Mago nashi is essential for spermatogenesis in Marsilea

Spermatogenesis in Marsilea vestita is a rapid process that is activated by placing dry microspores into water. Nine division cycles produce seven somatic cells and 32 spermatids, where size and position define identity. Spermatids undergo de novo formation of basal bodies in a particle known as a blepharoplast. We are interested in mechanisms responsible for spermatogenous initial formation. Mago nashi (Mv-mago) is a highly conserved gene present as stored mRNA and stored protein in the microspore. Mv-mago protein increases in abundance during development and it localizes at discrete cytoplasmic foci (Mago-dots). RNA interference experiments show that new Mv-mago protein is required for development. With Mv-mago silenced, asymmetric divisions become symmetric, cell fate is disrupted, and development stops. The alpha-tubulin protein distribution, centrin translation, and Mv-PRP19 mRNA distribution are no longer restricted to the spermatogenous cells. Centrin aggregations, resembling blepharoplasts, occur in jacket cells. Mago-dots are undetectable after the silencing of Mv-mago, Mv-Y14, or Mv-eIF4AIII, three core components of the exon junction complex (EJC), suggesting that Mago-dots are either EJCs in the cytoplasm, or Mv-mago protein aggregations dependent on EJCs. Mv-mago protein and other EJC components apparently function in cell fate determination in developing male gametophytes of M. vestita.     

The control of protein synthesis during heat shock in Drosophila cells involves altered polypeptide elongation rates

When Drosophila tissue culture cells are shifted from 25 to 36°C (heat shocked) the pre-existing mRNAs (25°C mRNAs) remain in the cytoplasm but their translation products are underrepresented relative to the induced heat shock proteins. Many of these undertranslated 25°C mRNAs are found in association with polysomes of similar size in heat-shocked and control cells. Furthermore, the messages encoding α-tubulin, β-tubulin, and actin are found associated with one-third to one-half as many total ribosomes in heat-shocked cells as in cells incubated at 25°C. Increased temperature should lead to increased output of protein per ribosome. However, the 25°C proteins are actually synthesized at less than 10% of 25°C levels in heat-shocked cells. Thus, the rates of both elongation and initiation of translation are significantly (15- to 30-fold) slower on 25°C mRNAs than they are on heat shock mRNAs in heat-shocked cells.     

DIVISION OF THE GENERATIVE CELL AND LATE DEVELOPMENT IN THE MALE GAMETOPHYTE OF GINKGO BILOBA

Division of the generative cell in the male gametophyte of Ginkgo biloba to yield the sterile cell and spermatogenous cell was examined in vivo and in vitro. Evidence is presented in support of a new interpretation of development in which the sterile cell and spermatogenous cell arise from an unusual anticlinal ringlike division of the generative cell. This type of cell division is only known to occur during antheridial development in leptosporangiate ferns and stomatal development among certain ferns in the Schizaeaceae and Polypodiaceae. The strong similarities in development and cell arrangement within the male gametophytes of cycads and Ginkgo suggest that division of the generative cell in cycads may be the same as in Ginkgo. Although the ringlike (conically annular) divisions in the antheridia of leptosporangiate ferns and the male gametophytes of Ginkgo (and probably cycads) are remarkably similar and result in the production of a central spermatogenous cell, it is conjectural as to whether these patterns represent a striking convergence or evolutionary homology.     

Activity-dependent polyadenylation in neurons

Activity-dependent changes in protein synthesis modify synaptic efficacy. One mechanism that regulates mRNA translation in the synapto-dendritic compartment is cytoplasmic polyadenylation, a process controlled by CPEB, the cytoplasmic polyadenylation element (CPE)-specific RNA binding protein. In neurons, very few mRNAs are known CPEB substrates, and none appear to be responsible for the effects on plasticity that are found in the CPEB knockout mouse. These results suggest that the translation of other mRNAs is regulated by CPEB. To identify them, we have developed a functional assay based on the polyadenylation of brain-derived mRNAs injected into Xenopus oocytes, a surrogate system that carries out this 3' end processing event in an efficient manner. The polyadenylated RNAs were isolated by binding to and thermal elution from poly(U) agarose and identified by microarray analysis. Selected sequences that were positive for polyadenylation were cloned and retested for polyadenylation by injection into oocytes. These sequences were then examined for activity-dependent polyadenylation in cultured hippocampal neurons. Finally, the levels of two proteins encoded by polyadenylated mRNAs were examined in glutamate-stimulated synaptoneurosomes. These studies show that many mRNAs undergo activity-dependent polyadenylation in neurons and that this process coincides with increased translation in the synapto-dendritic compartment.     

Autoregulation of Musashi1 mRNA translation during Xenopus oocyte maturation

The mRNA translational control protein, Musashi, plays a critical role in cell fate determination through sequence‐specific interactions with select target mRNAs. In proliferating stem cells, Musashi exerts repression of target mRNAs to promote cell cycle progression. During stem cell differentiation, Musashi target mRNAs are de‐repressed and translated. Recently, we have reported an obligatory requirement for Musashi to direct translational activation of target mRNAs during Xenopus oocyte meiotic cell cycle progression. Despite the importance of Musashi in cell cycle regulation, only a few target mRNAs have been fully characterized. In this study, we report the identification and characterization of a new Musashi target mRNA in Xenopus oocytes. We demonstrate that progesterone‐stimulated translational activation of the Xenopus Musashi1 mRNA is regulated through a functional Musashi binding element (MBE) in the Musashi1 mRNA 3′ untranslated region (3′ UTR). Mutational disruption of the MBE prevented translational activation of Musashi1 mRNA and its interaction with Musashi protein. Further, elimination of Musashi function through microinjection of inhibitory antisense oligonucleotides prevented progesterone‐induced polyadenylation and translation of the endogenous Musashi1 mRNA. Thus, Xenopus Musashi proteins regulate translation of the Musashi1 mRNA during oocyte maturation. Our results indicate that the hierarchy of sequential and dependent mRNA translational control programs involved in directing progression through meiosis are reinforced by an intricate series of nested, positive feedback loops, including Musashi mRNA translational autoregulation. These autoregulatory positive feedback loops serve to amplify a weak initiating signal into a robust commitment for the oocyte to progress through the cell cycle and become competent for fertilization.Mol. Reprod. Dev. 79: 553‐563, 2012. © 2012 Wiley Periodicals, Inc.