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This review describes the current models for how the subunit abundance of the Ndc80 complex, a key kinetochore component, is regulated in budding yeast and metazoan meiosis. The past decades of kinetochore research have established the Ndc80 complex to be a key microtubule interactor and a central hub for regulating chromosome segregation. Recent studies further demonstrate that Ndc80 is the limiting kinetochore subunit that dictates the timing of kinetochore activation in budding yeast meiosis. Here, we discuss the molecular circuits that regulate Ndc80 protein synthesis and degradation in budding yeast meiosis and compare the findings with those from metazoans. We envision the regulatory principles discovered in budding yeast to be conserved in metazoans, thereby providing guidance into future investigations on kinetochore regulation in human health and disease.

Sugar metabolism not only determines fruit sweetness and quality but also acts as signaling molecules to substantially connect with other primary metabolic processes and, therefore, modulates plant growth and development, fruit ripening, and stress response. The basic region/leucine zipper motif (bZIP) transcription factor family is ubiquitous in eukaryotes and plays a diverse array of biological functions in plants. Among the bZIP family members, the smallest bZIP subgroup, S1-bZIP, is a unique one, due to the conserved upstream open reading frames (uORFs) in the 5′ leader region of their mRNA. The translated small peptides from these uORFs are suggested to mediate Sucrose-Induced Repression of Translation (SIRT), an important mechanism to maintain sucrose homeostasis in plants. Here, we review recent research on the evolution, sequence features, and biological functions of this bZIP subgroup. S1-bZIPs play important roles in fruit quality, abiotic and biotic stress responses, plant growth and development, and other metabolite biosynthesis by acting as signaling hubs through dimerization with the subgroup C-bZIPs and other cofactors like SnRK1 to coordinate the expression of downstream genes. Direction for further research and genetic engineering of S1-bZIPs in plants is suggested for the improvement of quality and safety traits of fruit.

Toxoplasma gondiiis a common parasite of humans and animals, causing life-threatening disease in the immunocompromized, fetal abnormalities when contracted during gestation, and recurrent ocular lesions in some patients. Central to the prevalence and pathogenicity of this protozoan is its ability to adapt to a broad range of environments, and to differentiate between acute and chronic stages. These processes are underpinned by a major rewiring of gene expression, yet the mechanisms that regulate transcription in this parasite are only partially characterized. Deciphering these mechanisms requires a precise and comprehensive map of transcription start sites (TSSs); however,ToxoplasmaTSSs have remained incompletely defined. To address this challenge, we used 5′-end RNA sequencing to genomically assess transcription initiation in both acute and chronic stages ofToxoplasma. Here, we report an in-depth analysis of transcription initiation at promoters, and provide empirically-defined TSSs for 7603 (91%) protein-coding genes, of which only 1840 concur with existing gene models. Comparing data from acute and chronic stages, we identified instances of stage-specific alternative TSSs that putatively generate mRNA isoforms with distinct 5′ termini. Analysis of the nucleotide content and nucleosome occupancy around TSSs allowed us to examine the determinants of TSS choice, and outline features ofToxoplasmapromoter architecture. We also found pervasive divergent transcription atToxoplasmapromoters, clustered within the nucleosomes of highly-symmetrical phased arrays, underscoring chromatin contributions to transcription initiation. Corroborating previous observations, we asserted thatToxoplasma5′ leaders are among the longest of any eukaryote studied thus far, displaying a median length of approximately 800 nucleotides. Further highlighting the utility of a precise TSS map, we pinpointed motifs associated with transcription initiation, including the binding sites of the master regulator of chronic-stage differentiation, BFD1, and a novel motif with a similar positional arrangement present at 44% ofToxoplasmapromoters. This work provides a critical resource for functional genomics inToxoplasma, and lays down a foundation to study the interactions between genomic sequences and the regulatory factors that control transcription in this parasite.

Eukaryotic mRNAs contain a 5' leader preceding the main open reading frame (mORF) and, depending on the species, 20-50% of eukaryotic mRNAs harbor an upstream ORF (uORF) in the 5' leader. An unknown fraction of these uORFs encode sequence conserved peptides (conserved peptide uORFs, CPuORFs). Most experimentally validated CPuORFs demonstrated to regulate the translation of the downstream main ORF, usually in a metabolite concentration dependent manner. To this end, comparative genomic approaches have been used to identify novel CPuORFs, by comparing AUG initiating uORF sequences of the Arabidopsis genome or Arabidopsis ESTs. Previous research has shown that most CPuORFs possess a start codon context suboptimal for translation initiation, which turns out to be favorable for translational regulation. The suboptimal initiation context may even include non-AUG start codons, which makes CPuORFs hard to predict. For this reason, we developed a novel pipeline to identify CPuORFs unbiased of start codon using well annotated sequence data from 32 eudicot plant species and rice. Our new pipeline was able to identify 30 novel Arabidopsis CPuORFs, conserved across a wide variety of eudicot species of which 16 do not initiate with an AUG start codon. In addition to CPuORFs, the pipeline was able to find 14 conserved coding regions directly upstream and in frame with the main ORF, which likely initiate translation on a non-AUG start codon. Altogether, our pipeline identified highly conserved coding regions in the 5' leaders of Arabidopsis transcripts, including in genes with proven functional importance such as LHY, a key regulator of the circadian clock, and the RAPTOR1 subunit of the Target Of Rapamycin (TOR) kinase.

Background With the development of rapid and inexpensive DNA sequencing, the genome sequences of more than 100 fungal species have been made available. This dataset provides an excellent resource for comparative genomics analyses, which can be used to discover genetic elements, including noncoding RNAs (ncRNAs). Bioinformatics tools similar to those used to uncover novel ncRNAs in bacteria, likewise, should be useful for searching fungal genomic sequences, and the relative ease of genetic experiments with some model fungal species could facilitate experimental validation studies. Results We have adapted a bioinformatics pipeline for discovering bacterial ncRNAs to systematically analyze many fungal genomes. This comparative genomics pipeline integrates information on conserved RNA sequence and structural features with alternative splicing information to reveal fungal RNA motifs that are candidate regulatory domains, or that might have other possible functions. A total of 15 prominent classes of structured ncRNA candidates were identified, including variant HDV self-cleaving ribozyme representatives, atypical snoRNA candidates, and possible structured antisense RNA motifs. Candidate regulatory motifs were also found associated with genes for ribosomal proteins, S-adenosylmethionine decarboxylase (SDC), amidase, and HexA protein involved in Woronin body formation. We experimentally confirm that the variant HDV ribozymes undergo rapid self-cleavage, and we demonstrate that the SDC RNA motif reduces the expression of SAM decarboxylase by translational repression. Furthermore, we provide evidence that several other motifs discovered in this study are likely to be functional ncRNA elements. Conclusions Systematic screening of fungal genomes using a computational discovery pipeline has revealed the existence of a variety of novel structured ncRNAs. Genome contexts and similarities to known ncRNA motifs provide strong evidence for the biological and biochemical functions of some newly found ncRNA motifs. Although initial examinations of several motifs provide evidence for their likely functions, other motifs will require more in-depth analysis to reveal their functions. Electronic supplementary material The online version of this article (10.1186/s12864-017-4171-y) contains supplementary material, which is available to authorized users.

Background Campomelic dysplasia (CD) is a semilethal developmental disorder caused by mutations in and around SOX9. CD is characterized by multiple skeletal malformations including bending (campomelia) of long bones. Surviving patients frequently have the acampomelic form of CD (ACD). Methods This is a single case report on a patient with clinical and radiological features of ACD who has no mutation in the SOX9 protein‐coding sequence nor a translocation with breakpoint in the SOX9 regulatory domain. We include functional studies of the novel mutant protein in vitro and in cultured cells. Results The patient was found to have a de novo heterozygous mutation c.‐185G>A in the SOX9 5′UTR. The mutation creates an upstream translation start codon, uAUG, with a much better fit of its flanking sequence to the Kozak consensus than the wild‐type AUG. By in vitro transcription‐translation and transient transfection into COS‐7 cells, we show that the uAUG leads to translation of a short peptide from a reading frame that terminates just after the wild‐type AUG start codon. This results in reduced translation of the wild‐type protein, compatible with the milder phenotype of the patient. Conclusion Findings support the notion that more mildly affected, surviving CD/ACD patients carry mutant SOX9 alleles with residual expression of SOX9 wild‐type protein. Although rarely described in human genetic disease and for the first time here for CD, mutations creating upstream AUG codons may be more common than generally assumed.

It is generally accepted that the presence of ORFs in the 5′ untranslated region of eukaryotic transcripts modulates the production of proteins by controlling the translation initiation rate of the main CDS. In trypanosomatid parasites, which almost exclusively depend on post-transcriptional mechanisms to regulate gene expression, translation has been identified as a key step. However, the mechanisms of control of translation are not fully understood. In the present work, we have annotated the 5′UTRs of the Trypanosoma cruzi genome both in epimastigotes and metacyclic trypomastigotes and, using a stringent classification approach, we identified putative regulatory uORFs in about 9% of the analyzed 5′UTRs. The translation efficiency (TE) and translational levels of transcripts containing putative repressive uORFs were found to be significantly reduced. These findings are supported by the fact that proteomic methods only identify a low number of proteins coded by transcripts containing repressive uORF. We additionally show that AUG is the main translation initiator codon of repressive uORFs in T. cruzi. Interestingly, the decrease in TE is more pronounced when the uORFs overlaps the main CDS. In conclusion, we show that the presence of the uORF and features such as initiation codon and/or location of the uORFs may be acting to fine tune translation levels in these parasites.

ACAULIS5 (ACL5) encodes thermospermine synthase in Arabidopsis and its loss-of-function mutant acl5 shows excess xylem differentiation and severe dwarfism. SAC51 encodes a basic helix-loop-helix (bHLH) protein and was identified from sac51-d, a dominant suppressor mutant of acl5, which restores the wild-type phenotype without thermospermine. The 5' leader of the SAC51 mRNA contains multiple upstream open-reading frames (uORFs) and sac51-d has a premature stop codon in the fourth uORF. This uORF is conserved among SAC51 family genes in vascular plants. According to the GUS reporter assay, the SAC51 promoter was not responsive to thermospermine but the SAC51 5' leader fused to the constitutive 35S promoter enhanced the GUS activity in response to thermospermine. Disruption experiments of each start codon of the SAC51 uORFs revealed that uORF4 and uORF6 whose start codon corresponds to the second methionine codon of uORF4 had an inhibitory effect on the main ORF translation while the other four uORFs rather had a stimulatory effect. The response of the 5' leader to thermospermine was retained after disruption of each one of six start codons of these uORFs but abolished by mutating both uORF4 and uORF6 start codons, suggesting the importance of the C-terminal sequence shared by these uORFs in the action of thermospermine. We introduced GUS fusions with 5' leaders of SAC51 family genes from other angiosperm species into Arabidopsis and found that all 5' leaders responsive to thermospermine, so far examined, contained these two conserved, and overlapping uORFs.

The acaulis5 (acl5) mutant of Arabidopsis thaliana is defective in the biosynthesis of thermospermine and shows a dwarf phenotype associated with excess xylem differentiation. SAC51 was identified from a dominant suppressor of acl5, sac51-d, and encodes a basic helix-loop-helix protein. The sac51-d mutant has a premature termination codon in an upstream open reading frame (uORF) that is conserved among all four members of the SAC51 family, SAC51 and SACL1-SACL3 This suggests that thermospermine cancels the inhibitory effect of the uORF in main ORF translation. Another suppressor, sac57-d, has a mutation in the conserved uORF of SACL3 To define further the function of the SAC51 family in the thermospermine response, we analyzed T-DNA insertion mutants of each gene. Although sacl1-1 may not be a null allele, the quadruple mutant showed a semi-dwarf phenotype but with an increased level of thermospermine and decreased sensitivity to exogenous thermospermine that normally represses xylem differentiation. The sac51-1 sacl3-1 double mutant was also insensitive to thermospermine. These results suggest that SAC51 and SACL3 play a key role in thermospermine-dependent negative control of thermospermine biosynthesis and xylem differentiation. Using 5' leader-GUS (β-glucuronidase) fusion constructs, however, we detected a significant enhancement of the GUS activity by thermospermine only in SAC51 and SACL1 constructs. Furthermore, while acl5-1 sac51-1 showed the acl5 dwarf phenotype, acl5-1 sacl3-1 exhibited an extremely tiny-plant phenotype. These results suggest a complex regulatory network for the thermospermine response in which SAC51 and SACL3 function in parallel pathways.

To better understand the gene regulatory mechanisms that program developmental processes, we carried out simultaneous, genome-wide measurements of mRNA, translation and protein through meiotic differentiation in budding yeast. Surprisingly, we observed that the levels of several hundred mRNAs are anti-correlated with their corresponding protein products. We show that rather than arising from canonical forms of gene regulatory control, the regulation of at least 380 such cases—or over 8% of all measured genes—involves temporally regulated switching between production of a canonical, translatable transcript and a 5′ extended isoform that is not efficiently translated into protein. By this pervasive mechanism for the modulation of protein levels through a natural developmental program, a single transcription factor can coordinately activate and repress protein synthesis for distinct sets of genes. The distinction is not based on whether an mRNA is induced or not, but rather based on the type of transcript produced.

The boundaries of protein coding sequences are more difficult to define at the 5' end than at the 3' end due to potential multiple translation initiation sites (TISs). Even in the presence of phylogenetic data, the use of sequence information only may not be sufficient for the accurate identification of TISs. Traditional proteomics approaches may also fail because the N-termini of newly synthesized proteins are often processed. Thus ribosome profiling (ribo-seq), producing a snapshot of the ribosome distribution across the entire transcriptome, is an attractive experimental technique for the purpose of TIS location exploration. The GWIPS-viz (Genome Wide Information on Protein Synthesis visualized) browser (http://gwips.ucc.ie) provides free access to the genomic alignments of ribo-seq data and corresponding mRNA-seq data along with relevant annotation tracks. In this brief, we illustrate how GWIPS-viz can be used to explore the ribosome occupancy at the 5' ends of protein coding genes to assess the activity of AUG and non-AUG TISs responsible for the synthesis of proteoforms with alternative or heterogeneous N-termini. The presence of ribo-seq tracks for various organisms allows for cross-species comparison of orthologous genes and the availability of datasets from multiple laboratories permits the assessment of the technical reproducibility of the ribosome densities.

PTCH1 gene codes for a 12-pass transmembrane receptor with a negative regulatory role in the Hedgehog-Gli signaling pathway. PTCH1 germline mutations cause Gorlin syndrome, a disorder characterized by developmental abnormalities and tumor susceptibility. The autosomal dominant inheritance, and the evidence for PTCH1 haploinsufficiency, suggests that fine-tuning systems of protein patched homolog 1 (PTC1) levels exist to properly regulate the pathway. Given the role of 5' untranslated region (5'UTR) in protein expression, our aim was to thoroughly explore cis-regulatory elements in the 5'UTR of PTCH1 transcript 1b. The (CGG)n polymorphism was the main potential regulatory element studied so far but with inconsistent results and no clear association between repeat number and disease risk. Using luciferase reporter constructs in human cell lines here we show that the number of CGG repeats has no strong impact on gene expression, both at mRNA and protein levels. We observed variability in the length of 5'UTR and changes in abundance of the associated transcripts after pathway activation. We show that upstream AUG codons (uAUGs) present only in longer 5'UTRs could negatively regulate the amount of PTC1 isoform L (PTC1-L). The existence of an internal ribosome entry site (IRES) observed using different approaches and mapped in the region comprising the CGG repeats, would counteract the effect of the uAUGs and enable synthesis of PTC1-L under stressful conditions, such as during hypoxia. Higher relative translation efficiency of PTCH1b mRNA in HEK 293T cultured hypoxia was observed by polysomal profiling and Western blot analyses. All our results point to an exceptionally complex and so far unexplored role of 5'UTR PTCH1b cis-element features in the regulation of the Hedgehog-Gli signaling pathway.

International audience; A large body of evidence indicates that genome annotation pipelines have biased our view of coding sequences because they generally undersample small proteins and peptides. The recent development of genome-wide translation profiling reveals the prevalence of small/short open reading frames (smORFs or sORFs), which are scattered over all classes of transcripts, including both mRNAs and presumptive long noncoding RNAs. Proteomic approaches further confirm an unexpected variety of smORF-encoded peptides (SEPs), representing an overlooked reservoir of bioactive molecules. Indeed, functional studies in a broad range of species from yeast to humans demonstrate that SEPs can harbor key activities for the control of development, differentiation, and physiology. Here we summarize recent advances in the discovery and functional characterization of smORF/SEPs and discuss why these small players can no longer be ignored with regard to genome function.

AtHB1 is an Arabidopsis (Arabidopsis thaliana) homeodomain-leucine zipper transcription factor that participates in hypocotyl elongation under short-day conditions. Here, we show that its expression is posttranscriptionally regulated by an upstream open reading frame (uORF) located in its 5' untranslated region. This uORF encodes a highly conserved peptide (CPuORF) that is present in varied monocot and dicot species. The Arabidopsis uORF and its maize (Zea mays) homolog repressed the translation of the main open reading frame in cis, independent of the sequence of the latter. Published ribosome footprinting results and the analysis of a frame-shifted uORF, in which the repression capability was lost, indicated that the uORF causes ribosome stalling. The regulation exerted by the CPuORF was tissue specific and did not act in the absence of light. Moreover, a photosynthetic signal is needed for the CPuORF action, since plants with uncoupled chloroplasts did not show uORF-dependent repression. Plants transformed with the native AtHB1 promoter driving AtHB1 expression did not show differential phenotypes, whereas those transformed with a construct in which the uORF was mutated exhibited serrated leaves, compact rosettes, and, most significantly, short nondehiscent anthers and siliques containing fewer or no seeds. Thus, we propose that the uncontrolled expression of AtHB1 is deleterious for the plant and, hence, finely repressed by a translational mechanism. Fil: Ribone, Pamela Anahí. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Capella, Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Arce, Agustín Lucas. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Chan, Raquel Lia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina

Definition of functional genomic elements is one of the greater challenges of the genomic era. Traditionally, putative short open reading frames (sORFs) coding for less than 100 amino acids were disregarded due to computational and experimental limitations; however, it has become clear over the past several years that translation of sORFs is pervasive and serves diverse functions. The development of ribosome profiling, allowing identification of translated sequences genome wide, revealed wide spread, previously unidentified translation events. New computational methodologies as well as improved mass spectrometry approaches also contributed to the task of annotating translated sORFs in different organisms. Viruses are of special interest due to the selective pressure on their genome size, their rapid and confining evolution, and the potential contribution of novel peptides to the host immune response. Indeed, many functional viral sORFs were characterized to date, and ribosome profiling analyses suggest that this may be the tip of the iceberg. Our computational analyses of sORFs identified by ribosome profiling in DNA viruses demonstrate that they may be enriched in specific features implying that at least some of them are functional. Combination of systematic genome editing strategies with synthetic tagging will take us into the next step—elucidation of the biological relevance and function of this intriguing class of molecules.

Summary Intracellular sorting of mRNAs is an essential process for regulating gene expression and protein localization. Most mitochondrial proteins are nuclear-encoded and imported into the mitochondria through post-translational or co-translational processes. In the latter case, mRNAs are found to be enriched in the vicinity of mitochondria. A genome-scale analysis of mRNAs associated with mitochondria has been performed to determine plant cytosolic mRNAs targeted to the mitochondrial surface. Many messengers encoding mitochondrial proteins were found associated with mitochondria. These mRNAs correspond to particular functions and complexes, such as respiration or mitoribosomes, which indicates a coordinated control of mRNA localization within metabolic pathways. In addition, upstream AUGs in 5' untranslated regions (UTRs), which modulate the translation efficiency of downstream sequences, were found to negatively affect the association of mRNAs with mitochondria. A mutational approach coupled with in vivo mRNA visualization confirmed this observation. Moreover, this technique allowed the identification of 3'-UTRs as another essential element for mRNA localization at the mitochondrial surface. Therefore, this work offers new insights into the mechanism, function and regulation of the association of cytosolic mRNAs with plant mitochondria.

Astrophysicists use the term "dark matter" to describe the majority of the matter and/or energy in the universe that is hidden from view, and biologists now apply it to the new families of RNA they are uncovering. We review evidence for an analogous hidden world containing peptides. The critical experiments involved pulse-labeling human cells with tagged amino acids for periods as short as five seconds. Results are extraordinary in two respects: both nucleus and cytoplasm become labeled, and most signals disappear with a half-life of less than one minute. Just as the synthesis of each mature mRNA is regulated by the abortive production of hundreds of shorter transcripts that are quickly degraded, it seems that the synthesis of each full-length protein in the stable proteome is regulated by an apparently wasteful production and degradation of shorter peptides. Some of the nuclear synthesis is probably a byproduct of nuclear ribosomes proofreading newly-made RNA for inappropriately-placed termination codons (a process that triggers "nonsense-mediated decay"). We speculate that some "dark-matter" peptides will play other important roles in the cell.

Abundant evidence for translation within the 5′ leaders of many human genes is rapidly emerging, especially, because of the advent of ribosome profiling. In most cases, it is believed that the act of translation rather than the encoded peptide is important. However, the wealth of available sequencing data in recent years allows phylogenetic detection of sequences within 5′ leaders that have emerged under coding constraint and therefore allow for the prediction of functional 5′ leader translation. Using this approach, we previously predicted a CUG-initiated, 173 amino acid N-terminal extension to the human tumour suppressor PTEN. Here, a systematic experimental analysis of translation events in the PTEN 5′ leader identifies at least two additional non-AUG-initiated PTEN proteoforms that are expressed in most human cell lines tested. The most abundant extended PTEN proteoform initiates at a conserved AUU codon and extends the canonical AUG-initiated PTEN by 146 amino acids. All N-terminally extended PTEN proteoforms tested retain the ability to downregulate the PI3K pathway. We also provide evidence for the translation of two conserved AUG-initiated upstream open reading frames within the PTEN 5′ leader that control the ratio of PTEN proteoforms.

A growing body of work demonstrates the importance of post-transcriptional control, in particular translation initiation, in the overall regulation of gene expression. Here we focus on the contribution of regulatory elements within the 5’ and 3’ untranslated regions of mRNA to gene expression in eukaryotic cells including terminal oligopyrimidine tracts, internal ribosome entry segments, upstream open reading frames and cytoplasmic polyadenylation elements. These mRNA regulatory elements may adopt complex secondary structures and/or contain sequence motifs that allow their interaction with a variety of regulatory proteins, RNAs and RNA binding proteins, particularly hnRNPs. The resulting interactions are context-sensitive, and provide cells with a sensitive and fast response to cellular signals such as hormone exposure or cytotoxic stress. Importantly, an increasing number of diseases have been identified, particularly cancers and those associated with neurodegeneration, which originate either from mutation of these regulatory motifs, or from deregulation of their cognate binding partners.

The RNA helicase Rok1 plays a role in rRNA processing and in control of cell cycle progression in Saccharomyces cerevisiae. We identified two upstream open reading frames (uORFs) within the ROK1 5′ untranslated region, which inhibited Rok1 translation. Mutating uATG to uAAG or generation of a premature stop codon in the uORFs resulted in increased Rok1p levels. Rok1 protein levels oscillated during the cell cycle, declining at G1/S and increasing at G2. The uAAG1 mutation caused a constitutive level of Rok1 proteins throughout the cell cycle, resulting in significant delays in mitotic bud emergence and recovery from pheromone arrest. Our study reveals that the Rok1 protein level is regulated by uORFs, which is critical in cell cycle progression.

A translational regulatory mechanism for Tomato golden mosaic virus (TGMV) complementary-sense gene expression has been characterized. TGMV transcribes two mRNAs, AL-1935 and AL-1629 transcripts, both of which contain the AL2 and AL3 open reading frames. However, AL2 is only expressed from AL-1629 whereas AL3 is expressed from both. Three AUG translation initiation codons are located upstream of both the AL2 and AL3 coding regions, within the 5′-untranslated region (UTR) of the AL-1935 transcript. Translation can initiate at the first AUG, specifying the C-terminal 122 amino acids of the AL1 protein (cAL1). Initiation of translation at this AUG is inhibitory for the downstream expression of both AL2 and AL3. This is most likely due to the terminator codon of cAL1 being positioned after the AUG initiation codon for the AL2 ORF. The mechanism by which AL3 is expressed from AL-1935 is currently unknown but a gap between the cAL1 termination codon and the start of AL3 suggests that it may involve reinitiation and/or internal initiation. In contrast, expression of AL3 from AL-1629 most likely occurs via leaky ribosome scanning since the AL3 initiation codon occurs before the terminator codon of AL2. Mutation of the AUG encoding cAL1 in the curtovirus, Spinach curly top virus , leads to increased infectivity as measured by a shorter latent period. Together this suggests that geminiviruses use a post-translational regulatory mechanism to regulate the synthesis of viral proteins important for replication and suppression of host defenses.

Copyright © 2015 by Annual Reviews. All rights reserved. Herpesviral mRNAs are produced and translated by cellular machinery, rendering them susceptible to the network of regulatory events that impact translation. In response, these viruses have evolved to infiltrate and hijack translational control pathways as well as to integrate specialized host translation strategies into their own repertoire. They are robust systems to dissect mechanisms of mammalian translational regulation and continue to offer insight into cis-acting mRNA features that impact assembly and activity of the translation apparatus. Here, I discuss recent advances revealing the extent to which the three herpesvirus subfamilies regulate both host and viral translation, thereby dramatically impacting the landscape of protein synthesis in infected cells.

We show that a common polymorphic variant in the ERCC5 5′ untranslated region (UTR) generates an upstream ORF (uORF) that affects both the background expression of this protein and its ability to be synthesized following exposure to agents that cause bulky adduct DNA damage. Individuals that harbor uORF1 have a marked resistance to platinum-based agents, illustrated by the significantly reduced progression-free survival of pediatric ependymoma patients treated with such compounds. Importantly, inhibition of DNA-PKcs restores sensitivity to platinum-based compounds by preventing uORF1-dependent ERCC5 expression. Our data support a model in which a heritable 5′ noncoding mRNA element influences individuals’ responses to platinum-based chemotherapy.

The cAMP-dependent kinase (PKA) plays a crucial part in long-term memory formation in the honeybee (Apis mellifera). One of the putative substrates of the PKA activity is the cAMP response element binding protein (CREB), a transcription factor in the bZIP protein family. We searched the honeybee genome to characterize genes from the CREB/CREM and the PKA families. We identified two genes that encode regulatory subunits and three genes encode catalytic subunits of PKA. Eight genes code for bZIP proteins, but only one gene was found that encodes a member of the CREB/CREM family. The phylogenetic relationship of these genes was analysed with their Drosophila and human counterparts.

The sperm mitochondrial cysteine-rich protein (SMCP) is a rapidly evolving cysteine- and proline-rich protein that is localized in the mitochondrial capsule and enhances sperm motility. The sequences of the SMCP protein, gene, and mRNA in a variety of mammals have been compared to understand their evolution and regulation. SMCP can now be reliably identified by its tripartite structure including a short amino-terminal segment; a central segment containing short tandem repeats rich in cysteine, proline, glutamine, and lysine; and a C-terminal segment containing no repeats, few cysteines, and a C-terminal lysine. The SMCP gene is located in the epidermal differentiation complex (EDC), a large gene cluster that functions in forming epithelial barriers. Similarities in chromosomal location, molecular function, intron–exon structure, and protein organization argue that SMCP originated from an EDC gene and acquired spermatogenic cell-specific transcriptional and translational regulation and a novel cellular function in sperm motility. The SMCP 5′ UTR and 3′ UTR contain conserved elements and uORFs that may function in cytoplasmic regulation of gene expression, and the levels of SMCP mRNA in human are much lower than in other mammals, a feature of male-biased expression. The evolution of SMCP has been accompanied by changes in the sequence, number, and length of repeat units, including three alleles in dogs. The major proteins associated with the mitochondrial capsule, SMCP and phospholipid hydroperoxide glutathione peroxidase, provide outstanding examples of changes in cellular function driven by selective pressures on sperm motility, an important determinant of male reproductive success.

Background Ribosome profiling (ribo-seq) provides experimental data on the density of elongating or initiating ribosomes at the whole transcriptome level that can be potentially used for estimating absolute levels of translation initiation at individual Translation Initiation Sites (TISs). These absolute levels depend on the mutual organisation of TISs within individual mRNAs. For example, according to the leaky scanning model of translation initiation in eukaryotes, a strong TIS downstream of another strong TIS is unlikely to be productive, since only a few scanning ribosomes would be able to reach the downstream TIS. In order to understand the dependence of translation initiation efficiency on the surrounding nucleotide context, it is important to estimate the strength of TISs independently of their mutual organisation, i.e. to estimate with what probability a ribosome would initiate at a particular TIS. Results We designed a simple computational approach for estimating the probabilities of ribosomes initiating at individual start codons using ribosome profiling data. The method is based on the widely accepted leaky scanning model of translation initiation in eukaryotes which postulates that scanning ribosomes may skip a start codon if the initiation context is unfavourable and continue on scanning. We tested our approach on three independent ribo-seq datasets obtained in mammalian cultured cells. Conclusions Our results suggested that the method successfully discriminates between weak and strong TISs and that the majority of numerous non-AUG TISs reported recently are very weak. Therefore the high frequency of non-AUG TISs observed in ribosome profiling experiments is due to their proximity to mRNA 5′-ends rather than their strength. Detectable translation initiation at non-AUG codons downstream of AUG codons is comparatively infrequent. The leaky scanning method will be useful for the characterization of differences in start codon selection between tissues, developmental stages and in response to stress conditions. Electronic supplementary material The online version of this article (doi:10.1186/s12859-014-0380-4) contains supplementary material, which is available to authorized users.

Background Variability in protein levels is generated through intricate control of the different gene decoding phases. Presently little is known about the links between the various gene expression stages. Here we investigated the relationship between transcription and translation regulatory properties encoded in mammalian genes. Results We found that the TATA-box, a core promoter element known to enhance transcriptional output, is associated not only with higher mRNA levels but also with positive translation regulatory features and elevated translation efficiency. Further investigation revealed general association between transcription and translation regulatory trends. Specifically, translation inhibitory features such as the presence of upstream AUG (uAUG) and increased lengths of the 5′UTR, the coding sequence and the 3′UTR, are strongly associated with lower translation as well as lower transcriptional rate. Conclusions Our findings reveal that co-occurrence of several gene-encoded transcription and translation regulatory features with the same trend substantially contributes to the final mRNA and protein expression levels and enables their coordination. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-688) contains supplementary material, which is available to authorized users.

As part of the chromosome-centric human proteome project (C-HPP) initiative, we report our progress on the annotation of chromosome 22. Chromosome 22, spanning 51 million base pairs, was the first chromosome to be sequenced. Gene dosage alterations on this chromosome have been shown to be associated with a number of congenital anomalies. In addition, several rare but aggressive tumors have been associated with this chromosome. A number of important gene families including immunoglobulin lambda locus, Crystallin beta family, and APOBEC gene family are located on this chromosome. On the basis of proteomic profiling of 30 histologically normal tissues and cells using high-resolution mass spectrometry, we show protein evidence of 367 genes on chromosome 22. Importantly, this includes 47 proteins, which are currently annotated as "missing" proteins. We also confirmed the translation start sites of 120 chromosome 22-encoded proteins. Employing a comprehensive proteogenomics analysis pipeline, we provide evidence of novel coding regions on this chromosome which include upstream ORFs and novel exons in addition to correcting existing gene structures. We describe tissue-wise expression of the proteins and the distribution of gene families on this chromosome. These data have been deposited to ProteomeXchange with the identifier PXD000561.

Post-transcriptional control makes a major contribution to the overall regulation of gene expression pathway. Within the cytoplasm this is mediated by a combination of regulatory RNA motifs within the 5′ and 3′ untranslated regions of mRNAs and their interacting protein/RNA partners. One of the most common regulatory RNA elements in mammalian transcripts (present in approximately 40% of all mRNAs) are upstream open reading frames (uORFs). However, despite the prevalence of these RNA elements how they function is not well understood. In general, they act to repress translation of the physiological ORF under control conditions, and under certain pathophysiological stresses this repression can be alleviated. It is known that re-initiation following the translation of an uORF is utilised in some situations however there are numerous alternative mechanisms that control the synthesis of a protein whose mRNA contains uORFs. Moreover, the trans-acting factors that are also involved in this process are not well defined. In this review we summarise our current understanding of this area and highlight some common features of these RNA motifs that have been discovered to date.

Conserved upstream open reading frames (uORFs) are found within many eukaryotic transcripts and are known to regulate protein translation. Evidence from genetic and bioinformatic studies implicates disturbed uORF-mediated translational control in the etiology of human diseases. A genetic mouse model has recently provided proof-of-principle support for the physiological relevance of uORF-mediated translational control in mammals. The targeted disruption of the uORF initiation codon within the transcription factor CCAAT/enhancer binding protein β (C/EBPβ) gene resulted in deregulated C/EBPβ protein isoform expression, associated with defective liver regeneration and impaired osteoclast differentiation. The high prevalence of uORFs in the human transcriptome suggests that intensified search for mutations within 5′ RNA leader regions may reveal a multitude of alterations affecting uORFs, causing pathogenic deregulation of protein expression.

We investigated the translational regulation of SCL protein expression and its role in hematopoietic lineage choice. We show that the expression of different SCL protein isoforms is regulated by signal transduction pathways that modulate translation initiation factor (eIF) function. A conserved small upstream open reading frame (uORF) in SCL transcripts acts as acis-regulatory element for isoform expression. At the onset of erythroid differentiation, truncated SCL protein isoforms arise by alternative translation initiation and favor the erythroid lineage. In comparison, full-length SCL proteins are more efficient at enhancing the megakaryocyte lineage. Together, our studies unravel translational control as a novel mechanism regulating hematopoietic outcome.