piRNAs mediate posttranscriptional retroelement silencing and localization to pi-bodies in the Drosophila germline

Nuage, a well-conserved perinuclear organelle found in germline cells, is thought to mediate retroelement repression in Drosophila melanogaster by regulating the production of Piwi-interacting RNAs (piRNAs). In this study, we present evidence that the nuage–piRNA pathway components can be found in cytoplasmic foci that also contain retroelement transcripts, antisense piRNAs, and proteins involved in messenger RNA (mRNA) degradation. These mRNA degradation proteins, decapping protein 1/2 (DCP1/2), Me31B (maternal expression at 31B), and pacman (PCM), are normally thought of as components of processing bodies. In spindle-E (spn-E) and aubergine (aub) mutants that lack piRNA production, piRNA pathway proteins no longer overlap the mRNA degradation proteins. Concomitantly, spn-E and aub mutant ovaries show an accumulation of full-length retroelement transcripts and prolonged stabilization of HeT-A mRNA, supporting the role of piRNAs in mediating posttranscriptional retroelement silencing. HeT-A mRNA is derepressed in mRNA degradation mutants twin, dcp1, and ski3, indicating that these enzymes also aid in removing full-length transcripts and/or decay intermediates

A tudor domain protein SPINDLIN1 interacts with the mRNA-binding protein SERBP1 and is involved in mouse oocyte meiotic resumption

Mammalian oocytes are arrested at prophase I of meiosis, and resume meiosis prior to ovulation. Coordination of meiotic arrest and resumption is partly dependent on the post-transcriptional regulation of maternal transcripts. Here, we report that, SPINDLIN1 (SPIN1), a maternal protein containing Tudor-like domains, interacts with a known mRNA-binding protein SERBP1, and is involved in regulating maternal transcripts to control meiotic resumption. Mouse oocytes deficient for Spin1 undergo normal folliculogenesis, but are defective in resuming meiosis. SPIN1, via its Tudor-like domain, forms a ribonucleoprotein complex with SERBP1, and regulating mRNA stability and/or translation. The mRNA for the cAMP-degrading enzyme, PDE3A, is reduced in Spin1 mutant oocytes, possibly contributing to meiotic arrest. Our study demonstrates that Spin1 regulates maternal transcripts post-transcriptionally and is involved in meiotic resumption.Ting Gang Chew, Anne Peaston, Ai Khim Lim, Chanchao Lorthongpanich, Barbara B. Knowles, Davor Solte

Functional analysis of the nuage, a unique germline organelle, in Drosophila melanogaster

Ph.DDOCTOR OF PHILOSOPH

Impact of information technology on underwriting of direct insurers in Singapore.

This project identifies the impact of IT on underwriting of General Insurer in Singapore in terms of the job scope of underwriter, efficiency, effectivenes, behaviour and level of security risk. Besides, constraining factors of implementing the information technology in underwriting of Singapore has been discussed in the project

Assessing the usefulness of applied research project for business graduates

136 p.The business environment had been undergoing turbulent changes and development. This underpinned the need for organisations to take on changing roles and the need for managers to cope with increasingly complex duties. From the literature review, criticisms by various academics highlighted the skill deficiencies in management education. As a result, there was a need for business schools to teach relevant skills. A portfolio of nine skills necessary for entry-level jobs were identified based on the taxanomies of skills proposed by various authorsACCOUNTANC

Tudor domains are required for SPIN1 functions.

<p>(A) Structural alignment of three SPIN1 Tudor domains with the SMN Tudor domain, adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069764#pone.0069764-Zhao1" target="_blank">[15]</a>. Amino acid residues important for the structural fold of Tudor domain are highlighted in green. The glutamate residue involved in SMN protein-protein interactions is denoted by an asterisk. (B) Three-dimensional structure of human SPIN1 (PDB ID: 2NS2). The three tyrosine residues (Y98, Y177, Y254) of human SPIN1 are displayed. (C) Co-immunoprecipitation assay of SERBP1 with SPIN1 containing various mutations in the Tudor domains. HEK293T cells were transfected with MYC-tagged SPIN1 wild type or with the MYC-tagged SPIN1 point-mutants. The protein complexes were pulled down with MYC-antibody, and the endogenous SERBP1 was probed using SERBP1-antibody. (D) Luciferase reporter assay to measure the RNA-binding activity of SERBP1. HEK293T cells were transfected with luciferase reporters carrying the Serpine1 3′UTR, and were then co-transfected with the various plasmids. The RNA-binding activity of the endogenous SERBP1 was measured based on the relative expression of luciferase. Data are mean ± SEM, Student’s t-tests.</p

Yeast two-hybrid screening identifies hyaluronan/mRNA-binding protein family members: SERBP1 and HABP4 as binding proteins of SPIN1.

<p>(A) Yeast cells transformed with different plasmids were grown on selective medium under permissive condition (24°C) and restrictive condition (37°C). i and ii are positive controls, iv and vi are negative controls, iii and v are yeast cells with positive clones from the yeast two-hybrid screening, co-transformed with bait plasmid containing <i>Spin1</i>. (B) Summary of the yeast two-hybrid screening. (C) HA-tagged SERBP1 is co-immunoprecipitated with MYC-tagged SPIN1 using MYC-antibody and extract from HEK293T cells expressing HA-SERBP1 and MYC-SPIN1. (D) HA-tagged HABP4 is co-immunoprecipitated with MYC-SPIN1 using MYC-antibody and extract from HEK293T cells expressing HA-HABP4 and MYC-SPIN1.</p

Summary of the genetic crosses of Spin1-GT heterozygous.

<p>Based on analyses of 19 litters from 6 mating pairs, ∼4 pups/litter.</p

<i>Spin1</i> mutant undergoes normal folliculogenesis and oocyte growth, but exhibit defective meiotic resumption.

<p>(A) Haematoxylin and eosin staining of ovarian sections grafted under the kidney capsules. Left panel: wild type; right panel: <i>Spin1</i> mutant. Scale bar represents 500µm. (B) Fully grown oocytes harvested from wild type and <i>Spin1</i> mutant ovaries after stimulation by PMSG. Scale bar represents 50µm. (C) Immunofluorescence staining of fully grown oocytes by anti-SPIN1 (green) and anti-Tubulin (red) antibodies. DNA (blue) was visualized by Hoechst 33342 dye. Scale bar represents 50µm. (D) Quantification of the percentage of fully grown oocytes with GV after release from the phosphodiesterase inhibitor IBMX. Data are mean ± SEM, Student’s t-tests.</p