A conserved choreography of mRNAs at centrosomes reveals a localization mechanism involving active polysome transport

Abstract

Local translation allows for a spatial control of gene expression. Here, we used high-throughput smFISH to screen centrosomal protein-coding genes, and we describe 8 human mRNAs accumulating at centrosomes. These mRNAs localize at different stages during cell cycle with a remarkable choreography, indicating a finely regulated translational program at centrosomes. Interestingly, drug treatments and reporter analyses revealed a common translation-dependent localization mechanism requiring the nascent protein. Using ASPM and NUMA1 as models, single mRNA and polysome imaging revealed active movements of endogenous polysomes towards the centrosome at the onset of mitosis, when these mRNAs start localizing. ASPM polysomes associate with microtubules and localize by either motor-driven transport or microtubule pulling. Remarkably, the Drosophila orthologs of the human centrosomal mRNAs also localize to centrosomes and also require translation. These data identify a conserved family of centrosomal mRNAs that localize by active polysomes transport mediated by nascent proteins.

A choreography of centrosomal mRNAs reveals a conserved localization mechanism involving active polysome transport

“…Spot detection was performed with Big-FISH 62 (https://github.com/fish-quant/ big-fish), a python implementation of FISH-Quant 63 . It starts with a Laplacian of Gaussian filter (LoG) to enhance the spot signal.…”

Exon Junction Complex dependent mRNA localization is linked to centrosome organization during ciliogenesis

“…We used a high-throughput smFISH strategy (schematized in Fig. 5a; see also Safieddine et al 67 ) to screen about 700 mRNAs encoding centrosome- and cilium-related proteins (Supplementary Table 1). Briefly, we generated 50 to 100 distinct single-stranded RNA probes for each mRNA.…”

“…More recently, four transcripts encoding the central PCM component, Pericentrin (PCNT), Abnormal spindle-like microcephaly-associated protein (ASPM), the nuclear mitotic apparatus protein 1 (NUMA1) and the Hyaluronan Mediated Motility Receptor (HMMR) were detected by single molecule approaches around centrosome of HeLa cells during cell division 67,74,75 . Here, we identified BICD2 and NIN transcripts as two additional mRNAs concentrated around centrosome at the base of cilia in quiescent RPE1 cells.…”

“…In this case, the cytosolic translation of transcripts is arrested after translation of a signal sequence that mediates the transport of the ribosome-bound mRNP to the endoplasmic reticulum where translation resumes after translocation of the nascent polypeptide 85 . The delivery of PCNT and ASPM mRNAs to centrosomes requires active polysomes as well as microtubules and dynein activity 74,74 , and direct visualization of single polysomes in live cells with the SunTag showed that the ASPM and NUMA1 polysomes are actively transported to mitotic centrosomes 67 . Recently, a large dual protein-mRNA screen in human cells revealed that the majority of the transcripts displaying specific cytoplasmic locations reach their destination in a translationdependent manner 75 .…”

Improved methods for protein and single-molecule RNA detection inC. elegansembryos

“…As smFISH has become more widely utilized, novel methods of analysis beyond spot counting are rapidly developing. For instance, FISH-quant has been ported from Matlab to an open-source implementation in Python and successfully applied to two large-scale screen projects 29,30 . This package includes methods for detecting, deconvolving overlapping RNAs to increase the counting accuracy of highly abundant or clustered RNAs 5,30 , measuring the signal-to-noise ratio of an image (https://github.com/fish-quant), and even identifying diverse subcellular localization patterns of RNA 30,31 .…”