The coordinated localization of mRNA to centrosomes facilitates error-free mitosis
Posted on: 14 April 2020
Preprint posted on 10 April 2020
Article now published in Journal of Cell Biology at http://dx.doi.org/10.1083/jcb.202004101
Cen marks the spot: Localization of cen mRNA to centrosomes regulates mitotic success in the Drosophila embryo
Selected by Grace Lim
Categories: cell biology, developmental biology
Background
RNA localization is emerging as an important layer of post-transcriptional regulation. Targeting RNA transcripts to specific subcellular components is thought to play an important role in concentrating interacting RNAs performing a common function, promoting efficiency of local protein synthesis, and coordinating translation of multiple mRNAs (Chin and Lécuyer, 2017; Suter, 2018). In development, the asymmetric localization of mRNAs and other non-coding RNAs has been implicated in symmetry breaking that drives lineage segregation in the early embryo (Bashirullah et al., 1998).
The Drosophila embryo is perhaps one of the most widely studied model system for RNA localization, with a prominent example being bicoid, a key fate determinant specifying the anterior pole of the embryo (Berleth et al., 1988). Over the decades, a growing list of RNAs has been identified with distinct localization patterns within the embryo (Lécuyer et al., 2007). In this study, Ryder et al. focused on the role of centrocortin (cen) RNA localization to centrosomes, in the context of cell divisions within the early Drosophila embryo. Although RNAs localizing specifically to centrosomal proteins have been identified in many systems (Lambert and Nagy, 2002; Lécuyer et al., 2007), it remains unclear how and when these RNAs are targeted to the centrosome, and what downstream effects result from this centrosome-specific localization.
Key findings
To better characterize RNA localization to centrosomes within the early Drosophila embryo, the authors utilized a quantitative image analysis strategy to measure distances between GFP-Centrosomin (Cnn) fluorescence signals (as a marker for centrosomes) and single molecule fluorescence in situ hybridization (smFISH) signals for each of the RNAs of interest. This allowed them to carefully dissect the proportion of RNAs in close proximity to centrosomes (with 1 micron as the threshold set for the definition of “pericentrosomal” or “centrosome-enriched”), the proportion of RNAs organized into higher order granular structures, and how these proportions change at different phases of the cell cycle (interphase versus metaphase) or developmental stages (NC 10 versus NC 13). Interestingly, centrosome-localized RNAs displayed common features including a higher enrichment at centrosomes and higher incidence of granule-formation during interphase, as compared to metaphase, suggesting that centrosomal RNA localization is regulated in a cell-cycle dependent manner. These features were especially evident for centrocortin (cen) RNA, where at least 80% of transcripts were detected in the pericentrosomal region during interphase, mostly in the form of micron-scale granules, as illustrated in Figure 1A. This proportion drops to approximately 40% in metaphase (Figure 1B). Strikingly, cen RNA also localized in an asymmetric manner to one centrosome (Figure 1A).
Figure 1: cen RNA localization in interphase and metaphase embryos
(adapted from Fig. 2A, B of Ryder et al).
The authors next investigated how these cen RNA granules formed and localized to centrosomes. First, an intact centrosomal scaffold is required, since disrupting the centrosome scaffold by abolishing the interaction between Cnn and Cen proteins prevented proper centrosomal localization of cen RNA. Second, localization of cen RNA is negatively regulated by the RNA-binding protein fragile X mental retardation protein (FMRP), identified in this study as an interacting partner of both cen RNA and Cen protein. Frm1-null embryos displayed higher levels of cen granule formation in interphase, along with increased cen RNA and Cen protein levels. Given that the early Drosophila embryo is transcriptionally inactive, these changes in RNA and protein levels are likely regulated at the post-transcriptional level, for instance via RNA stability or turnover.
Finally, how do the levels and localization of cen RNA to centrosomes affect mitosis? As shown in Figure 2, prominent spindle defects were observed in both cen-null (loss of cen) and fmr1-null (upregulation of cen) embryos, compared to a wildtype control. These spindle defects appear to be partially rescued in cen hemizygous embryos in a fmr1-null mutant background, suggesting that cen dosage is an important regulator of mitosis. Similarly, when transgenic cen RNA incorporating the bicoid 3’UTR sequence was expressed to target cen RNA to the anterior pole of the embryo, embryos displayed elevated levels of spindle defects, both at the anterior pole (receiving excessive levels of cen) and the posterior pole (with reduced levels of cen). Together, the results support a model whereby dysregulation of wildtype cen RNA levels or mislocalization of cen RNA alters spindle assembly and mitotic success.
Figure 2: Mitotic cells in wildtype embryos (F), cen-null embryos (G), fmr1-null embryos (H), and hemizygous cen embryos in fmr1-null background (I). Embryos labelled for DAPI (blue),
alpha-tubulin (red) and Cnn (green) (adapted from Fig. 5F-I of Ryder et al).
What I like about this preprint
Combining biochemistry, imaging of both RNA and protein localization patterns, and quantitative image analysis strategies, this preprint uses an impressive and diverse suite of methods to comprehensively dissect the role of cen RNA localization to centrosomes in the early Drosophila embryo. These new findings build upon a body of existing research identifying specific RNAs with unique spatial distributions within the embryo, including a number with tight associations with the centrosome, and the emerging roles that RNA localization plays in the regulation of key cellular activities.
Future directions and questions for the authors
- RNA localization was quantitatively tracked in different embryos at interphase and metaphase to analyze cell cycle-dependent changes in RNA distribution relative to centrosomal location. Apart from the changes in RNA localization described in this study, do centrosomes themselves display different organizations during interphase and metaphase? If so, how do these structural changes compare with RNA localization patterns?
- The asymmetric localization of cen RNA preferentially to one centrosome is an interesting phenotype that was not explored in this study. Is this asymmetric localization important in the regulation of spindle assembly / mitotic success? How does this asymmetric localization change in the fmr1-null and hemizygous cen embryos?
References
Bashirullah, A., Cooperstock, R.L., and Lipshitz, H.D. (1998). RNA LOCALIZATION IN DEVELOPMENT. Annual Review of Biochemistry 67, 335–394.
Berleth, T., Burri, M., Thoma, G., Bopp, D., Richstein, S., Frigerio, G., Noll, M., and Nüsslein-Volhard, C. (1988). The role of localization of bicoid RNA in organizing the anterior pattern of the Drosophila embryo. The EMBO Journal 7, 1749–1756.
Chin, A., and Lécuyer, E. (2017). RNA localization: Making its way to the center stage. Biochimica et Biophysica Acta (BBA) – General Subjects 1861, 2956–2970.
Lambert, J.D., and Nagy, L.M. (2002). Asymmetric inheritance of centrosomally localized mRNAs during embryonic cleavages. Nature 420, 682–686.
Lécuyer, E., Yoshida, H., Parthasarathy, N., Alm, C., Babak, T., Cerovina, T., Hughes, T.R., Tomancak, P., and Krause, H.M. (2007). Global Analysis of mRNA Localization Reveals a Prominent Role in Organizing Cellular Architecture and Function. Cell 131, 174–187.
Suter, B. (2018). RNA localization and transport. Biochimica et Biophysica Acta (BBA) – Gene Regulatory Mechanisms 1861, 938–951.
doi: https://doi.org/10.1242/prelights.18504
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