , Flow Cytometry , Mitochondria/genetics , Mitochondria/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolismABSTRACT
Paraspeckles are mammalian-specific nuclear bodies built on the long noncoding RNA NEAT1_2 The molecular mechanisms of paraspeckle formation have been mainly studied using human or mouse cells, and it is not known if the same molecular components are involved in the formation of paraspeckles in other mammalian species. We thus investigated the expression pattern of NEAT1_2 in naked mole-rats (nNEAT1_2), which exhibit extreme longevity and lower susceptibility to cancer. In the intestine, nNEAT1_2 is widely expressed along the entire intestinal epithelium, which is different from the expression of mNeat1_2 that is restricted to the cells of the distal tip in mice. Notably, the expression of FUS, a FET family RNA binding protein, essential for the formation of paraspeckles both in humans and mice, was absent in the distal part of the intestinal epithelium in naked mole-rats. Instead, mRNAs of other FET family proteins EWSR1 and TAF15 were expressed in the distal region. Exogenous expression of these proteins in Fus-deficient murine embryonic fibroblast cells rescued the formation of paraspeckles. These observations suggest that nNEAT1_2 recruits a different set of RNA binding proteins in a cell type-specific manner during the formation of paraspeckles in different organisms.
Subject(s)
Paraspeckles , RNA, Long Noncoding , Animals , Humans , Intestinal Mucosa/metabolism , Mice , Mole Rats/genetics , Mole Rats/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , RNA-Binding Proteins/geneticsABSTRACT
Plants activate immunity upon recognition of pathogen-associated molecular patterns. Although phytopathogens have evolved a set of effector proteins to counteract plant immunity, some effectors are perceived by hosts and induce immune responses. Here, we show that two secreted ribonuclease effectors, SRN1 and SRN2, encoded in a phytopathogenic fungus, Colletotrichum orbiculare, induce cell death in a signal peptide- and catalytic residue-dependent manner, when transiently expressed in Nicotiana benthamiana. The pervasive presence of SRN genes across Colletotrichum species suggested the conserved roles. Using a transient gene expression system in cucumber (Cucumis sativus), an original host of C. orbiculare, we show that SRN1 and SRN2 potentiate host pattern-triggered immunity responses. Consistent with this, C. orbiculare SRN1 and SRN2 deletion mutants exhibited increased virulence on the host. In vitro analysis revealed that SRN1 specifically cleaves single-stranded RNAs at guanosine, leaving a 3'-end phosphate. Importantly, the potentiation of C. sativus responses by SRN1 and SRN2, present in the apoplast, depends on ribonuclease catalytic residues. We propose that the pathogen-derived apoplastic guanosine-specific single-stranded endoribonucleases lead to immunity potentiation in plants.
Subject(s)
Cucumis sativus , Ribonucleases , Cucumis sativus/microbiology , Fungi , Plants , Immunity , Plant Diseases/microbiology , Plant ImmunityABSTRACT
Proteins are typically denatured and aggregated by heating at near-boiling temperature. Exceptions to this principle include highly disordered and heat-resistant proteins found in extremophiles, which help these organisms tolerate extreme conditions such as drying, freezing, and high salinity. In contrast, the functions of heat-soluble proteins in non-extremophilic organisms including humans remain largely unexplored. Here, we report that heat-resistant obscure (Hero) proteins, which remain soluble after boiling at 95°C, are widespread in Drosophila and humans. Hero proteins are hydrophilic and highly charged, and function to stabilize various "client" proteins, protecting them from denaturation even under stress conditions such as heat shock, desiccation, and exposure to organic solvents. Hero proteins can also block several different types of pathological protein aggregations in cells and in Drosophila strains that model neurodegenerative diseases. Moreover, Hero proteins can extend life span of Drosophila. Our study reveals that organisms naturally use Hero proteins as molecular shields to stabilize protein functions, highlighting their biotechnological and therapeutic potential.
Subject(s)
Drosophila Proteins/metabolism , Animals , Animals, Genetically Modified , Argonaute Proteins/chemistry , Argonaute Proteins/genetics , Argonaute Proteins/metabolism , DNA-Binding Proteins/metabolism , Desiccation , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Eye/pathology , HEK293 Cells , Hot Temperature , Humans , Hydrophobic and Hydrophilic Interactions , L-Lactate Dehydrogenase/chemistry , L-Lactate Dehydrogenase/metabolism , Longevity , Male , Motor Neurons/pathology , Motor Neurons/physiology , Protein Stability , Retinal Degeneration/genetics , Retinal Degeneration/pathology , SolubilityABSTRACT
Although ribosome-profiling and translation initiation sequencing (TI-seq) analyses have identified many noncanonical initiation codons, the precise detection of translation initiation sites (TISs) remains a challenge, mainly because of experimental artifacts of such analyses. Here, we describe a new method, TISCA (TIS detection by translation Complex Analysis), for the accurate identification of TISs. TISCA proved to be more reliable for TIS detection compared with existing tools, and it identified a substantial number of near-cognate codons in Kozak-like sequence contexts. Analysis of proteomics data revealed the presence of methionine at the NH2-terminus of most proteins derived from near-cognate initiation codons. Although eukaryotic initiation factor 2 (eIF2), eIF2A and eIF2D have previously been shown to contribute to translation initiation at near-cognate codons, we found that most noncanonical initiation events are most probably dependent on eIF2, consistent with the initial amino acid being methionine. Comprehensive identification of TISs by TISCA should facilitate characterization of the mechanism of noncanonical initiation.
Subject(s)
Codon, Initiator , Eukaryotic Initiation Factor-2/metabolism , Peptide Chain Initiation, Translational , Computational Biology/methods , Eukaryotic Initiation Factor-3/metabolism , HEK293 Cells , Humans , Open Reading Frames , Protein Footprinting , Proteomics , Sequence Analysis, RNAABSTRACT
Oxytocin is a central neuromodulator required for facilitating mate preferences for familiar individuals in a monogamous rodent (prairie vole), irrespective of sex. While the role of oxytocin in mate choice is only understood in a few monogamous species, its function in nonmonogamous species, comprising the vast majority of vertebrate species, remains unclear. To address this issue, we evaluated the involvement of an oxytocin homolog (isotocin, referred herein as oxt) in mate choice in medaka fish (Oryzias latipes). Female medaka prefer to choose familiar mates, whereas male medaka court indiscriminately, irrespective of familiarity. We generated mutants of the oxt ligand (oxt) and receptor genes (oxtr1 and oxtr2) and revealed that the oxt-oxtr1 signaling pathway was essential for eliciting female mate preference for familiar males. This pathway was also required for unrestricted and indiscriminate mating strategy in males. That is, either oxt or oxtr1 mutation in males decreased the number of courtship displays toward novel females, but not toward familiar females. Further, males with these mutations exhibited enhanced mate-guarding behaviors toward familiar females, but not toward novel females. In addition, RNA-sequencing (seq) analysis revealed that the transcription of genes involved in gamma-amino butyric acid metabolism as well as those encoding ion-transport ATPase are up-regulated in both oxt and oxtr1 mutants only in female medaka, potentially explaining the sex difference of the mutant phenotype. Our findings provide genetic evidence that oxt-oxtr1 signaling plays a role in the mate choice for familiar individuals in a sex-specific manner in medaka fish.
Subject(s)
Mating Preference, Animal/physiology , Oryzias/genetics , Oryzias/physiology , Oxytocin/genetics , Oxytocin/physiology , Reproduction/physiology , Animals , Courtship , Female , Male , Mutation , Oxytocin/analogs & derivatives , Phenotype , Receptors, Oxytocin/genetics , Receptors, Oxytocin/metabolism , Recognition, Psychology , Reproduction/genetics , Sex Characteristics , Sexual Behavior, Animal/physiologyABSTRACT
Rocaglamide A (RocA) typifies a class of protein synthesis inhibitors that selectively kill aneuploid tumour cells and repress translation of specific messenger RNAs. RocA targets eukaryotic initiation factor 4A (eIF4A), an ATP-dependent DEAD-box RNA helicase; its messenger RNA selectivity is proposed to reflect highly structured 5' untranslated regions that depend strongly on eIF4A-mediated unwinding. However, rocaglate treatment may not phenocopy the loss of eIF4A activity, as these drugs actually increase the affinity between eIF4A and RNA. Here we show that secondary structure in 5' untranslated regions is only a minor determinant for RocA selectivity and that RocA does not repress translation by reducing eIF4A availability. Rather, in vitro and in cells, RocA specifically clamps eIF4A onto polypurine sequences in an ATP-independent manner. This artificially clamped eIF4A blocks 43S scanning, leading to premature, upstream translation initiation and reducing protein expression from transcripts bearing the RocA-eIF4A target sequence. In elucidating the mechanism of selective translation repression by this lead anti-cancer compound, we provide an example of a drug stabilizing sequence-selective RNA-protein interactions.
Subject(s)
Benzofurans/pharmacology , Eukaryotic Initiation Factor-4A/metabolism , Protein Biosynthesis/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Repressor Proteins/metabolism , 5' Untranslated Regions/genetics , Adenosine Triphosphate/metabolism , DEAD-box RNA Helicases/chemistry , DEAD-box RNA Helicases/metabolism , Eukaryotic Initiation Factor-4A/chemistry , HEK293 Cells , Humans , Hydrolysis , Nucleic Acid Conformation , Nucleotide Motifs , Open Reading Frames/genetics , Peptide Chain Initiation, Translational/drug effects , Protein Binding/drug effects , Protein Stability/drug effects , RNA, Messenger/chemistry , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Repressor Proteins/chemistry , Substrate Specificity/drug effects , ThermodynamicsABSTRACT
Protein synthesis stands at the last stage of the central dogma of molecular biology, providing a final regulatory layer for gene expression. Reacting to environmental cues and internal signals, the translation machinery can quickly tune the translatome from a pre-existing pool of RNAs, before the transcriptome changes. Although the translation reaction itself has been known since the 1950s, the quantitative or even qualitative measurement of its efficacy in cells has posed experimental and analytic hurdles. In this review, we outline the array of state-of-the-art methods that have emerged to tackle the hidden aspects of translational control.