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The synthesis of most proteins begins at AUG codons, yet a small number of non-AUG initiated proteoforms are also known. Here we analyse a large number of publicly available Ribo-seq datasets to identify novel, previously uncharacterised non-AUG proteoforms using Trips-Viz implementation of a novel algorithm for detecting translated ORFs. In parallel we analyse genomic alignment of 120 mammals to identify evidence of protein coding evolution in sequences encoding potential extensions. Unexpectedly we find that the number of non-AUG proteoforms identified with ribosome profiling data greatly exceeds those with strong phylogenetic support suggesting their recent evolution. Our study argues that the protein coding potential of human genome greatly exceeds that detectable through comparative genomics and exposes the existence of multiple proteins encoded by the same genomic loci.Analysis of a large number of Ribo-seq datasets and genomic alignments led to detection of novel non-AUG proteoforms. Unexpectedly the number of non-AUG proteoforms identified with Ribo-seq greatly exceeds those with strong phylogenetic support. [ABSTRACT FROM AUTHOR]

Activated ghrelin receptor GHS-R1α triggers cell signalling pathways that modulate energy homeostasis and biosynthetic processes. However, the effects of ghrelin on mRNA translation are unknown. Using various reporter assays, here we demonstrate a rapid elevation of protein synthesis in cells within 15–30 min upon stimulation of GHS-R1α by ghrelin. We further show that ghrelin-induced activation of translation is mediated, at least in part, through the de-phosphorylation (de-suppression) of elongation factor 2 (eEF2). The levels of eEF2 phosphorylation at Thr56 decrease due to the reduced activity of eEF2 kinase, which is inhibited via Ser366 phosphorylation by rpS6 kinases. Being stress-susceptible, the ghrelin-mediated decrease in eEF2 phosphorylation can be abolished by glucose deprivation and mitochondrial uncoupling. We believe that the observed burst of translation benefits rapid restocking of neuropeptides, which are released upon GHS-R1α activation, and represents the most time- and energy-efficient way of prompt recharging the orexigenic neuronal circuitry. [ABSTRACT FROM AUTHOR]

Ribosome profiling (riboseq) has opened the possibilities for the genome-wide studies of translation in all living organisms. This method is based on deep sequencing of mRNA fragments protected by the ribosomes from hydrolysis by ribonucleases, the so-called ribosomal footprints (RFPs). Ribosomal profiling together with RNA sequencing allows not only to identify with a reasonable accuracy translated reading frames in the transcriptome, but also to track changes in gene expression in response to various stimuli. Notably, ribosomal profiling in its classical version has certain limitations. The size of the selected mRNA fragments is 25-35 nts, while RFPs of other sizes are usually omitted from analysis. Also, ribosomal profiling "averages" the data from all ribosomes and does not allow to study specific ribosomal complexes associated with particular translation factors. However, recently developed modifications of ribosomal profiling provide answers to a number of questions. Thus, it has become possible to analyze not only elongating, but also scanning and reinitiating ribosomes, to study events associated with the collision of ribosomes during mRNA translation, to discover new ways of cotranslational assembly of multisubunit protein complexes during translation, and to selectively isolate ribosomal complexes associated with certain protein factors. New data obtained using these modified approaches provide a better understanding of the mechanisms of translation regulation and the functional roles of translational apparatus components. [ABSTRACT FROM AUTHOR]

An addendum to an article published in Nature Communications states that the parameters used for calculating the PhyloCSF score in the original article were obtained from an alignment other than the intended 120 mammals. This resulted in a less sensitive analysis. Recalculation of the PhyloCSF score with the correct parameters revealed a larger dataset of 177 genes, with 58 of the original 60 genes included. This does not change the main conclusion of the article, which highlights the prevalence of ribo-seq-supported non-AUG-initiated N-terminal extensions. However, it does indicate that the number of N-terminally extended proteoforms satisfying the criteria for protein-coding evolution is three times greater than originally reported. The corrected parameters are now available on the PhyloCSF github page. Supplementary data is also provided. [Extracted from the article]

In addition to acting as template for protein synthesis, messenger RNA (mRNA) often contains sensory sequence elements that regulate this process. Here we report a new mechanism that limits the number of complete protein molecules that can be synthesized from a single mRNA molecule of the human AMD1 gene encoding adenosylmethionine decarboxylase 1 (AdoMetDC). A small proportion of ribosomes translating AMD1 mRNA stochastically read through the stop codon of the main coding region. These readthrough ribosomes then stall close to the next in-frame stop codon, eventually forming a ribosome queue, the length of which is proportional to the number of AdoMetDC molecules that were synthesized from the same AMD1 mRNA. Once the entire spacer region between the two stop codons is filled with queueing ribosomes, the queue impinges upon the main AMD1 coding region halting its translation. Phylogenetic analysis suggests that this mechanism is highly conserved in vertebrates and existed in their common ancestor. We propose that this mechanism is used to count and limit the number of protein molecules that can be synthesized from a single mRNA template. It could serve to safeguard from dysregulated translation that may occur owing to errors in transcription or mRNA damage. [ABSTRACT FROM AUTHOR]

Unlike bacteria, a specialized eukaryotic initiation factor (eIF)-2, in the form of the ternary complex eIF2–GTP–Met-tRNAi Met, is used to deliver the initiator tRNA to the ribosome in all eukaryotic cells. Here we show that the hepatitis C virus (HCV) internal ribosome entry site (IRES) can direct translation without eIF2 and its GTPase-activating protein eIF5. In addition to the general eIF2- and eIF5-dependent pathway of 80S complex assembly, the HCV IRES makes use of a bacterial-like pathway requiring as initiation factors only eIF5B (an analog of bacterial IF2) and eIF3. The switch from the conventional eukaryotic mode of translation initiation to the eIF2-independent mechanism occurs when eIF2 is inactivated by phosphorylation under stress conditions. [ABSTRACT FROM AUTHOR]

mRNAs lacking 5′ untranslated regions (leaderless mRNAs) are molecular relics of an ancient translation initiation pathway. Nevertheless, they still represent a significant portion of transcriptome in some taxons, including a number of eukaryotic species. In bacteria and archaea, the leaderless mRNAs can bind non-dissociated 70 S ribosomes and initiate translation without protein initiation factors involved. Here we use the Fleeting mRNA Transfection technique (FLERT) to show that translation of a leaderless reporter mRNA is resistant to conditions when eIF2 and eIF4F, two key eukaryotic translation initiation factors, are inactivated in mammalian cells. We report an unconventional translation initiation pathway utilized by the leaderless mRNA in vitro, in addition to the previously described 80S-, eIF2-, or eIF2D-mediated modes. This mechanism is a bacterial-like eIF5B/IF2-assisted initiation that has only been reported for hepatitis C virus-like internal ribosome entry sites (IRESs). Therefore, the leaderless mRNA is able to take any of four different translation initiation pathways in eukaryotes. [ABSTRACT FROM AUTHOR]