Addendum to the main text (i) ATE1-/-embryos from ATE1 +/-intercrosses were present at the expected (~25%) frequency up to ~E13.5, but virtually no ATE1 -/-embryos were recovered alive by E17. Specifically, no ATE1 -/-mice were recovered amongst either 954 F 2 -generation pups of the C57BL/6J-129SvEv (mixed) background or 267 F 2 -generation pups of the 129SvEv (inbred) background. Timed intercrosses of ATE1 +/-mice were used to determine that ATE1 -/-embryos were present at approximately the expected (25%) frequency up to ~E13.5, but no ATE1 -/-embryos were recovered alive by E17.Until E12.5, ATE1 -/-embryos appeared to be morphologically normal; however, their growth stopped during E13.5-E15.5. By E14.5-E15.5, ~50% of ATE1 -/-embryos were still alive, but growth-retarded. Live E14.5-E15.5 embryos were capable of opening their mouths and flexing their bodies, suggesting the absence of gross neuromuscular defects. Sections through E13.5 ATE1-/-embryos indicated the presence and apparently normal appearance of major organs, except for the phenotypes described in the main text and below.(ii) We examined the expression of ATE1 mRNA during embryogenesis using Northern hybridization with total RNA from +/+ embryos ranging in age from E4.5 to E18.5. ATE1 mRNA was present at least as early as E4.5, and a strong spike of ATE1 expression was observed during E7.5-9.5 (Fig. S1E). The ~2 kb ATE1 transcript detected in adult mouse testis (1) was also clearly present during the spike of ATE1 expression in E7.5-E9.5 embryos (Fig. S1E). The ATE1 -allele was marked with NLS-β-galactosidase (hereafter βgal), expressed from the ATE1 promoter

Chromosome segregation during mitosis depends on the action of the mitotic spindle, a self-organizing, bipolar protein machine which uses microtubules (MTs) and their associated motors. Members of the BimC subfamily of kinesin-related MT-motor proteins are believed to be essential for the formation and functioning of a normal bipolar spindle. Here we report that KRP130, a homotetrameric BimC-related kinesin purified from Drosophila melanogaster embryos, has an unusual ultrastructure. It consists of four kinesin-related polypeptides assembled into a bipolar aggregate with motor domains at opposite ends, analogous to a miniature myosin filament. Such a bipolar 'minifilament' could crosslink spindle MTs and slide them relative to one another. We do not know of any other MT motors that have a bipolar structure.

Posttranslational arginylation is critical for mouse embryogenesis, cardiovascular development, and angiogenesis, but its molecular effects and the identity of proteins arginylated in vivo are unknown. We found that beta-actin was arginylated in vivo to regulate actin filament properties, beta-actin localization, and lamella formation in motile cells. Arginylation of beta-actin apparently represents a critical step in the actin N-terminal processing needed for actin functioning in vivo. Thus, posttranslational arginylation of a single protein target can regulate its intracellular function, inducing global changes on the cellular level, and may contribute to cardiovascular development and angiogenesis.

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.

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.

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.

Many of the best-studied actin regulatory proteins use non-covalent means to modulate the properties of actin. Yet, actin is also susceptible to covalent modifications of its amino acids. Recent work is increasingly revealing that actin processing and its covalent modifications regulate important cellular processes. In addition, numerous pathogens express enzymes that specifically use actin as a substrate to regulate their hosts cells. Actin post-translational alterations have been linked to different normal and disease processes and the effects associated with metabolic and environmental stressors. Herein, we highlight specific co- and post-translational modifications of actin and discuss the role that these modifications play in regulating actin dynamics.

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 mouse Ate1 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 in ate1⌬ Saccharomyces cerevisiae in the presence of test substrates that included Asp-␤gal (␤-galactosidase) and Cys-␤gal. Both forms of the mouse R-transferase conferred instability on Asp-␤gal (but not on Cys-␤gal) through the arginylation of its Nterminal 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 ϳ0.1 in the skeletal muscle, ϳ0.25 in the spleen, ϳ3.3 in the liver and brain, and ϳ10 in the testis, suggesting that the two R-transferases are functionally distinct.