eIF3 and Its mRNA-Entry-Channel Arm Contribute to the Recruitment of mRNAs With Long 5′-Untranslated Regions

Translation initiation in eukaryotes is a multi-step pathway and the most regulated phase of translation. Eukaryotic initiation factor 3 (eIF3) is the largest and most complex of the translation initiation factors, and it contributes to events throughout the initiation pathway. In particular, eIF3 appears to play critical roles in mRNA recruitment. More recently, eIF3 has been implicated in driving the selective translation of specific classes of mRNAs. However, unraveling the mechanism of these diverse contributions—and disentangling the roles of the individual subunits of the eIF3 complex—remains challenging. We employed ribosome profiling of budding yeast cells expressing two distinct mutations targeting the eIF3 complex. These mutations either disrupt the entire complex or subunits positioned near the mRNA-entry channel of the ribosome and which appear to relocate during or in response to mRNA binding and start-codon recognition. Disruption of either the entire eIF3 complex or specific targeting of these subunits affects mRNAs with long 5′-untranslated regions and whose translation is more dependent on eIF4A, eIF4B, and Ded1 but less dependent on eIF4G, eIF4E, and PABP. Disruption of the entire eIF3 complex further affects mRNAs involved in mitochondrial processes and with structured 5′-untranslated regions. Comparison of the suite of mRNAs most sensitive to both mutations with those uniquely sensitive to disruption of the entire complex sheds new light on the specific roles of individual subunits of the eIF3 complex.

Comparative Analysis of the Complete Mitochondrial Genomes of Five Species of Ricaniidae (Hemiptera: Fulgoromorpha) and Phylogenetic Implications

Ricaniidae is a relatively small planthopper family with about 69 genera and 442 species worldwide. Members of this family occur throughout the warm temperate and tropical regions. Some species cause devastating damage to major agricultural and economic plants. However, the relationship between Ricaniidae and other families of Fulgoroidea needs to be further explored. The morphological definitions of the two biggest genera, Pochazia Amyot & Serville, 1843 and Ricania Germar, 1818 (the type genus of Ricaniidae) remain controversial. In this study, mitogenomes of five representatives in these two genera were decoded using the next-generation sequence method and genome assembly. Results showed that their complete mitogenomes are circular DNA molecules with 15,457 to 16,411 bp. All protein-coding genes (PCGs) begin with the start codon ATN, GTG or TTG and end with TAA, TAG, an incomplete stop codon single T or an incomplete stop codon single A. A lost DHU arm was discovered in the trnS gene of the five mitogenomes and the trnV gene within Pochaziaconfusa, Pochazia guttifera and Ricania simulans. The remnant tRNAs folded into clover-leaf structures. The sliding window, genetic distance, and Ka/Ks analyses indicated that the cox1 gene is the slowest evolving and is relatively conserved. The phylogenetic tree topologies support (Delphacidae + (((Issidae + (Lophopidae + Caliscelidae)) + (Flatidae + Ricaniidae)) + (Achilidae + (Dictyopharidae + Fulgoridae)))) as the best topology, as recognized by both PhyloBayes, RAxML and MrBayes based on four data sets (PCG, PCGRNA, PCG12, PCG12RNA). The monophyly of Ricaniidae and the sister group status of two families Flatidae and Ricaniidae are supported, but all analyses failed to support the monophyly of Pochazia and Ricania. The diagnoses between these two genera cannot be resolved until more evidence is acquired.

Mitogenomes of Nine Asian Skipper Genera and Their Phylogenetic Position (Lepidoptera: Hesperiidae: Pyrginae)

In this study, complete mitochondrial genomes of nine species representing three tribes in the subfamily Pyrginae sensu lato were newly sequenced. The mitogenomes are closed double-stranded circular molecules, with the length ranging from 15,232 bp to 15,559 bp, which all encode 13 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and a control region. The orientation and gene order of these nine mitogenomes are identical to the inferred ancestral arrangement of insects. All PCGs exhibit the typical start codon ATN except for cox1 (using CGA) and cox2 (using TTG) in Mooreana trichoneura. Most of the PCGs terminate with a TAA stop codon, while cox1, cox2, nad4, and nad5 end with the incomplete codon single T. For the different datasets, we found that the one comprising all 37 genes of the mitogenome produced the highest nodal support, indicating that the inclusion of RNAs improves the phylogenetic signal. This study re-confirmed the status of Capila, Pseudocoladenia, and Sarangesa; namely, Capila belongs to the tribe Tagiadini, and Pseudocoladenia and Sarangesa to the tribe Celaenorrhini. Diagnostic characters distinguishing the two tribes, the length of the forewing cell and labial palpi, are no longer significant. Two populations of Pseudocoladenia dan fabia from China and Myanmar and P. dan dhyana from Thailand are confirmed as conspecific.

Variability in an effector gene promoter of a necrotrophic fungal pathogen dictates epistasis and effector-triggered susceptibility in wheat

The fungus Parastagonospora nodorum uses proteinaceous necrotrophic effectors (NEs) to induce tissue necrosis on wheat leaves during infection, leading to the symptoms of septoria nodorum blotch (SNB). The NEs Tox1 and Tox3 induce necrosis on wheat possessing the dominant susceptibility genes Snn1 and Snn3B1/Snn3D1, respectively. We previously observed that Tox1 is epistatic to the expression of Tox3 and a quantitative trait locus (QTL) on chromosome 2A that contributes to SNB resistance/susceptibility. The expression of Tox1 is significantly higher in the Australian strain SN15 compared to the American strain SN4. Inspection of the Tox1 promoter region revealed a 401 bp promoter genetic element in SN4 positioned 267 bp upstream of the start codon that is absent in SN15, called PE401. Analysis of the world-wide P. nodorum population revealed that a high proportion of Northern Hemisphere isolates possess PE401 whereas the opposite was observed in representative P. nodorum isolates from Australia and South Africa. The presence of PE401 removed the epistatic effect of Tox1 on the contribution of the SNB 2A QTL but not Tox3. PE401 was introduced into the Tox1 promoter regulatory region in SN15 to test for direct regulatory roles. Tox1 expression was markedly reduced in the presence of PE401. This suggests a repressor molecule(s) binds PE401 and inhibits Tox1 transcription. Infection assays also demonstrated that P. nodorum which lacks PE401 is more pathogenic on Snn1 wheat varieties than P. nodorum carrying PE401. An infection competition assay between P. nodorum isogenic strains with and without PE401 indicated that the higher Tox1-expressing strain rescued the reduced virulence of the lower Tox1-expressing strain on Snn1 wheat. Our study demonstrated that Tox1 exhibits both ‘selfish’ and ‘altruistic’ characteristics. This offers an insight into a complex NE-NE interaction that is occurring within the P. nodorum population. The importance of PE401 in breeding for SNB resistance in wheat is discussed.

Analysis of genetic diversity in Prunus sibirica L. in inner Mongolia using SCoT molecular markers

Population genetic diversity contributes to the protection and utilization of germplasm resources, especially via genetic breeding. In the present study, start codon targeted polymorphism (SCoT) molecular markers were used to study the genetic diversity of 278 individuals from 10 Prunus sibirica L. populations in Inner Mongolia. A total of 289 polymorphic bands were amplified with 23 SCoT primers, showing a polymorphism percentage of 98.87% and an average of 12.6 polymorphic bands per primer. The SCoT21, SCoT32, and SCoT53 primers amplified up to 17 bands, and the polymorphism percentage was 100%. The minimum number of bands amplified by SCoT25 was 9, and the polymorphism percentage was 90%. Therefore, SCoT molecular markers were shown to be highly polymorphic and suitable for genetic diversity studies of P. sibirica in Inner Mongolia. The analysis of molecular variance showed that 39% of the observed genetic differentiation occurred among populations and 61% occurred within populations, indicating that the genetic differentiation within populations was greater than that among populations. The results of the unweighted pair-group method with an arithmetic cluster analysis, principal coordinate analysis and STRUCTURE analysis were basically the same and divided the 278 individuals from the 10 populations into 2 groups. The results indicated that the efficient SCoT molecular marker-based genetic diversity analysis of P. sibirica in Inner Mongolia can provide a reference for P. sibirica variety breeding and resource development.

Case Report: Meningoencephalitis With Thrombotic Occlusive Vasculopathy in a Young EBV-Naïve Boy Is Associated With a Novel SH2D1A Mutation

X-linked lymphoproliferative disease (XLP1) is a combined immunodeficiency characterized by severe immune dysregulation caused by mutations in the SH2D1A/SAP gene. Loss or dysfunction of SH2D1A is associated with the inability in clearing Epstein-Barr-Virus (EBV) infections. Clinical manifestation is diverse and ranges from life-threatening hemophagocytic lymphohistiocytosis (HLH) and fulminant infectious mononucleosis (FIM) to lymphoma and antibody deficiency. Rare manifestations include aplastic anemia, chronic gastritis and vasculitis. Herein, we describe the case of a previously healthy eight-year old boy diagnosed with XLP1 presenting with acute non-EBV acute meningoencephalitis with thrombotic occlusive vasculopathy. The patient developed multiple cerebral aneurysms leading to repeated intracerebral hemorrhage and severe cerebral damage. Immunological examination was initiated after development of a susceptibility to infections with recurrent bronchitis and one episode of severe pneumonia and showed antibody deficiency with pronounced IgG1-3-4 subclass deficiency. We could identify a novel hemizygous SH2D1A point mutation affecting the start codon. Basal levels of SAP protein seemed to be detectable in CD8+ and CD4+ T- and CD56+ NK-cells of the patient what indicated an incomplete absence of SAP. In conclusion, we could demonstrate a novel SH2D1A mutation leading to deficient SAP protein expression and a rare clinical phenotype of non-EBV associated acute meningoencephalitis with thrombotic occlusive vasculopathy.

Conformational rearrangements upon start codon recognition in human 48S translation initiation complex

Selection of the translation start codon is a key step during protein synthesis in human cells. We obtained cryo-EM structures of human 48S initiation complexes and characterized the intermediates of codon recognition by kinetic methods using eIF1A as a reporter. Both approaches capture two distinct ribosome populations formed on an mRNA with a cognate AUG codon in the presence of eIF1, eIF1A, eIF2–GTP–Met-tRNAiMet, and eIF3. The ‘open’ 40S subunit conformation differs from the human 48S scanning complex and represents an intermediate preceding the codon recognition step. The ‘closed’ form is similar to reported structures of complexes from yeast and mammals formed upon codon recognition, except for the orientation of eIF1A, which is unique in our structure. Kinetic experiments show how various initiation factors mediate the population distribution of open and closed conformations until 60S subunit docking. Our results provide insights into the timing and structure of human translation initiation intermediates and suggest the differences in the mechanisms of start codon selection between mammals and yeast.

Genome-wide promoter analysis, homology modeling and protein interaction network of Dehydration Responsive Element Binding (DREB) gene family in Solanum tuberosum

Dehydration Responsive Element Binding (DREB) regulates the expression of numerous stress-responsive genes, and hence plays a pivotal role in abiotic stress responses and tolerance in plants. The study aimed to develop a complete overview of the cis-acting regulatory elements (CAREs) present in S. tuberosum DREB gene promoters. A total of one hundred and four (104) cis-regulatory elements (CREs) were identified from 2.5kbp upstream of the start codon (ATG). The in-silico promoter analysis revealed variable sets of cis-elements and functional diversity with the predominance of light-responsive (30%), development-related (20%), abiotic stress-responsive (14%), and hormone-responsive (12%) elements in StDREBs. Among them, two light-responsive elements (Box-4 and G-box) were predicted in 64 and 61 StDREB genes, respectively. Two development-related motifs (AAGAA-motif and as-1) were abundant in StDREB gene promoters. Most of the DREB genes contained one or more Myeloblastosis (MYB) and Myelocytometosis (MYC) elements associated with abiotic stress responses. Hormone-responsive element i.e. ABRE was found in 59 out of 66 StDREB genes, which implied their role in dehydration and salinity stress. Moreover, six proteins were chosen corresponding to A1-A6 StDREB subgroups for secondary structure analysis and three-dimensional protein modeling followed by model validation through PROCHECK server by Ramachandran Plot. The predicted models demonstrated >90% of the residues in the favorable region, which further ensured their reliability. The present study also anticipated pocket binding sites and disordered regions (DRs) to gain insights into the structural flexibility and functional annotation of StDREB proteins. The protein association network determined the interaction of six selected StDREB proteins with potato proteins encoded by other gene families such as MYB and NAC, suggesting their similar functional roles in biological and molecular pathways. Overall, our results provide fundamental information for future functional analysis to understand the precise molecular mechanisms of the DREB gene family in S. tuberosum.

Combinations of slow-translating codon clusters can increase mRNA half-life in Saccharomyces cerevisiae

The presence of a single cluster of nonoptimal codons was found to decrease a transcript’s half-life through the interaction of the ribosome-associated quality control machinery with stalled ribosomes in Saccharomyces cerevisiae. The impact of multiple nonoptimal codon clusters on a transcript’s half-life, however, is unknown. Using a kinetic model, we predict that inserting a second nonoptimal cluster near the 5′ end can lead to synergistic effects that increase a messenger RNA’s (mRNA’s) half-life in S. cerevisiae. Specifically, the 5′ end cluster suppresses the formation of ribosome queues, reducing the interaction of ribosome-associated quality control factors with stalled ribosomes. We experimentally validate this prediction by introducing two nonoptimal clusters into three different genes and find that their mRNA half-life increases up to fourfold. The model also predicts that in the presence of two clusters, the cluster closest to the 5′ end is the primary determinant of mRNA half-life. These results suggest the “translational ramp,” in which nonoptimal codons are located near the start codon and increase translational efficiency, may have the additional biological benefit of allowing downstream slow-codon clusters to be present without decreasing mRNA half-life. These results indicate that codon usage bias plays a more nuanced role in controlling cellular protein levels than previously thought.

Distinct mechanoreceptor pezo-1 isoforms modulate food intake in the nematode Caenorhabditis elegans

Two PIEZO mechanosensitive cation channels, PIEZO1 and PIEZO2, have been identified in mammals, where they are involved in numerous sensory processes. While structurally similar, PIEZO channels are expressed in distinct tissues and exhibit unique properties. How different PIEZOs transduce force, how their transduction mechanism varies, and how their unique properties match the functional needs of the tissues they are expressed in remain all-important unanswered questions. The nematode Caenorhabditis elegans has a single PIEZO ortholog (pezo-1) predicted to have twelve isoforms. These isoforms share many transmembrane domains but differ in those that distinguish PIEZO1 and PIEZO2 in mammals. We used transcriptional and translational reporters to show that putative promoter sequences immediately upstream of the start codon of long pezo-1 isoforms predominantly drive GFP expression in mesodermally derived tissues (such as muscle and glands). In contrast, sequences upstream of shorter pezo-1 isoforms resulted in GFP expression primarily in neurons. Putative promoters upstream of different isoforms drove GFP expression in different cells of the same organs of the digestive system. The observed unique pattern of complementary expression suggests that different isoforms could possess distinct functions within these organs. We used mutant analysis to show that pharyngeal muscles and glands require long pezo-1 isoforms to respond appropriately to the presence of food. The number of pezo-1 isoforms in C. elegans, their putative differential pattern of expression, and roles in experimentally tractable processes make this an attractive system to investigate the molecular basis for functional differences between members of the PIEZO family of mechanoreceptors.