Microprocessor Recruitment to Elongating RNA Polymerase II Is Required for Differential Expression of MicroRNAs.

The cellular abundance of mature microRNAs (miRNAs) is dictated by the efficiency of nuclear processing of primary miRNA transcripts (pri-miRNAs) into pre-miRNA intermediates. The Microprocessor complex of Drosha and DGCR8 carries this out, but it has been unclear what controls Microprocessor's differential processing of various pri-miRNAs. Here, we show that Drosophila DGCR8 (Pasha) directly associates with the C-terminal domain of the RNA polymerase II elongation complex when it is phosphorylated by the Cdk9 kinase (pTEFb). When association is blocked by loss of Cdk9 activity, a global change in pri-miRNA processing is detected. Processing of pri-miRNAs with a UGU sequence motif in their apical junction domain increases, while processing of pri-miRNAs lacking this motif decreases. Therefore, phosphorylation of RNA polymerase II recruits Microprocessor for co-transcriptional processing of non-UGU pri-miRNAs that would otherwise be poorly processed. In contrast, UGU-positive pri-miRNAs are robustly processed by Microprocessor independent of RNA polymerase association.

Functionally diverse microRNA effector complexes are regulated by extracellular signaling.

Because microRNAs (miRNAs) influence the expression of many genes in cells, discovering how the miRNA pathway is regulated is an important area of investigation. We found that the Drosophila miRNA-induced silencing complex (miRISC) exists in multiple forms. A constitutive form, called G-miRISC, is comprised of Ago1, miRNA, and GW182. Two distinct miRISC complexes that lack GW182 are regulated by mitogenic signaling. Exposure of cells to serum, lipids, or the tumor promoter PMA suppressed formation of these complexes. P-miRISC is comprised of Ago1, miRNA, and Loqs-PB, and it associates with mRNAs assembled into polysomes. The other regulated Ago1 complex associates with membranous organelles and is likely an intermediate in miRISC recycling. The formation of these complexes is correlated with a 5- to 10-fold stronger repression of target gene expression inside cells. Taken together, these results indicate that mitogenic signaling regulates the miRNA effector machinery to attenuate its repressive activities.

A pre-embedding immunogold approach reveals localization of myosin VI at the ultrastructural level in the actin cones that mediate Drosophila spermatid individualization.

Stable actin structures play important roles in the development and specialization of differentiated cells. How these structures form, are organized, and are used to mediate physiological processes is not well understood in most cases. In Drosophila testis, stable actin structures, called actin cones, mediate spermatid individualization, a large-scale cellular remodeling process. These actin cones are composed of two structural domains, a front meshwork and a rear region of parallel bundles. Myosin VI is an important player in proper actin cone organization and function. Myosin VI localizes to the cones' fronts and its specific localization is required for proper actin cone formation and function during individualization. To understand how these structures are organized and assembled, ultrastructural studies are important to reveal both organization of actin and the precise localization of actin regulators relative to regions with different filament organizations. In the present work, we have developed a novel pre-embedding immunogold-silver labeling method for high-resolution analysis of protein distribution in actin structures which allowed both satisfactory antibody labeling and good ultrastructural preservation. Electron microscopic studies revealed that myosin VI accumulated at the extreme leading edge of the actin cone and preferentially localized throughout the front meshwork of the cone where branched actin filaments were most concentrated. No myosin VI labeling was found adjacent to the membranes along the length of the cone or connecting neighboring cones. This method has potential to reveal important information about precise relationships between actin-binding proteins, membranes, and different types of actin structures.

Myosin VI regulates actin structure specialization through conserved cargo-binding domain sites.

Actin structures are often stable, remaining unchanged in organization for the lifetime of a differentiated cell. Little is known about stable actin structure formation, organization, or maintenance. During Drosophila spermatid individualization, long-lived actin cones mediate cellular remodeling. Myosin VI is necessary for building the dense meshwork at the cones' fronts. We test several ideas for myosin VI's mechanism of action using domain deletions or site-specific mutations of myosin VI. The head (motor) and globular tail (cargo-binding) domains were both needed for localization at the cone front and dense meshwork formation. Several conserved partner-binding sites in the globular tail previously identified in vertebrate myosin VI were critical for function in cones. Localization and promotion of proper actin organization were separable properties of myosin VI. A vertebrate myosin VI was able to localize and function, indicating that functional properties are conserved. Our data eliminate several models for myosin VI's mechanism of action and suggest its role is controlling organization and action of actin assembly regulators through interactions at conserved sites. The Drosophila orthologues of interaction partners previously identified for vertebrate myosin VI are likely not required, indicating novel partners mediate this effect. These data demonstrate that generating an organized and functional actin structure in this cell requires multiple activities coordinated by myosin VI.

Coiled-coil-mediated dimerization is not required for myosin VI to stabilize actin during spermatid individualization in Drosophila melanogaster.

Myosin VI is a pointed-end-directed actin motor that is thought to function as both a transporter of cargoes and an anchor, capable of binding cellular components to actin for long periods. Dimerization via a predicted coiled coil was hypothesized to regulate activity and motor properties. However, the importance of the coiled-coil sequence has not been tested in vivo. We used myosin VI's well-defined role in actin stabilization during Drosophila spermatid individualization to test the importance in vivo of the predicted coiled coil. If myosin VI functions as a dimer, a forced dimer should fully rescue myosin VI loss of function defects, including actin stabilization, actin cone movement, and cytoplasmic exclusion by the cones. Conversely, a molecule lacking the coiled coil should not rescue at all. Surprisingly, neither prediction was correct, because each rescued partially and the molecule lacking the coiled coil functioned better than the forced dimer. In extracts, no cross-linking into higher molecular weight forms indicative of dimerization was observed. In addition, a sequence required for altering nucleotide kinetics to make myosin VI dimers processive is not required for myosin VI's actin stabilization function. We conclude that myosin VI does not need to dimerize via the predicted coiled coil to stabilize actin in vivo.

The localization of LAP2 beta during pronuclear formation in bovine oocytes after fertilization or activation.

We have shown that the assembly of lamin-associated polypeptide (LAP) 2beta was detected surrounding the chromatin mass around the time of extrusion of the second polar body (PB) in some fertilized oocytes, but not in most activated oocytes, by using A23187 and cycloheximide (CaA + CH). Here, we immunohistologically analysed the correlation between LAP2beta assembly and chromatin condensation in fertilized and activated oocytes during the second meiosis. In bovine cumulus cells, the onset of LAP2beta assembly was observed around anaphase chromosomes with strongly phosphorylated histone H3. No LAP2beta assembled around the chromosomes in the first and second polar bodies and the alternative oocyte chromatin (oCh) if histone H3 was phosphorylated. Only histone H3 of oCh was completely dephosphorylated during the telophase II/G1 transition (Tel II/G1), and then LAP2beta assembled around only the oCh without phosphorylated histone H3. In the oocytes activated by CaA + CH, LAP2beta did not assemble around the condensed oCh during the Tel II/G1 transition, although their histone H3 dephosphorylation occurred rather rapidly compared with that of the fertilized oocytes. The patterns of histone H3 dephosphorylation and LAP2beta assembly in oocytes activated by CaA alone showed greater similarity to those in fertilized oocytes than to those in oocytes activated by CaA + CH. These results show that LAP2beta assembles around only oCh after complete dephosphorylation of histone H3 after fertilization and activation using CaA alone, and that the timing of histone H3 dephosphorylation and LAP2beta assembly in these oocytes is different from that of somatic cells. The results also indicate that CH treatment inhibits LAP2beta assembly around oCh but not histone H3 dephosphorylation.

The localization of LAP2beta in bovine oocytes after in vitro activation and fertilization.

The present study was designed to clarify the localization of LAP2beta and to compare it with those of lamins A/C and B in bovine oocytes after activation and in vitro fertilization (IVF). After fertilization, LAP2beta was not found until telophase II, and was observed around condensed chromatin after the extrusion of the second polar body, but not in activated oocytes. Although the reaction of LAP2beta was temporally negative or weak on the membrane of the growing small pronuclei, it became strong on the fully grown pronuclei of both activated and fertilized oocytes. Examination of the timing of DNA synthesis using bromodeoxyuridine revealed that the expression of LAP2beta on the pronuclear membrane became strong around the end of the DNA synthesis in both activated and fertilized oocytes. Both male and female pronuclei exhibited the same reactivity to all nuclear proteins examined. It was also shown that LAP2beta first assembled around condensed chromatin, followed by the integration of lamins B and A/C as in somatic cells. LAP2beta staining was maintained on the nuclear membrane of the embryonic cells at interphase until the later stage of preimplantational development. There were no differences between parthenogenetic and fertilized embryos in the expression and localization of LAP2beta from the PN-stage oocyte to the blastocyst. The assembly of LAP2beta was observed around the telophase chromatin of both blastocyst and cumulus cells. Thus, it was shown that the timing of the aggregation of LAP2beta at the second meiosis was different from that in the mitosis of blastocyst and somatic cells. LAP2beta was constantly expressed in the nuclear membrane in in vitro fertilized and parthenogenetic embryos as was lamin B, and lamin A/C was expressed stage-dependently in both types of embryos. Lamin A/C was positive in some inner cell mass cells of parthenogenetic blastocysts, but not those of in vitro fertilized embryos.

Stage-specific effect of growth hormone on developmental competence of bovine embryos produced in-vitro.

Many efforts have been made to develop effective culture conditions for the production of bovine blastocysts. Growth hormone (GH) and glucose are known to affect in vitro embryo development. To improve in vitro culture conditions, the culture medium containing fetal calf serum (FCS) or bovine serum albumin (BSA) was supplemented with GH at various periods of development, and the effects of GH on the rate of development and the quality of the blastocysts were studied. Then, starting at the morula stage, the effect of glucose and GH on the rate of development was studied. In all experimental periods, FCS was more effective than BSA at improving the development rate and increasing the cell number of blastocysts. Adding GH to the culture medium between 18 and 48 h after fertilization (1-8 cell stage embryo) did not affect either the rate of blastulation or the cell number regardless of the serum protein (FCS or BSA). From 48 to 120 h after fertilization (5-cell to morula stage) GH increased the cell number of the blastocysts in the presence of BSA, but not in the presence of FCS. From 120 to 192 h after fertilization (morula to blastocyst stage), GH improved the developmental rate and cell number in the presence of FCS, although there was no significant difference when BSA was used instead of FCS as the serum protein. When cows were implanted with blastocysts developed in the presence of GH from the morula stage, their pregnancy rate did not differ from that of the control. Increasing the glucose concentration in the medium from 1.5 mM to 3 mM starting at the morula stage (120 h after fertilization) slightly decreased the rate of development, but on the other hand, decreasing the glucose concentration to 0 mM did not affect either the rate of development or the cell number. Also, then GH had no effect on the developmental rate or the cell number in the absence of glucose. In conclusion, when the medium was supplemented with serum, GH improved embryo development from the morula stage, but an increased concentration of glucose decreased embryo development. Furthermore, GH did not improve the pregnancy rate of blastocysts developed in vitro.