The mammalian cytoskeletal proteins β- and γ-actin are highly homologous, but only β-actin is N-terminally arginylated, which regulates its function. Here we examined the metabolic fate of exogenously expressed arginylated and non-arginylated actin isoforms. Arginylated γ-actin, unlike β-, was highly unstable and was selectively ubiquitinated and degraded in vivo. This instability was regulated by the differences in the coding sequence between the two actin isoforms, which conferred different translation rates. γ-actin was translated more slowly than β-actin, and this slower processing resulted in the exposure of a normally hidden lysine residue for ubiquitination, leading to the preferential degradation of γ-actin upon arginylation. This degradation mechanism, coupled to nucleotide coding sequence, may regulate protein arginylation in vivo.

Summary Posttranslational arginylation mediated by arginyltransferase (ATE1) plays an important role in cardiovascular development, cell motility and regulation of cytoskeleton and metabolic enzymes. This protein modification was discovered decades ago, however, the arginylation reaction and the functioning of ATE1 remained poorly understood due to the lack of good biochemical models. Here we report the development of an in vitro arginylation system, in which ATE1 function and molecular requirements can be tested using purified recombinant ATE1 isoforms supplemented with a controlled number of components. Our results show that arginylation reaction is a self-sufficient, ATP-independent process that can affect different sites in a polypeptide, and that arginyltransferases form different molecular complexes in vivo, associate with components of the translation machinery, and have distinct, partially overlapping subsets of substrates, suggesting that these enzymes play different physiological functions.

Japanese encephalitis virus or Rabies virus results in the activation of a gene encoding a novel, non-coding RNA (ncRNA) in the mouse central nervous system. This transcript, named virus-inducible ncRNA (VINC), is identical to a 3?18 kb transcript expressed in mouse neonate skin (GenBank accession no. AK028745) that, together with a number of unannotated cDNAs and expressed sequence tags, is grouped in the mouse unigene cluster Mm281895. VINC is expressed constitutively in early mouse embryo and several adult non-neuronal mouse tissues, as well as a murine renal adenocarcinoma (RAG) cell line. Northern blotting of nuclear and cytoplasmic RNAs revealed that VINC is localized primarily in the nucleus of RAG cells and is thus a novel member of the nuclear ncRNA family. Non-protein-coding eukaryotic genome sequences, often referred to as 'junk DNA', are estimated to encode several non-coding RNAs (ncRNAs), which may account for nearly 98 % of all genomic output in humans (http://research. imb.uq.edu.au/rnadb). In addition to the classical ncRNAs, such as rRNA, tRNA and small nucleolar RNAs, the eukaryotic genome encodes two distinct categories of ncRNAs, referred to as small ncRNAs and long mRNA-like ncRNAs. The long ncRNAs, which are transcribed by RNA polymerase II, spliced and polyadenylated, are implicated in a number of regulatory processes, such as imprinting, X-chromosome inactivation, DNA demethylation, transcription, RNA interference, chromatin-structure dynamics and antisense regulation. In addition, long mRNA-like ncRNAs such as MALAT-1, BC-1 and BC-200 serve as prognostic markers for cancer, whilst the prion-associated RNAs LIT-1, SCA-8 etc. are implicated in a number of neurological disorders (Costa, 2005). Thus, identification and characterization of novel ncRNAs and constant updating of the mammalian RNome are essential for the complete deciphering of genome biology and understanding mammalian gene regulation.

Actin arginylation regulates lamella formation in motile fibroblasts, but the underlying molecular mechanisms are unknown. Here, we found that actin regulation by arginylation affects its biochemical properties and binding of actin-associated proteins, modulating the overall structural organization of actin filaments in the cell.

Coordinated cell migration during development is crucial for morphogenesis and largely relies on cells of the neural crest lineage that migrate over long distances to give rise to organs and tissues throughout the body. Recent studies of protein arginylation implicated this poorly understood posttranslational modification in the functioning of actin cytoskeleton and in cell migration in culture. Knockout of arginyltransferase (Ate1) in mice leads to embryonic lethality and severe heart defects that are reminiscent of cell migration–dependent phenotypes seen in other mouse models. To test the hypothesis that arginylation regulates cell migration during morphogenesis, we produced Wnt1-Cre Ate1 conditional knockout mice (Wnt1-Ate1), with Ate1 deletion in the neural crest cells driven by Wnt1 promoter. Wnt1-Ate1 mice die at birth and in the first 2–3 weeks after birth with severe breathing problems and with growth and behavioral retardation. Wnt1-Ate1 pups have prominent defects, including short palate and altered opening to the nasopharynx, and cranial defects that likely contribute to the abnormal breathing and early death. Analysis of neural crest cell movement patterns in situ and cell motility in culture shows an overall delay in the migration of Ate1 knockout cells that is likely regulated by intracellular mechanisms rather than extracellular signaling events. Taken together, our data suggest that arginylation plays a general role in the migration of the neural crest cells in development by regulating the molecular machinery that underlies cell migration through tissues and organs during morphogenesis.

Posttranslational modifications constitute a major field of emerging biological significance as mounting evidence demonstrates their key role in multiple physiological processes. Following in the footsteps of protein phosphorylation studies, new modification are being shown to regulate protein properties and functions in vivo. Among such modifications, an important role belongs to protein arginylation – posttranslational tRNA-mediated addition of arginine, mediated by arginyltransferase, Ate1. Recent studies show that arginylation is essential for embryogenesis in many organisms and that it regulates such important processes as heart development, angiogenesis, and tissue morphogenesis in mammals. This review summarizes the key data in the protein arginylation field since its original discovery to date.

A proteolytic fragment of talin is regulated by arginylation and promotes cadherin-dependent cell–cell adhesion.

Protein arginylation mediated by arginyltransferase (ATE1) is essential for heart formation during embryogenesis, however its cell-autonomous role in cardiomyocytes and the differentiated heart muscle has never been investigated. To address this question, we generated cardiac muscle-specific Ate1 knockout mice, in which Ate1 deletion was driven by α-myosin heavy chain promoter (αMHC-Ate1 mouse). These mice were initially viable, but developed severe cardiac contractility defects, dilated cardiomyopathy, and thrombosis over time, resulting in high rates of lethality after 6 months of age. These symptoms were accompanied by severe ultrastructural defects in cardiac myofibrils, seen in the newborns and far preceding the onset of cardiomyopathy, suggesting that these defects were primary and likely underlay the development of the future heart defects. Several major sarcomeric proteins were arginylated in vivo. Moreover, Ate1 deletion in the hearts resulted in a significant reduction of active and passive myofibril forces, suggesting that arginylation is critical for both myofibril structural integrity and contractility. Thus, arginylation is essential for maintaining the heart function by regulation of the major myofibril proteins and myofibril forces, and its absence in the heart muscle leads to progressive heart failure through cardiomyocyte-specific defects.