In vivo mapping of a dynamic ribonucleoprotein granule interactome in early Drosophila embryos

Abstract

Macromolecular complexes and organelles play crucial roles within cells, but their native architectures are often unknown. Here, we use an evolutionarily conserved germline organelle, the germ granule, as a paradigm. In Drosophila embryos, we map one of its interactomes using a novel in vivo crosslinking approach that employs two interacting granule proteins and determines their common neighbor molecules. We identified an in vivo granule assembly of Tudor, Aubergine, motor and metabolic proteins, and RNA helicases, and provide evidence for direct interactions within this assembly using purified components. Our study indicates that germ granules contain efficient biochemical reactors involved in post‐transcriptional gene regulation.

An in vivo proteomic analysis of the Me31B interactome in Drosophila germ granules

“…Dynein and kinesin motors are required for the motility, anchoring, and distribution of germ plasm components during oogenesis , and dynein‐dependent transport ensures their segregation into forming germ cells . Furthermore, Dhc was shown to colocalize with Vas in the germ plasm, and, together with Khc, was found to complex with core germ plasm proteins Aub and Tud , consistent with our model that Me31B interacts with Aub and Tud (see below and Fig. ).…”

“…In accordance with our data, PyK and PgK were found in polar granules by immuno-EM [22]. Furthermore, PyK and Eno are both found in Tud and Aub complexes [23], and PyK directly binds to Tud and contains sDMA [22]. Considering that Me31B may bind to Tud in a similar way (see below), we propose a model where the glycolytic enzymes are linked to Me31B via scaffold protein Tud (Fig.…”

Phase transitioned nuclear Oskar promotes cell division of Drosophila primordial germ cells

“…Flies were maintained on cornmeal molasses/yeast medium at 25°C using standard procedures. The following fly lines were used: Wild-type:w −1118 (Bloomington (BDSC); Stock #3605), y,w; P[E GFP-vas w + ] cyIII ( Trcek et al, 2015 ), UAS–vasa–KO ( Cinalli and Lehmann, 2013 ; Trcek et al, 2015 ), pFlyFos-Osk ( Jambor et al, 2015 ; Trcek et al, 2015 ), UASp-GFP-Aub ( Webster et al, 2015 ), GFP-Piwi ( Le Thomas et al, 2013 ), GFP-Tud ( Zheng et al, 2016 ), ;;P{GAL4::VP16-nos.UTR}/TM3Ser ( Pae et al, 2017 ), w[*]; P { w[+mC]=His2Av-mRFP1 } II.2 (Bloomington (BDSC); Stock #23651), w; matα-gal4;PrDr/TM3 ( Pae et al, 2017 ), w; P(EPgy2)gcl EY09611 /CyO; nosGal4VP16 (w+)/TM3 ( Cinalli and Lehmann, 2013 ), Df(3R)Pxt103/TM3 Ser Sb and osk54 st ry ss/TM3Ser ( Lehmann and Nüsslein-Volhard, 1991 ).…”

piRNAs and PIWI proteins: regulators of gene expression in development and stem cells

“…1C). Strikingly, Aub is involved in germ cell mRNA decay in the soma but is also a core component of polar granules in the germ plasm (Little et al, 2015;Thomson et al, 2008;Trcek et al, 2015;Zheng et al, 2016). This raises the intriguing issue of whether Aub might be involved in two opposite functions depending on its localization: mRNA decay in the somatic part of the embryo and mRNA localization in the germ plasm (Barckmann et al, 2015).…”