Co-Administration of Myostatin-Targeting siRNA and ActRIIB-Fc Fusion Protein Increases Masseter Muscle Mass and Fiber Size.

Myostatin, a member of the TGF-ß superfamily, is a negative regulator of skeletal muscle cell growth and differentiation, and binds with high affinity to the activin type IIB receptor (ActRIIB). The soluble ligand-binding domain of ActRIIB fused to the Fc domain of IgG (ActRIIB-Fc) potently binds and inhibits TGF-ß family members in muscle, leading to rapid and marked muscle growth. The present study was designed to assess the effectiveness of the co-delivery of myostatin-targeting siRNA (Mstn-siRNA) and ActRIIB-Fc into skeletal muscle as a potential treatment of atrophic myopathies. Eleven-week-old, male C57BL/6 mice were injected with atelocollagen (ATCOL)-mediated Mstn-siRNA with/without ActRIIB-Fc locally into the masseter muscle twice a week. Inhibition of myostatin function by the combination of Mstn-siRNA and ActRIIB-Fc increased muscle weight and myofibril size in murine masseter muscle. Real-time RT-PCR analysis revealed significant downregulation of myostatin mRNA expression in both the Mstn-siRNA-treated and the combination treatment group. Furthermore, myogenin mRNA expression was upregulated in the combination treatment group, while MuRF-1 and Atrogin-1 mRNA expression was downregulated compared to administration of each compound alone. These findings suggest that double inhibition of myostatin is a potentially useful treatment strategy to increase muscle mass and fiber size and could be a useful treatment of patients with various muscle atrophies, including muscular dystrophy.

Translational Regulation in the Mammalian Oocyte.

Fully grown oocytes arrest meiosis at prophase I and deposit maternal RNAs. A subset of maternal transcripts is stored in a dormant state in the oocyte, and the timely driven translation of specific mRNAs guides meiotic progression, the oocyte-embryo transition, and early embryo development. In the absence of transcription, the regulation of gene expression in oocytes is controlled almost exclusively at the level of transcriptome and proteome stabilization and at the level of protein synthesis.This chapter focuses on the recent findings on RNA distribution related to the temporal and spatial translational control of the meiotic cycle progression in mammalian oocytes. We discuss the most relevant mechanisms involved in the organization of the oocyte's maternal transcriptome storage and localization, and the regulation of translation, in correlation with the regulation of oocyte meiotic progression.

The mRNAs of maternally and paternally inherited mtDNAs of the mussel Mytilus galloprovincialis: start/end points and polycistronic transcripts.

Transcription of the mitochondrial genomes of the Mediterranean mussel Mytilus galloprovincialis has been studied by RT-PCR and RNA circularization. This species has an egg-transmitted (F) and a sperm-transmitted (M) mitochondrial genome, in accordance with the doubly uniparental inheritance (DUI) pattern of mtDNA transmission. The primary transcript is cleaved into ten transcripts, eight of which are monocistronic, one is tricistronic and one is most likely, but not certainly, bicistronic. The start/end points of these transcripts have been determined. In the majority of cases cleavage is mediated according to the "tRNA punctuation" model. However, we have identified four cases of cleavage that do not coincide with the presence of a tRNA. In these cases transcription starts immediately or only a few bases from the end point of the preceding gene and cleavage is, most likely, mediated by a stem-loop structure formed at the start point of the gene. The identification of a tricistronic transcript is a novel finding for metazoan mtDNA. We propose that its evolution has been facilitated by the fact that all coding genes are transcribed from the same DNA strand and that co-transcription is sustained by selection emanating from the fact that proteins derived from all three co-transcribed genes participate in the formation of the same oxidative phosphorylation complex.

Zygotic amplification of secondary piRNAs during silkworm embryogenesis.

PIWI-interacting RNAs (piRNAs) are 23-30-nucleotide-long small RNAs that act as sequence-specific silencers of transposable elements in animal gonads. In flies, genetics and deep sequencing data have led to a hypothesis for piRNA biogenesis called the ping-pong cycle, where antisense primary piRNAs initiate an amplification loop to generate sense secondary piRNAs. However, to date, the process of the ping-pong cycle has never been monitored at work. Here, by large-scale profiling of piRNAs from silkworm ovary and embryos of different developmental stages, we demonstrate that maternally inherited antisense-biased piRNAs trigger acute amplification of secondary sense piRNA production in zygotes, at a time coinciding with zygotic transcription of sense transposon mRNAs. These results provide on-site evidence for the ping-pong cycle.

Localization and anchorage of maternal mRNAs to cortical structures of ascidian eggs and embryos using high resolution in situ hybridization.

In several species, axis formation and tissue differentiation are the result of developmental cascades which begin with the localization and translation of key maternal mRNAs in eggs. Localization and anchoring of mRNAs to cortical structures can be observed with high sensitivity and resolution by fluorescent in situ hybridization coupled with labeling of membranes and macromolecular complexes. Oocytes and embryos of ascidians--marine chordates closely related to vertebrates--are ideal models to understand how maternal mRNAs pattern the simple ascidian tadpole. More than three dozen cortically localized maternal mRNAs have been identified in ascidian eggs. They include germ cell markers such as vasa or pem-3 and determinants of axis (pem-1), unequal cleavage (pem-1), and muscle cells (macho-1). High resolution localization of mRNAs, proteins, and lipids in whole eggs and embryos and their cortical fragments shows that maternal mRNA determinants pem-1 and macho-1 are anchored to cortical endoplasmic reticulum and segregate with it into small posterior somatic cells. In contrast, mRNAs such as vasa are associated with granular structures which are inherited by the same somatic cells plus adjacent germ cell precursors. In this chapter, we provide detailed protocols for simultaneous localization of mRNAs and proteins to determine their association with cellular structures in eggs and embryos. Using preparations of isolated cortical fragments with intact membranous structures allows unprecedented high resolution analysis and identification of cellular anchoring sites for key mRNAs. This information is necessary for understanding the mechanisms for localizing mRNAs and partitioning them into daughter cells after cleavage.

Identification and sequencing of remnant messenger RNAs found in domestic swine (Sus scrofa) fresh ejaculated spermatozoa.

The existence of specific messenger RNA remnants contained within freshly ejaculated spermatozoa was described in several species. Those investigations, using high-throughput techniques to screen the population of transcripts in ejaculated spermatozoa, were limited to the probes which mostly derived from nucleic acids of testicular tissues of either human or mice. The objective of this study was to investigate mRNA remnants from ejaculated spermatozoa of the domestic swine (Sus scrofa), a valuable model for biomedical research. A non-redundant 5'-end complementary DNA library was generated from swine ejaculated spermatozoa. After sequence quality verification, 4562 clones remained. These clones were then clustered and assembled into 514 unique sequences including 188 contigs (36.58%) and 326 singletons (63.42%), representing those clusters containing at least two clones and those clusters without having enough similarity with other clones. These unique gene sequences were annotated in Gene Ontology (GO) hierarchy; they included biological processes (38.7%), molecular functions (39.1%) and cellular components (40.3%). Based on the analysis, a broad spectrum of messenger RNAs existed in swine ejaculated spermatozoa and was closely correlated with nucleic acid binding, structural modifications, and transcriptional regulation. All of these categories are considered to have profound effects on the male reproductive system. Therefore, our work provides initial results on potential spermatozoal gene expression for future studies regarding the tightly regulated spermiogenic processes and later fertilization events.

The functional analysis of Type I postplasmic/PEM mRNAs in embryos of the ascidian Halocynthia roretzi.

Maternal factors, such as a muscle determinant macho-1 mRNA that is localized to the posterior-vegetal cortex (PVC) of fertilized ascidian eggs, are crucial for embryonic axis formation and cell fate specification. Maternal mRNAs that show an identical posterior localization pattern to that of macho-1 in eggs and embryos are called Type I postplasmic/PEM mRNAs. We investigated the functions of five of the nine Type I mRNAs so far known in Halocynthia roretzi: Hr-Wnt-5, Hr-GLUT, Hr-PEM3, Hr-PEN1, and Hr-PEN2. Suppression of their functions with specific antisense morpholino oligonucleotides (MOs) had effects on the formation of various tissues: Hr-Wnt-5 on notochord, muscle, and mesenchyme, although zygotic function of Hr-Wnt-5 is responsible for notochord formation; Hr-GLUT on notochord, mesenchyme, and endoderm; and Hr-PEN2 on muscle, mesenchyme, and endoderm. On the other hand, Hr-PEM3 and Hr-PEN1 MOs seemed to have no effect. We conclude that the functions of at least some localized maternal Type I postplasmic/PEM mRNAs are necessary for early embryonic patterning in ascidians.

The mitochondrial ribosome-specific MrpL55 protein is essential in Drosophila and dynamically required during development.

We report on the essential Drosophila mRpL55 gene conserved exclusively in metazoans. Null mRpL55 mutants did not grow after hatching, moved slowly and died as first instar larvae. MrpL55 is similar to mammalian MRPL55, a protein that, in a large-scale mass spectrometry study, has been found as a mitoribosome-specific large subunit protein. We showed that MrpL55 was localised to the mitochondrion in S2 cells and tissues and was enriched in cells with a higher protein synthesis activity. The MrpL55 protein contains a KOW-like motif present in proteins with a role in transcriptional anti-termination and regulation of translation. Modulation of mRpL55 expression level is critical for development. Somatic clonal analysis showed that MrpL55 was not required in larval eye imaginal discs but required in pupal discs apparently during the second mitotic wave. Therefore, our results showed that the MrpL55 protein acts dynamically in the cell during development. We propose that MrpL55 is involved in Drosophila mitochondrial biogenesis and G2/M phase cell cycle progression.