To New Beginnings: Riboproteogenomics Discovery of N-Terminal Proteoforms in Arabidopsis Thaliana

Alternative translation initiation is a widespread event in biology that can shape multiple protein forms or proteoforms from a single gene. However, the respective contribution of alternative translation to protein complexity remains largely enigmatic. By complementary ribosome profiling and N-terminal proteomics (i.e., riboproteogenomics), we provide clear-cut evidence for ~90 N-terminal proteoform pairs shaped by (alternative) translation initiation in Arabidopsis thaliana. Next to several cases additionally confirmed by directed mutagenesis, identified alternative protein N-termini follow the enzymatic rules of co-translational N-terminal protein acetylation and initiator methionine removal. In contrast to other eukaryotic models, N-terminal acetylation in plants cannot generally be considered as a proxy of translation initiation because of its posttranslational occurrence on mature proteolytic neo-termini (N-termini) localized in the chloroplast stroma. Quantification of N-terminal acetylation revealed differing co- vs. posttranslational N-terminal acetylation patterns. Intriguingly, our data additionally hints to alternative translation initiation serving as a common mechanism to supply protein copies in multiple cellular compartments, as alternative translation sites are often in close proximity to cleavage sites of N-terminal transit sequences of nuclear-encoded chloroplastic and mitochondrial proteins. Overall, riboproteogenomics screening enables the identification of (differential localized) N-terminal proteoforms raised upon alternative translation.

RUFY4 exists as two translationally regulated isoforms, that localize to the mitochondrion in activated macrophages

We report here that RUFY4, a newly characterized member of the ‘RUN and FYVE domain-containing’ family of proteins previously associated with autophagy enhancement, is highly expressed in alveolar macrophages (AM). We show that RUFY4 interacts with mitochondria upon stimulation by microbial-associated molecular patterns of AM and dendritic cells. RUFY4 interaction with mitochondria and other organelles is dependent on a previously uncharacterized OmpH domain located immediately upstream of its C-terminal FYVE domain. Further, we demonstrate that rufy4 messenger RNA can be translated from an alternative translation initiation codon, giving rise to a N-terminally truncated form of the molecule lacking most of its RUN domain and with enhanced potential for its interaction with mitochondria. Our observations point towards a role of RUFY4 in selective mitochondria clearance in activated phagocytes.

Regulation of translation by one-carbon metabolism in bacteria and the eukaryotic organelles

Protein synthesis is an energetically costly cellular activity. It is therefore important that the process of mRNA translation remains in excellent synchrony with cellular metabolism and its energy reserves. Unregulated translation could lead to the production of incomplete, mistranslated, or misfolded proteins, squandering the energy needed for cellular sustenance, and causing cytotoxicity. One-carbon metabolism (OCM), an integral part of cellular intermediary metabolism, produces a number of one-carbon unit intermediates (formyl, methylene, methenyl, methyl). These OCM intermediates are required for the production of amino acids like methionine, and biomolecules such as purines, thymidylate, and redox regulators. In this review, we discuss how OCM impacts the translation apparatus (composed of ribosome, tRNA, mRNA, and translation factors) and regulates crucial steps in protein synthesis. More specifically, we address how the OCM metabolites regulate the fidelity and rate of translation initiation in bacteria and eukaryotic organelles such as mitochondria. Modulation of the fidelity of translation initiation by OCM opens new avenues to understand alternative translation mechanisms involved in stress tolerance and drug resistance.

Identification and characterization of a new isoform of small GTPase RhoE

The Rho family of GTPases consists of 20 members including RhoE. Here, we discover the existence of a short isoform of RhoE designated as RhoEα, the first Rho GTPase isoform generated from alternative translation. Translation of this new isoform is initiated from an alternative start site downstream of and in-frame with the coding region of the canonical RhoE. RhoEα exhibits a similar subcellular distribution while its protein stability is higher than RhoE. RhoEα contains binding capability to RhoE effectors ROCK1, p190RhoGAP and Syx. The distinct transcriptomes of cells with the expression of RhoE and RhoEα, respectively, are demonstrated. The data propose distinctive and overlapping biological functions of RhoEα compared to RhoE. In conclusion, this study reveals a new Rho GTPase isoform generated from alternative translation. The discovery provides a new scope of understanding the versatile functions of small GTPases and underlines the complexity and diverse roles of small GTPases.

A Novel Cis-Acting RNA Structural Element Embedded in the Core Coding Region of the Hepatitis C Virus Genome Directs Internal Translation Initiation of the Overlapping Core+1 ORF

Hepatitis C virus (HCV) genome translation is initiated via an internal ribosome entry site (IRES) embedded in the 5′-untranslated region (5′UTR). We have earlier shown that the conserved RNA stem-loops (SL) SL47 and SL87 of the HCV core-encoding region are important for viral genome translation in cell culture and in vivo. Moreover, we have reported that an open reading frame overlapping the core gene in the +1 frame (core+1 ORF) encodes alternative translation products, including a protein initiated at the internal AUG codons 85/87 of this frame (nt 597–599 and 603–605), downstream of SL87, which is designated core+1/Short (core+1/S). Here, we provide evidence for SL47 and SL87 possessing a novel cis-acting element that directs the internal translation initiation of core+1/S. Firstly, using a bicistronic dual luciferase reporter system and RNA-transfection experiments, we found that nucleotides 344–596 of the HCV genotype-1a and -2a genomes support translation initiation at the core+1 frame AUG codons 85/87, when present in the sense but not the opposite orientation. Secondly, site-directed mutagenesis combined with an analysis of ribosome–HCV RNA association elucidated that SL47 and SL87 are essential for this alternative translation mechanism. Finally, experiments using cells transfected with JFH1 replicons or infected with virus-like particles showed that core+1/S expression is independent from the 5′UTR IRES and does not utilize the polyprotein initiation codon, but it requires intact SL47 and SL87 structures. Thus, SL47 and SL87, apart from their role in viral polyprotein translation, are necessary elements for mediating the internal translation initiation of the alternative core+1/S ORF.

PROBLEMS OF EQUIVALENCE AND ADEQUACY IN THE TRANSLATION OF THE NOVEL “NAVOI” BY AYBEK

Some inappropriatenesses and defects on the issue of equivalence and adequacy in the translated version of the novel “Navoi” by Aybek are revealed in this article. These inappropriatenesses and defects are described in great detail with the help of examples and alternative translation variants are suggested, the problems of equivalence and adequacy in translation studies are researched from the scientific point of view, as well as, views and comments of the Uzbek and foreign translators and scientists are provided on theissue of the two concepts

Mammalian Alternative Translation Initiation Is Mostly Nonadaptive

Alternative translation initiation (ATLI) refers to the existence of multiple translation initiation sites per gene and is a widespread phenomenon in eukaryotes. ATLI is commonly assumed to be advantageous through creating proteome diversity or regulating protein synthesis. We here propose an alternative hypothesis that ATLI arises primarily from nonadaptive initiation errors presumably due to the limited ability of ribosomes to distinguish sequence motifs truly signaling translation initiation from similar sequences. Our hypothesis, but not the adaptive hypothesis, predicts a series of global patterns of ATLI, all of which are confirmed at the genomic scale by quantitative translation initiation sequencing in multiple human and mouse cell lines and tissues. Similarly, although many codons differing from AUG by one nucleotide can serve as start codons, our analysis suggests that using non-AUG start codons is mostly disadvantageous. These and other findings strongly suggest that ATLI predominantly results from molecular error, requiring a major revision of our understanding of the precision and regulation of translation initiation.