Localization of mRNAs to Centrosomes Requires the Pericentriolar Material Scaffold Pubblico

Abstract

The pericentriolar material (PCM) is a proteinaceous matrix surrounding the centrioles that promotes centrosome microtubule-nucleating activity. We and others previously showed the pericentriolar components Centrosomin (Cnn) and Pericentrin-like protein (PLP) functionally cooperate to organize a PCM scaffold required for centrosome structure and microtubule organization. We recently showed that several centrosomal mRNAs are localized to the centrosome and contribute to centrosome function under the regulation of RNA-binding proteins. However, whether the PCM is required to recruit or anchor mRNA to the centrosome remains incompletely understood. We hypothesize that PCM functions as a scaffold to anchor centrosomal mRNAs at centrosomes. To test our hypothesis, we used a classic cnn allele to disrupt PCM organization and investigated consequences to the localization of centrocortin (cen) and plp mRNAs. We used super-resolution imaging and quantitative single-molecule fluorescence in situ hybridization (smFISH) to visualize and quantify mRNA enrichments at centrosomes in syncytial Drosophila embryos. We also adapted our customizable Subcellular Distribution computational pipeline and compared mRNA distributions as quantified from either the centroid or surface of centrioles to permit quantification using a variety of centrosome markers. Upon disrupting PCM organization, our data indicate that the enrichment of cen mRNAs at centrosomes and the distribution of cen mRNA within granules are significantly decreased. Similarly, we observed a significant decrease in plp mRNA localization to centrosomes in cnn mutants. These data demonstrate that the PCM scaffold is needed for cen and plp mRNA localization to centrosomes and for packaging cen mRNA into higher-order RNA granules. Our work helps elucidate mechanisms of mRNA localization to centrosomes.

Table of Contents

1 Localization of mRNAs to Centrosomes Requires the PCM Scaffold 1

1.1 Introduction………………………………………………………………………………..................................................1

1.2 Results……………………………………………………………………………………....................................................5

1.2.1 cen mRNA Localization to Centrosomes and Granule Formation Decreases upon PCM Disruption…….…....5

1.2.2 Quantification of the cen mRNA Enrichment at Centrosomes in WT vs. cnnB4 Mutants……………….….......8

1.2.3 Localization of plp mRNA to Centrosomes Decreases upon PCM Disruption…..........................................11

1.2.4 Decrease in RNA Localization is Not Caused by Whole-cell RNA Level Reduction……………….…………......13

1.3 Discussion……………………………….…………………………………………….....................................................15

1.4 Materials and Experimental Procedures……………….……………………………...............................................17

2 Exploring the Regulation of plp mRNA Localization to Centrosomes by FMRP 21

3 Consequences of plp mRNA Mislocalization at Centrosomes 23

4 Examination of RNA Localization Relative to the Centrosome Centroid 26

References 29

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Cell
Parola chiave	Drosophila centrosome RNA localization
Committee Chair / Thesis Advisor	Lerit, Dorothy, Emory University
Committee Members	Rieder, Leila, Emory University McDonald, Frank, Emory University

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