Signed, sealed, and delivered: RNA localization and translation at centrosomes

Abstract

Protein localization is intrinsic to cellular function and specialized activities, such as migration or proliferation. Many localized proteins enrich at defined organelles, forming subdomains of functional activity further specified by interacting protein assemblies. One well-studied organelle showing dynamic, functional changes in protein composition is the centrosome. Centrosomes are microtubule-organizing centers with diverse cellular functions largely defined by the composition of the pericentriolar material, an ordered matrix of proteins organized around a central pair of centrioles. Also localizing to the pericentriolar material are mRNAs. Although RNA was identified at centrosomes decades ago, the characterization of specific RNA transcripts and their functional contributions to centrosome biology remained largely unstudied. While the identification of RNA localized to centrosomes accelerated with the development of high-throughput screening methods, this discovery still outpaces functional characterization. Recent work indicates RNA localized to centrosomes is biologically significant and further implicates centrosomes as sites for local protein synthesis. Distinct RNA localization and translational activities likely contribute to the diversity of centrosome functions within cells.

FIGURE 1:

The centrosome as a microtubule-organizing center. (A) Cartoon depicts microtubules composed of α- (pale green) and β-tubulin (dark green) heterodimers nucleated from the PCM (gray sphere). Microtubules are polarized structures with minus ends (−) embedded in the PCM and plus ends (+) extending into the cytosol. (B) Structured-illumination microscopy image of a mitotic Drosophila embryo centrosome labeled with pericentrin-like protein (PLP; yellow; Martinez-Campos et al., 2004), γTubulin (γTub; blue; Joshi et al., 1992), and centrosomin (Cnn; magenta; Megraw et al., 1999; Vaizel-Ohayon and Schejter, 1999) antibodies. Image courtesy of Nasser Rusan. Dashed arrow shows the direction of the line scan used in (C) to measure the intensity distributions of centrosome proteins. The peak intensity of each protein was normalized to 100 and the distribution is plotted relative to the center of the centriole (0 nm). (D) Artistic rendition of the subconcentric organization of the centrosome, where each color represents the distribution of a centrosome protein. (1) The inner zone (yellow) represents centriolar proteins (e.g., PLP), (2) the midzone (blue) represents PCM proteins residing closer to the centriole, such as γTub, while (3) the outer zone PCM (red) defines proteins at the outer margin of the centrosome, such as Cnn. (E) Organization of mitotic spindles within a Drosophila embryo. Magenta centrosomes are marked by Asterless (Asl; Varmark et al., 2007); microtubules (green), actin (red), and DNA (blue) are displayed. Image courtesy of Elías Castro. Bars: (B) 500 nm and (E) 5 μm.

FIGURE 2:

The intimate association of the translational machinery with microtubules and centrosomes. (A) Repro­duction of “Biosites: Cytoplasm, 2005” illustrated by David S. Goodsell; available online (doi: 10.2210/rcsb_pdb/goodsell-gallery-006). Illustration shows a microtubule filament (light blue, left) juxtaposed to ribosomes (dark blue) synthesizing proteins (pink). (B) Electron micrograph of a rat lymphocyte showing abundant polyribosomes (PR) clustered near the duplicated centrioles from Murray et al. (1965) originally published in Journal of Cell Biology and reprinted with permission from Rockefeller University Press.

FIGURE 3:

Mistargeting Cen mRNA in syncytial Drosophila embryos impairs centrosome function. Schematic shows Cen mRNA and protein localization and associated centrosome-related phenotypes in (A) control and (B) Cen-bcd-3′UTR embryos. In control embryos, Cen mRNA and protein colocalize as pericentrosomal granules asymmetrically enriched at the mother centrosome. Expression of Cen-bcd-3′UTR within otherwise Cen null embryos, however, results in the ectopic localization of Cen mRNA and protein to the anterior cortex. Mislocalized Cen mRNA and protein also form massive centrosome-enriched granules. In contrast, Cen-bcd-3′UTR embryos lack Cen mRNA or protein at more distal centrosomes near the embryo midregion or posterior. Mistargeting Cen mRNA to the anterior cortex significantly disrupts centrosome function, resulting in defects in centrosome position and centrosome–nucleus tethering. More severe phenotypes consistent with mitotic errors are apparent near the anterior, including disorganized microtubules, supernumerary centrosomes, enlarged and dysmorphic nuclei, as well as nuclear fallout, the ejection of damaged nuclei from the syncytial blastoderm cortex. Consequently, Cen-bcd-3′UTR embryos also show elevated rates of embryonic lethality. Taken together, these observations indicate the local concentration of Cen mRNA is important for centrosome function and mitotic integrity.