Synonymous codon substitutions modulate transcription and translation of a divergent upstream gene by modulating antisense RNA production.

Synonymous codons were originally viewed as interchangeable, with no phenotypic consequences. However, substantial evidence has now demonstrated that synonymous substitutions can perturb a variety of gene expression and protein homeostasis mechanisms, including translational efficiency, translational fidelity, and cotranslational folding of the encoded protein. To date, most studies of synonymous codon-derived perturbations have focused on effects within a single gene.

Remnants of the Balbiani body are required for formation of RNA transport granules in oocytes.

The Balbiani body (Bb), an organelle comprised of mitochondria, ER, and RNA, is found in the oocytes of most organisms. In , the structure is initially positioned immediately adjacent to the nucleus, extends toward the vegetal pole, and eventually disperses, leaving behind a region highly enriched in mitochondria. This area is later transversed by RNP complexes that are being localized to the vegetal cortex.

High-Throughput, Comprehensive Single-Cell Proteomic Analysis of Embryos at the 50-Cell Stage Using a Microplate-Based MICROFASP System.

We report both the design of a high-throughput MICROFASP (a miniaturized filter aided sample preparation) system and its use for the comprehensive proteomic analysis of single blastomeres isolated from 50-cell stage embryos (∼200 ng of yolk-free protein/blastomere). A single run of the MICROFASP system was used to process 146 of these blastomeres in parallel. Three samples failed to generate signals presumably due to membrane clogging.

Quantitative capillary zone electrophoresis-mass spectrometry reveals the N-glycome developmental plan during vertebrate embryogenesis.

Glycans are known to be involved in many biological processes, while little is known about the expression of N-glycans during vertebrate development. We now report the first quantitative studies of both the expression of N-linked glycans at six early development stages and the expression of N-glycosylated peptides at two early development stages in Xenopus laevis, the African clawed frog. N-Glycans were labeled with isobaric tandem mass tags, pooled, separated by capillary electrophoresis, and characterized using tandem mass spectrometry.

Miniaturized Filter-Aided Sample Preparation (MICRO-FASP) Method for High Throughput, Ultrasensitive Proteomics Sample Preparation Reveals Proteome Asymmetry in Embryos.

We report a miniaturized filter aided sample preparation method (micro-FASP) for low-loss preparation of submicrogram proteomic samples. The method employs a filter with ∼0.1 mm surface area, reduces the total volume of reagents to <10 μL, and requires only two sample transfer steps.

MALDI-imaging of early stage Xenopus laevis embryos.

Xenopus laevis is an important model organism for vertebrate development. An extensive literature has developed on changes in transcript expression during development of this organism, and there is a growing literature on the corresponding protein expression changes during development. In contrast, there is very little information on changes in metabolite expression during development.

A deficiency in SUMOylation activity disrupts multiple pathways leading to neural tube and heart defects in Xenopus embryos.

Background: Adenovirus protein, Gam1, triggers the proteolytic destruction of the E1 SUMO-activating enzyme. Microinjection of an empirically determined amount of Gam1 mRNA into one-cell Xenopus embryos can reduce SUMOylation activity to undetectable, but nonlethal, levels, enabling an examination of the role of this post-translational modification during early vertebrate development.

Results: We find that SUMOylation-deficient embryos consistently exhibit defects in neural tube and heart development.

Time-lapse imaging of cell death in cell culture and whole living organisms using turn-on deep-red fluorescent probes.

Cell death is a central process in developmental biology and also an important indicator of disease status and treatment efficacy. Two related fluorescent probes are described that are molecular conjugates of one or two zinc dipicolylamine (ZnDPA) coordination complexes with an appended solvatochromic benzothiazolium squaraine dye. The probes were designed to target the anionic phospholipid, phosphatidylserine (PS), that is exposed on the surface of dead and dying cells.

Author Correction: Phosphorylation Dynamics Dominate the Regulated Proteome during Early Xenopus Development.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Developing Practical Therapeutic Strategies that Target Protein SUMOylation.

Post-translational modification by small ubiquitin-like modifier (SUMO) has emerged as a global mechanism for the control and integration of a wide variety of biological processes through the regulation of protein activity, stability and intracellular localization. As SUMOylation is examined in greater detail, it has become clear that the process is at the root of several pathologies including heart, endocrine, and inflammatory disease, and various types of cancer. Moreover, it is certain that perturbation of this process, either globally or of a specific protein, accounts for many instances of congenital birth defects.

Phosphorylation Dynamics Dominate the Regulated Proteome during Early Xenopus Development.

The earliest stages of animal development are largely controlled by changes in protein phosphorylation mediated by signaling pathways and cyclin-dependent kinases. In order to decipher these complex networks and to discover new aspects of regulation by this post-translational modification, we undertook an analysis of the X. laevis phosphoproteome at seven developmental stages beginning with stage VI oocytes and ending with two-cell embryos.

Single Cell Proteomics Using Frog (Xenopus laevis) Blastomeres Isolated from Early Stage Embryos, Which Form a Geometric Progression in Protein Content.

Single cell analysis is required to understand cellular heterogeneity in biological systems. We propose that single cells (blastomeres) isolated from early stage invertebrate, amphibian, or fish embryos are ideal model systems for the development of technologies for single cell analysis. For these embryos, although cell cleavage is not exactly symmetric, the content per blastomere decreases roughly by half with each cell division, creating a geometric progression in cellular content.

Nearly 1000 Protein Identifications from 50 ng of Xenopus laevis Zygote Homogenate Using Online Sample Preparation on a Strong Cation Exchange Monolith Based Microreactor Coupled with Capillary Zone Electrophoresis.

A sulfonate-silica hybrid strong cation exchange monolith microreactor was synthesized and coupled to a linear polyacrylamide coated capillary for online sample preparation and capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) bottom-up proteomic analysis. The protein sample was loaded onto the microreactor in an acidic buffer. After online reduction, alkylation, and digestion with trypsin, the digests were eluted with 200 mM ammonium bicarbonate at pH 8.

Proteomics of Xenopus development.

Modern mass spectrometry-based methods provide an exciting opportunity to characterize protein expression in the developing embryo. We have employed an isotopic labeling technology to quantify the expression dynamics of nearly 6000 proteins across six stages of development in Xenopus laevis from the single stage zygote through the mid-blastula transition and the onset of organogenesis. Approximately 40% of the proteins show significant changes in expression across the development stages.

Tetrahydrofuranyl and tetrahydropyranyl protection of amino acid side-chains enables synthesis of a hydroxamate-containing aminoacylated tRNA.

The ability to specifically engineer metal binding sites into target proteins has far-reaching consequences ranging from the development of new biocatalysts and imaging reagents to the production of proteins with increased stability. We report the efficient tRNA-mediated incorporation of the hydroxamate containing amino acid, N(ε)-acetyl-N(ε)-hydroxy-L-lysine, into a transcription factor (TFIIIA). Because this amino acid is compact, hydrophilic, and uncharged at physiological pH, it should have little or no effect on protein folding or solubility.

Small ubiquitin-like modifier (SUMO)-mediated repression of the Xenopus Oocyte 5 S rRNA genes.

The 5 S rRNA gene-specific transcription factor IIIA (TFIIIA) interacts with the small ubiquitin-like modifier (SUMO) E3 ligase PIAS2b and with one of its targets, the transcriptional corepressor, XCtBP. PIAS2b is restricted to the cytoplasm of Xenopus oocytes but relocates to the nucleus immediately after fertilization. Following the midblastula transition, PIAS2b and XCtBP are present on oocyte-type, but not somatic-type, 5 S rRNA genes up through the neurula stage, as is a limiting amount of TFIIIA.

Quantitative proteomics of Xenopus laevis embryos: expression kinetics of nearly 4000 proteins during early development.

While there is a rich literature on transcription dynamics during the development of many organisms, protein data is limited. We used iTRAQ isotopic labeling and mass spectrometry to generate the largest developmental proteomic dataset for any animal. Expression dynamics of nearly 4,000 proteins of Xenopus laevis was generated from fertilized egg to neurula embryo.

RNA localization in Xenopus oocytes uses a core group of trans-acting factors irrespective of destination.

The 3' untranslated region of mRNA encoding PHAX, a phosphoprotein required for nuclear export of U-type snRNAs, contains cis-acting sequence motifs E2 and VM1 that are required for localization of RNAs to the vegetal hemisphere of Xenopus oocytes. However, we have found that PHAX mRNA is transported to the opposite, animal, hemisphere. A set of proteins that cross-link to the localization elements of vegetally localized RNAs are also cross-linked to PHAX and An1 mRNAs, demonstrating that the composition of RNP complexes that form on these localization elements is highly conserved irrespective of the final destination of the RNA.

Binding site for Xenopus ribosomal protein L5 and accompanying structural changes in 5S rRNA.

The structure of the eukaryotic L5-5S rRNA complex was investigated in protection and interference experiments and is compared with the corresponding structure (L18-5S rRNA) in the Haloarcula marismortui 50S subunit. In close correspondence with the archaeal structure, the contact sites for the eukaryotic ribosomal protein are located primarily in helix III and loop C and secondarily in loop A and helix V. While the former is unique to L5, the latter is also a critical contact site for transcription factor IIIA (TFIIIA), accounting for the mutually exclusive binding of these two proteins to 5S RNA.

Detection of protein-RNA complexes in Xenopus oocytes.

There is a remarkable variety of mechanisms for controlling post-transcriptional gene expression that is achieved through the formation of ribonucleoprotein (RNP) complexes on specific cis-acting regions of mRNA. These complexes regulate splicing, nuclear and cytoplasmic polyadenylation, stability, localization, and translation. Thus, it is important to be able to detect the association of specific proteins with specific RNAs within the context of these RNP complexes.

Interactions of 40LoVe within the ribonucleoprotein complex that forms on the localization element of Xenopus Vg1 mRNA.

Proline rich RNA-binding protein (Prrp), which associates with mRNAs that employ the late pathway for localization in Xenopus oocytes, was used as bait in a yeast two-hybrid screen of an expression library. Several independent clones were recovered that correspond to a paralog of 40LoVe, a factor required for proper localization of Vg1 mRNA to the vegetal cortex. 40LoVe is present in at least three alternatively spliced isoforms; however, only one, corresponding to the variant identified in the two-hybrid screen, can be crosslinked to Vg1 mRNA.

A manganese-dependent ribozyme in the 3'-untranslated region of Xenopus Vg1 mRNA.

The smallest catalytic RNA identified to date is a manganese-dependent ribozyme that requires only a complex between GAAA and UUU to effect site-specific cleavage. We show here that this ribozyme occurs naturally in the 3'-UTR of Vg1 and beta-actin mRNAs. In accord with earlier studies with model RNAs, cleavage occurs only in the presence of manganese or cadmium ions and proceeds optimally near 30 degrees C and physiological pH.

Deposition of DNA rafts on cationic SAMs on silicon [100].

We demonstrate a guided self-assembly approach to the fabrication of DNA nanostructures on silicon substrates. DNA oligonucleotides self-assemble into "rafts" 8 x 37 x 2 nm in size. The rafts bind to cationic SAMs on silicon wafers.

Restricted specificity of Xenopus TFIIIA for transcription of somatic 5S rRNA genes.

Xenopus transcription factor IIIA (TFIIIA) is phosphorylated on serine-16 by CK2. Replacements with alanine or glutamic acid were made at this position in order to address the question of whether phosphorylation possibly influences the function of this factor. Neither substitution has an effect on the DNA or RNA binding activity of TFIIIA.