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Rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) is an important economic cold-water fish that is susceptible to heat stress. To date, the heat stress response in rainbow trout is more widely understood at the transcriptional level, while little research has been conducted at the translational level. To reveal the translational regulation of heat stress in rainbow trout, in this study, we performed a ribosome profiling assay of rainbow trout liver under normal and heat stress conditions. Comparative analysis of the RNA-seq data with the ribosome profiling data showed that the folding changes in gene expression at the transcriptional level are moderately correlated with those at the translational level. In total, 1213 genes were significantly altered at the translational level. However, only 32.8% of the genes were common between both levels, demonstrating that heat stress is coordinated across both transcriptional and translational levels. Moreover, 809 genes exhibited significant differences in translational efficiency (TE), with the TE of these genes being considerably affected by factors such as the GC content, coding sequence length, and upstream open reading frame (uORF) presence. In addition, 3468 potential uORFs in 2676 genes were identified, which can potentially affect the TE of the main open reading frames. In this study, Ribo-seq and RNA-seq were used for the first time to elucidate the coordinated regulation of transcription and translation in rainbow trout under heat stress. These findings are expected to contribute novel data and theoretical insights to the international literature on the thermal stress response in fish. [ABSTRACT FROM AUTHOR]

Viruses are obligate, intracellular parasites that co-opt host cell machineries for propagation. Critical among these machineries are those that translate RNA into protein and their mechanisms of control. Most regulatory mechanisms effectuate their activity by targeting sequence or structural features at the RNA termini, i.e., at the 5′ or 3′ ends, including the untranslated regions (UTRs). Translation of most eukaryotic mRNAs is initiated by 5′ cap-dependent scanning. In contrast, many viruses initiate translation at internal RNA regions at internal ribosome entry sites (IRESs). Eukaryotic mRNAs often contain upstream open reading frames (uORFs) that permit condition-dependent control of downstream major ORFs. To offset genome compression and increase coding capacity, some viruses take advantage of out-of-frame overlapping uORFs (oORFs). Lacking the essential machinery of protein synthesis, for example, ribosomes and other translation factors, all viruses utilize the host apparatus to generate virus protein. In addition, some viruses exhibit RNA elements that bind host regulatory factors that are not essential components of the translation machinery. SARS-CoV-2 is a paradigm example of a virus taking advantage of multiple features of eukaryotic host translation control: the virus mimics the established human GAIT regulatory element and co-opts four host aminoacyl tRNA synthetases to form a stimulatory binding complex. Utilizing discontinuous transcription, the elements are present and identical in all SARS-CoV-2 subgenomic RNAs (and the genomic RNA). Thus, the virus exhibits a post-transcriptional regulon that improves upon analogous eukaryotic regulons, in which a family of functionally related mRNA targets contain elements that are structurally similar but lacking sequence identity. This "thrifty" virus strategy can be exploited against the virus since targeting the element can suppress the expression of all subgenomic RNAs as well as the genomic RNA. Other 3′ end viral elements include 3′-cap-independent translation elements (3′-CITEs) and 3′-tRNA-like structures. Elucidation of virus translation control elements, their binding proteins, and their mechanisms can lead to novel therapeutic approaches to reduce virus replication and pathogenicity. [ABSTRACT FROM AUTHOR]

BackgroundThe eukaryotic parasite Plasmodium falciparum causes millions of malarial infections annually while drug resistance to common anti-malarials is further confounding eradication efforts. Translation is an attractive therapeutic target that will benefit from a deeper mechanistic understanding. As the rate limiting step of translation, initiation is a primary driver of translational efficiency. It is a complex process regulated by both cis and trans acting factors, providing numerous potential targets. Relative to model organisms and humans, P. falciparum mRNAs feature unusual 5' untranslated regions suggesting cis-acting sequence complexity in this parasite may act to tune levels of protein synthesis through their effects on translational efficiency.MethodsHere, in vitro translation is deployed to compare the role of cis-acting regulatory sequences in P. falciparum and humans. Using parasite mRNAs with high or low translational efficiency, the presence, position, and termination status of upstream "AUG"s, in addition to the base composition of the 5' untranslated regions, were characterized.ResultsThe density of upstream "AUG"s differed significantly among the most and least efficiently translated genes in P. falciparum, as did the average "GC" content of the 5' untranslated regions. Using exemplars from highly translated and poorly translated mRNAs, multiple putative upstream elements were interrogated for impact on translational efficiency. Upstream "AUG"s were found to repress translation to varying degrees, depending on their position and context, while combinations of upstream "AUG"s had non-additive effects. The base composition of the 5' untranslated regions also impacted translation, but to a lesser degree. Surprisingly, the effects of cis-acting sequences were remarkably conserved between P. falciparum and humans.ConclusionsWhile translational regulation is inherently complex, this work contributes toward a more comprehensive understanding of parasite and human translational regulation by examining the impact of discrete cis-acting features, acting alone or in context.

The yield and quality of potatoes, an important staple crop, are seriously threatened by high temperature and drought stress. In order to deal with this adverse environment, plants have evolved a series of response mechanisms. However, the molecular mechanism of potato's response to environmental changes at the translational level is still unclear. In this study, we performed transcriptome- and ribosome-profiling assays with potato seedlings growing under normal, drought, and high-temperature conditions to reveal the dynamic translational landscapes for the first time. The translational efficiency was significantly affected by drought and heat stress in potato. A relatively high correlation (0.88 and 0.82 for drought and heat stress, respectively) of the fold changes of gene expression was observed between the transcriptional level and translational level globally based on the ribosome-profiling and RNA-seq data. However, only 41.58% and 27.69% of the different expressed genes were shared by transcription and translation in drought and heat stress, respectively, suggesting that the transcription or translation process can be changed independently. In total, the translational efficiency of 151 (83 and 68 for drought and heat, respectively) genes was significantly changed. In addition, sequence features, including GC content, sequence length, and normalized minimal free energy, significantly affected the translational efficiencies of genes. In addition, 28,490 upstream open reading frames (uORFs) were detected on 6463 genes, with an average of 4.4 uORFs per gene and a median length of 100 bp. These uORFs significantly affected the translational efficiency of downstream major open reading frames (mORFs). These results provide new information and directions for analyzing the molecular regulatory network of potato seedlings in response to drought and heat stress. [ABSTRACT FROM AUTHOR]

Viruses are obligate, intracellular parasites that co-opt host cell machineries for propagation. Critical among these machineries are those that translate RNA into protein and their mechanisms of control. Most regulatory mechanisms effectuate their activity by targeting sequence or structural features at the RNA termini, i.e., at the 5′ or 3′ ends, including the untranslated regions (UTRs). Translation of most eukaryotic mRNAs is initiated by 5′ cap-dependent scanning. In contrast, many viruses initiate translation at internal RNA regions at internal ribosome entry sites (IRESs). Eukaryotic mRNAs often contain upstream open reading frames (uORFs) that permit condition-dependent control of downstream major ORFs. To offset genome compression and increase coding capacity, some viruses take advantage of out-of-frame overlapping uORFs (oORFs). Lacking the essential machinery of protein synthesis, for example, ribosomes and other translation factors, all viruses utilize the host apparatus to generate virus protein. In addition, some viruses exhibit RNA elements that bind host regulatory factors that are not essential components of the translation machinery. SARS-CoV-2 is a paradigm example of a virus taking advantage of multiple features of eukaryotic host translation control: the virus mimics the established human GAIT regulatory element and co-opts four host aminoacyl tRNA synthetases to form a stimulatory binding complex. Utilizing discontinuous transcription, the elements are present and identical in all SARS-CoV-2 subgenomic RNAs (and the genomic RNA). Thus, the virus exhibits a post-transcriptional regulon that improves upon analogous eukaryotic regulons, in which a family of functionally related mRNA targets contain elements that are structurally similar but lacking sequence identity. This “thrifty” virus strategy can be exploited against the virus since targeting the element can suppress the expression of all subgenomic RNAs as well as the genomic RNA. Other 3′ end viral elements include 3′-cap-independent translation elements (3′-CITEs) and 3′-tRNA-like structures. Elucidation of virus translation control elements, their binding proteins, and their mechanisms can lead to novel therapeutic approaches to reduce virus replication and pathogenicity.

Abstract Background The eukaryotic parasite Plasmodium falciparum causes millions of malarial infections annually while drug resistance to common anti-malarials is further confounding eradication efforts. Translation is an attractive therapeutic target that will benefit from a deeper mechanistic understanding. As the rate limiting step of translation, initiation is a primary driver of translational efficiency. It is a complex process regulated by both cis and trans acting factors, providing numerous potential targets. Relative to model organisms and humans, P. falciparum mRNAs feature unusual 5′ untranslated regions suggesting cis-acting sequence complexity in this parasite may act to tune levels of protein synthesis through their effects on translational efficiency. Methods Here, in vitro translation is deployed to compare the role of cis-acting regulatory sequences in P. falciparum and humans. Using parasite mRNAs with high or low translational efficiency, the presence, position, and termination status of upstream “AUG”s, in addition to the base composition of the 5′ untranslated regions, were characterized. Results The density of upstream “AUG”s differed significantly among the most and least efficiently translated genes in P. falciparum, as did the average “GC” content of the 5′ untranslated regions. Using exemplars from highly translated and poorly translated mRNAs, multiple putative upstream elements were interrogated for impact on translational efficiency. Upstream “AUG”s were found to repress translation to varying degrees, depending on their position and context, while combinations of upstream “AUG”s had non-additive effects. The base composition of the 5′ untranslated regions also impacted translation, but to a lesser degree. Surprisingly, the effects of cis-acting sequences were remarkably conserved between P. falciparum and humans. Conclusions While translational regulation is inherently complex, this work contributes toward a more comprehensive understanding of parasite and human translational regulation by examining the impact of discrete cis-acting features, acting alone or in context.

The yield and quality of potatoes, an important staple crop, are seriously threatened by high temperature and drought stress. In order to deal with this adverse environment, plants have evolved a series of response mechanisms. However, the molecular mechanism of potato’s response to environmental changes at the translational level is still unclear. In this study, we performed transcriptome- and ribosome-profiling assays with potato seedlings growing under normal, drought, and high-temperature conditions to reveal the dynamic translational landscapes for the first time. The translational efficiency was significantly affected by drought and heat stress in potato. A relatively high correlation (0.88 and 0.82 for drought and heat stress, respectively) of the fold changes of gene expression was observed between the transcriptional level and translational level globally based on the ribosome-profiling and RNA-seq data. However, only 41.58% and 27.69% of the different expressed genes were shared by transcription and translation in drought and heat stress, respectively, suggesting that the transcription or translation process can be changed independently. In total, the translational efficiency of 151 (83 and 68 for drought and heat, respectively) genes was significantly changed. In addition, sequence features, including GC content, sequence length, and normalized minimal free energy, significantly affected the translational efficiencies of genes. In addition, 28,490 upstream open reading frames (uORFs) were detected on 6463 genes, with an average of 4.4 uORFs per gene and a median length of 100 bp. These uORFs significantly affected the translational efficiency of downstream major open reading frames (mORFs). These results provide new information and directions for analyzing the molecular regulatory network of potato seedlings in response to drought and heat stress.

The MYCC and MYCN loci are each associated with two upstream open reading frames (uORFs) potentially encoding small proteins (9-21 kDa). We previously demonstrated that uORFs mrtl and MYCHEX1 of MYCC are translated, and their protein products may function to regulate the expression of the "parent" oncogene. We hypothesized that a similar relationship might exist between MYCN and its two uORFs: MYCNOT and MNOP, and investigated the uORF-encoded proteins associated with MYCN to confirm their expression and intracellular location in neuroblastoma and medulloblastoma cells and tissues. MNOP, MYCNOT, mrtl, and MYCHEX1 were readily detected via reverse transcription polymerase chain reaction and Western blot analysis in tumor cell lines. In tumor tissue, MNOP protein expression was confirmed; however, MCYNOT generated from alternative splicing MYCNΔ1b mRNA was not detected. Immunofluorescence staining of MYCNOT displayed multiple bright foci in the nucleus and diffuse staining in the cytoplasm, suggesting that this small protein may function in both the nucleus and cytoplasm. Upon JQ1 treatment, MYCN, MYCNOT, and mrtl decreased substantially or disappeared completely in three different tumor cell lines. Significant levels of apoptosis were observed in each pediatric embryonal tumor cell line but not T47D breast carcinoma cells, suggesting that response to JQ1 transcriptional inhibition is greatest in tumor cells, which depend on MYC to maintain an undifferentiated phenotype. In conclusion, both MYCN uORF-encoded proteins MNOP and MYCNOT, together with the two MYCC uORF-encoded proteins mrtl and MYCHEX1 were detected simultaneously in tumor cell lines and tumor tissues. These four distinct proteins are translated from the "5'-untranslated region" of MYCN or MYCC mRNA and display consistent distribution patterns within the cell. Additional studies to further elucidate the physiological and pathological roles of these uORF-encoded proteins are warranted, as insights gained could inform new strategies for modulating MYC-family oncogenes by targeting their uORFs., (© 2023 Wiley Periodicals LLC.)