Anna Kashina

Differential Arginylation of Actin Isoforms Is Regulated by Coding Sequence–Dependent Degradation

Making Modifications Arginylation of β-actin regulates cell motility and the actin cytoskeleton, but how differential arginylation of the two highly similar actin isoforms—β and γ—is achieved in vivo is unclear. Zhang et al. (p. 1534; see the Perspective by Weygand-Durasevic and Ibba) describe a cotranslational mechanism that selectively regulates the arginylation of proteins through degradation and is dependent on the nucleotide coding sequence coupled to the translation speed. The work provides an explanation for the different N-terminal arginylation states of β- and γ-actin in vivo and suggests translation rate affected by nucleotide coding sequence confers different posttranslational states to proteins and selectively regulates protein degradation. The translational speed of proteins influences whether they are cotranslationally degraded after arginylation. The mammalian cytoskeletal proteins β- and γ-actin are highly homologous, but only β-actin is amino-terminally arginylated in vivo, which regulates its function. We examined the metabolic fate of exogenously expressed arginylated and nonarginylated 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 nucleotide 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.

Sounds of silence: synonymous nucleotides as a key to biological regulation and complexity

Messenger RNA is a key component of an intricate regulatory network of its own. It accommodates numerous nucleotide signals that overlap protein coding sequences and are responsible for multiple levels of regulation and generation of biological complexity. A wealth of structural and regulatory information, which mRNA carries in addition to the encoded amino acid sequence, raises the question of how these signals and overlapping codes are delineated along non-synonymous and synonymous positions in protein coding regions, especially in eukaryotes. Silent or synonymous codon positions, which do not determine amino acid sequences of the encoded proteins, define mRNA secondary structure and stability and affect the rate of translation, folding and post-translational modifications of nascent polypeptides. The RNA level selection is acting on synonymous sites in both prokaryotes and eukaryotes and is more common than previously thought. Selection pressure on the coding gene regions follows three-nucleotide periodic pattern of nucleotide base-pairing in mRNA, which is imposed by the genetic code. Synonymous positions of the coding regions have a higher level of hybridization potential relative to non-synonymous positions, and are multifunctional in their regulatory and structural roles. Recent experimental evidence and analysis of mRNA structure and interspecies conservation suggest that there is an evolutionary tradeoff between selective pressure acting at the RNA and protein levels. Here we provide a comprehensive overview of the studies that define the role of silent positions in regulating RNA structure and processing that exert downstream effects on proteins and their functions.

Alternative splicing results in differential expression, activity, and localization of the two forms of arginyl-tRNA-protein transferase, a component of the N-end rule pathway.

ABSTRACT The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The underlying ubiquitin-dependent proteolytic system, called the N-end rule pathway, is organized hierarchically: N-terminal aspartate and glutamate (and also cysteine in metazoans) are secondary destabilizing residues, in that they function through their conjugation, by arginyl-tRNA-protein transferase (R-transferase), to arginine, a primary destabilizing residue. We isolated cDNA encoding the 516-residue mouse R-transferase, ATE1p, and found two species, termed Ate1-1 and Ate1-2. The Ate1 mRNAs are produced through a most unusual alternative splicing that retains one or the other of the two homologous 129-bp exons, which are adjacent in the mouseAte1 gene. Human ATE1 also contains the alternative 129-bp exons, whereas the plant (Arabidopsis thaliana) and fly (Drosophila melanogaster)Ate1 genes encode a single form of ATE1p. A fusion of ATE1-1p with green fluorescent protein (GFP) is present in both the nucleus and the cytosol, whereas ATE1-2p–GFP is exclusively cytosolic. Mouse ATE1-1p and ATE1-2p were examined by expressing them inate1Δ Saccharomyces cerevisiae in the presence of test substrates that included Asp-βgal (β-galactosidase) and Cys-βgal. Both forms of the mouse R-transferase conferred instability on Asp-βgal (but not on Cys-βgal) through the arginylation of its N-terminal Asp, the ATE1-1p enzyme being more active than ATE1-2p. The ratio of Ate1-1 to Ate1-2 mRNA varies greatly among the mouse tissues; it is ∼0.1 in the skeletal muscle, ∼0.25 in the spleen, ∼3.3 in the liver and brain, and ∼10 in the testis, suggesting that the two R-transferases are functionally distinct.

Altered Activity, Social Behavior, and Spatial Memory in Mice Lacking the NTAN1p Amidase and the Asparagine Branch of the N-End Rule Pathway

ABSTRACT The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. N-terminal asparagine and glutamine are tertiary destabilizing residues, in that they are enzymatically deamidated to yield secondary destabilizing residues aspartate and glutamate, which are conjugated to arginine, a primary destabilizing residue. N-terminal arginine of a substrate protein is bound by theUbr1-encoded E3α, the E3 component of the ubiquitin-proteasome-dependent N-end rule pathway. We describe the construction and analysis of mouse strains lacking the asparagine-specific N-terminal amidase (NtN-amidase), encoded by the Ntan1 gene. In wild-type embryos,Ntan1 was strongly expressed in the branchial arches and in the tail and limb buds. The Ntan1 −/− mouse strains lacked the NtN-amidase activity but retained glutamine-specific NtQ-amidase, indicating that the two enzymes are encoded by different genes. Among the normally short-lived N-end rule substrates, only those bearing N-terminal asparagine became long-lived in Ntan1 −/− fibroblasts. TheNtan1 −/− mice were fertile and outwardly normal but differed from their congenic wild-type counterparts in spontaneous activity, spatial memory, and a socially conditioned exploratory phenotype that has not been previously described with other mouse strains.

Cell Biology of Embryonic Migration

Cell migration is an evolutionarily conserved mechanism that underlies the development and functioning of uni- and multicellular organisms and takes place in normal and pathogenic processes, including various events of embryogenesis, wound healing, immune response, cancer metastases, and angiogenesis. Despite the differences in the cell types that take part in different migratory events, it is believed that all of these migrations occur by similar molecular mechanisms, whose major components have been functionally conserved in evolution and whose perturbation leads to severe developmental defects. These mechanisms involve intricate cytoskeleton-based molecular machines that can sense the environment, respond to signals, and modulate the entire cell behavior. A big question that has concerned the researchers for decades relates to the coordination of cell migration in situ and its relation to the intracellular aspects of the cell migratory mechanisms. Traditionally, this question has been addressed by researchers that considered the intra- and extracellular mechanisms driving migration in separate sets of studies. As more data accumulate researchers are now able to integrate all of the available information and consider the intracellular mechanisms of cell migration in the context of the developing organisms that contain additional levels of complexity provided by extracellular regulation. This review provides a broad summary of the existing and emerging data in the cell and developmental biology fields regarding cell migration during development.

Global Analysis of Posttranslational Protein Arginylation

Posttranslational arginylation is critical for embryogenesis, cardiovascular development, and angiogenesis, but its molecular effects and the identity of proteins arginylated in vivo are largely unknown. Here we report a global analysis of this modification on the protein level and identification of 43 proteins arginylated in vivo on highly specific sites. Our data demonstrate that unlike previously believed, arginylation can occur on any N-terminally exposed residue likely defined by a structural recognition motif on the protein surface, and that it preferentially affects a number of physiological systems, including cytoskeleton and primary metabolic pathways. The results of our study suggest that protein arginylation is a general mechanism for regulation of protein structure and function and outline the potential role of protein arginylation in cell metabolism and embryonic development.

Centering of a radial microtubule array by translocation along microtubules spontaneously nucleated in the cytoplasm

Positioning of a radial array of microtubules (MTs) in the cell centre is crucial for cytoplasmic organization, but the mechanisms of such centering are difficult to study in intact cells that have pre-formed radial arrays. Here, we use cytoplasmic fragments of melanophores, and cytoplasts of BS-C-1 cells to study MT centering mechanisms. Using live imaging and computer modelling, we show that the MT aster finds a central location in the cytoplasm by moving along spontaneously nucleated non-astral MTs towards a point at which MT nucleation events occur equally on all sides. We hypothesize that similar mechanisms, in the presence of the centrosome, contribute to this centering mechanism and ensure the robustness of cytoplasmic organization.

Posttranslational arginylation as a global biological regulator.

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 modifications 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, to proteins 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.

Arginylation-Dependent Neural Crest Cell Migration Is Essential for Mouse Development

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.

Arginylation regulates intracellular actin polymer level by modulating actin properties and binding of capping and severing proteins.

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.