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

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.


Introduction
Messenger RNA localization is a post-transcriptional process by which cells target certain mRNAs to specific subcellular compartments. The trafficking of mRNA molecules is linked to its metabolism and function (Kejiou and Palazzo, 2017). Indeed, the subcellular localization of a transcript can influence its maturation, translation, and degradation. On one hand, mRNAs can be stored in a translationally repressed state in dedicated structures such as P-bodies (Hubstenberger et al., 2017). On the other hand, some mRNAs can localize to be translated locally. Such a local protein synthesis can be used to localize the mature polypeptide, and in this case it can contribute to a wide range of functions such as cell migration, cell polarity, synaptic plasticity, asymmetric cell divisions, embryonic patterning and others (Cody et al., 2013;Chin and Lécuyer, 2017;Ryder and Lerit, 2018 for reviews). Recently, local translation has also been linked to the metabolism of the nascent protein, rather than to localize the mature polypeptide. This is for instance the case for mRNAs translated in distinct foci termed translation factories, which correspond to small cytoplasmic aggregates containing multiple mRNA molecules of a given gene (Pichon et al., 2016;Chouaib et al., 2019).
Specific sub-cellular localization of mRNA molecules can be achieved by several mechanisms. Passive diffusion coupled with local entrapment and/or selective local protection from degradation are two strategies that can establish specific distributions of mRNA molecules (reviewed in Chin and Lécuyer, 2017). In most cases however, mRNA transport and localization occurs via motor-driven transport on the cytoskeleton (Bertrand et al., 1998;Bullock, 2007;Buxbaum et al., 2015). Molecular elements that regulate and control mRNA localization include cis-and trans-acting elements. Cis-acting elements are referred to as zip-codes and are often found within the 3'UTR of the transcript (Trcek and Singer, 2010;Kannaiah and Amster-Choder, 2014;Kim et al., 2015 Safieddine et al. 4 for reviews). Many types of zip-codes have been described based on primary sequence, number, redundancy, and secondary/tertiary structure. Zip-codes are defined by their ability to carry sufficient information for localizing the transcript. They bind one or several trans-acting RNA-binding proteins (RBPs), which mediate diverse aspects of RNA metabolism such as motor binding and translational regulation (reviewed in Chin and Lécuyer, 2017). Indeed, mRNAs in transit are often subjected to a spatial control of translation (reviewed in Besse and Ephrussi, 2008). A long-standing notion in the field is that the transport of localized mRNAs occurs in a translationally repressed state, which serves to spatially restrict protein synthesis (Vazquez-Pianzola and Suter, 2012;Vazquez-Pianzola et al., 2016). Local translational de-repression occurs once the transcript has reached its destination, for instance by phosphorylation events and/or competition with pre-existing local proteins (Hüttelmaier et al., 2005;Paquin et al., 2007;Götze et al., 2017) . While active transport of transcripts through RNA zip-codes appears to be a frequent mechanism, mRNA localization can also involve the nascent polypeptide, as in the case of secreted proteins. Here, the signal recognition particle (SRP) binds the nascent signal peptide, inhibits translation elongation, and mediates anchoring of the nascent polysome to the SRP receptor on the endoplasmic reticulum, where translation elongation resumes (Gilmore et al., 1982;Blobel, 1980, 1982). Recently, a few hints, such as puromycin sensitivity, suggested that translation may play a role in the localization of some other types of mRNAs (Sepulveda et al., 2018;Chouaib et al, 2019). Whether this is indeed the case and the mechanisms involved remain however unknown.
Centrosomes are ancient and evolutionary conserved organelles that function as microtubule (MT) organizing centers in most animal cells. They play key roles in cell division, signaling, polarity and motility (Wu and Akhmanova, 2017;Breslow and Holland, 2019; for reviews). A centrosome is composed of two centrioles and their surrounding pericentriolar material (PCM). In cycling cells, centriole duplication is tightly coupled to the cell cycle to ensure a constant number of centrioles in each cell after mitosis (reviewed in Nigg and Holland, 2018). Briefly, G1 cells contain one centrosome with two centrioles connected by a linker. At the beginning of S phase, each parental centriole orthogonally assembles one new procentriole. This configuration is termed engagement and prevents reduplication of the parental centrioles. Procentrioles elongate as the cell is progressing through S and G2. In G2, the two centriolar pairs mature and PCM expands, in preparation of mitotic spindle formation (Breslow and Holland, 2019). The G2/M transition marks the disruption of the centriole linker and centrosome separation. The first clues suggesting the importance of mRNA localization and local translation at the centrosomes were discovered almost 20 years ago in Xenopus early embryos (Groisman et al., 2000). It was found that cyclin B mRNAs concentrated on the mitotic spindle, and that this localization was dependent on the ability of CPEB to associate with microtubules and centrosomes. A more global view was obtained in Drosophila embryos where a systematic analysis of RNA localization was performed (Lécuyer et al., 2007). Although this study did not reach single molecule sensitivity, it revealed that 6 mRNAs localized at centrosomes across different stages of early Drosophila development. In a following study, 13 mRNAs were annotated as enriched on Drosophila centrosomes in at least one stage/tissue over the full course of embryogenesis (Wilk et al., 2016). In humans, four mRNAs were recently found to localize at centrosomes (PCNT, ASPM, NUMA1 and HMMR;Sepulveda et al., 2018;Chouaib et al., 2019). All these mRNAs all code for centrosomal proteins, suggesting that they are translated locally.
Here, we performed a systematic single molecule Fluorescent in situ hybridization (smFISH) screen of almost all human mRNAs coding for centrosomal proteins and we Safieddine et al.
6 described a total of 8 transcripts localizing at centrosomes. Remarkably, all 8 mRNAs required synthesis of the nascent protein to localize and, by imaging single ASPM and NUMA1 mRNAs and polysomes, we demonstrate that localization occurs by active transport of polysomes. Moreover, the Drosophila orthologs of the human centrosomal mRNAs also localized to centrosomes in a similar translation-dependent manner. This work thus identifies a conserved family of centrosomal mRNAs that become localized by active polysome transport. 7

Results
Screening genes encoding centrosomal proteins reveals a total of 8 human mRNAs localizing at the centrosome In order to acquire a global view of centrosomal mRNA localization in human cells, we developed a high-throughput smFISH technique (HT-smFISH) and screened genes encoding centrosomal and mitotic spindle proteins. The experimental pipeline is described in Figure 1A. Briefly, we designed 50 to 100 individual probes against each mRNA of the screen. The probes were then generated from complex pools of oligonucleotides (92,000), first by using gene-specific primers to PCR out the probes of single genes, followed by a second round of PCR to add a T7 promoter and in vitro transcription to generate single-stranded RNA probes ( Figure 1A; see Materials and Methods). The probes were designed such that each contained a gene-specific sequence flanked by two overhangs common to all probes (flaps X and Y). A pre-hybridization step then labeled the overhangs with fluorescently labeled locked nucleic acid (LNA) oligonucleotides, and the heteroduplexes were hybridized on cells as in the smiFISH technique (Tsanov et al., 2016), except that cells were grown and hybridized on 96-well plates. This approach is cost-effective because the probes are generated from an oligonucleotide pool. In addition, the probes can be used individually or combined in different colors, allowing a flexible experimental design.
We screened a total of 602 genes using HeLa cells stably expressing a Centrin1-GFP fusion to label centrosomes. High-throughput spinning-disk microscopy was used to acquire full 3D images at high resolution (200 nm lateral and 600 nm axial), and two sets of images were recorded, to image either interphase or mitotic cells (see Materials and Methods). Centrosomal RNA enrichment was assessed by manual annotations of the images. These analyses yielded several localized mRNAs, including six that concentrated Safieddine et al. 8 near centrosomes (Table 1 and Table S1). The localization of these mRNAs was then confirmed by performing low-throughput smiFISH. The results confirmed that the six candidate mRNAs accumulated at centrosomes during interphase and/or mitosis. These transcripts included PCNT and NUMA1 mRNA that were also recently identified by us and others (Sepulveda et al., 2018;Chouaib et al., 2019), as well as several new ones: NIN, BICD2, CCDC88C and CEP350 ( Figure 1B, Table 1). Taking into account ASPM and HMMR that we also recently identified (Chouaib et al., 2019), a total of 8 mRNAs thus localize at centrosomes in human cells. These transcripts encode proteins that regulate various aspects of centrosome maturation, spindle positioning, and MT dynamics. Interestingly, the localization of these mRNAs varied during the cell cycle. CCDC88C mRNA localized during interphase but not mitosis. PCNT, NIN, BICD2, HMMR and CEP350 mRNAs localized during interphase and early mitosis, but delocalized at later mitotic stages ( Figure 1C, and see below). In contrast, NUMA1 and ASPM mRNAs only localized during mitosis (Chouaib et al., 2019;see below). Since all these mRNA code for centrosomal proteins, centrosomes thus appear to have a dedicated translational program that is regulated during the cell cycle.

ASPM, NUMA1 and HMMR proteins localize to centrosome at specific cell cycle stage
We then focused on ASPM, NUMA1 and HMMR, and analyzed in more detail their expression and localization. We first analyzed the expression of their respective proteins during the cell cycle. For this, we took advantage of HeLa Kyoto cell lines that stably express bacterial artificial chromosomes (BAC) containing the entire genomic sequences of the genes of interest, and carrying a C-terminal GFP tag (Poser et al., 2008;Chouaib et al., 2019). These BACs contain all the gene regulatory sequences and are expressed at near-endogenous levels and with the proper spatio-temporal pattern (Poser et al., 2008).

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Time-lapse microscopy of single cells revealed that ASPM-GFP and HMMR-GFP expression rose progressively during interphase to culminate just before mitosis, while that of NUMA1-GFP appeared constant during the cell cycle. Interestingly, ASPM-GFP and NUMA1-GFP had similar localization patterns. Both proteins were mainly nucleoplasmic during interphase and precisely initiated centrosomal localization in prophase. During cell division, they accumulated at the spindle pole with a weak labeling of the proximal spindle fibers ( Figure S1A-B). In contrast, HMMR-GFP accumulated on the entire spindle throughout mitosis and furthermore concentrated on cytokinetic bridges in telophase.
During interphase, it labeled microtubules (MTs) and localized to centrosomes several hours before cell division ( Figure S1C).

ASPM, NUMA1, and HMMR mRNAs localize to centrosome at the same time as their proteins
We next determined the localization of ASPM, NUMA1 and HMMR mRNAs during the different mitotic phases. We again used the GFP-tagged BAC HeLa cell lines to correlate protein and mRNA localization. smFISH was performed against the GFP RNA sequence using a set of 44 Cy3 labeled oligonucleotide probes. In interphase, ASPM-GFP and NUMA1-GFP mRNAs and proteins did not localize to centrosomes as previously reported (Chouaib et al., 2019), while HMMR-GFP mRNAs and its protein co-localized to centrosomes in a fraction of the cells. During mitosis, ASPM-GFP mRNAs and protein were enriched together on mitotic centrosomes across all phases of cell division (Figure 2A and 2B). In contrast, NUMA1 and HMMR mRNAs only accumulated at centrosomes during the early stages of cell division, prophase and prometaphase, where they co-localized with their protein. A random mRNA distribution was seen during metaphase and anaphase, although both proteins still remained on the mitotic spindle ( Figure 2C and 2D). Unlike NUMA1, the centrosomal localization of HMMR mRNA and protein was re-established in telophase, where they accumulated together at the cytokinetic bridges ( Figure 2E and 2F).
To detail these findings, we performed two-color smFISH experiments detecting one BAC-GFP mRNA in Cy3 and an endogenous mRNA in Cy5. We analyzed all pairwise combinations of ASPM, NUMA1 and HMMR mRNAs. To gain more precision, we divided each of prophase, prometaphase and telophase into two sub-phases, early and late (see Materials and Methods). During early prophase, NUMA1 and HMMR mRNAs could be seen on centrosomes but not ASPM mRNAs that only joined during late prophase ( Figure S2).
During early prometaphase, all three mRNAs were enriched on centrosomes. However, the centrosomal localization of NUMA1 and HMMR mRNAs became much less frequent starting at late prometaphase, while that of ASPM mRNA could still be observed in metaphase and anaphase ( Figure S2 and S3). Finally, ASPM but not HMMR mRNAs accumulated on centrosomes during early telophase, while the opposite was observed at late telophase. Interestingly, the three mRNAs never perfectly co-localized on centrosomes at any of the cell-cycle stages: certain peri-centrosomal regions were occupied by one transcript while others contained the other mRNA (see Figures S2 and S3).
We recently showed that two translation factors, eIF4E and a phosphorylated form of RPS6, accumulated at centrosomes in prophase (Chouaib et al., 2019). Here, we examined their localization during the other mitotic phases. The centrosomal localization of these factors was strongest in prophase (unpublished data), and thus mirrored the bulk of centrosomal mRNAs, which, except for CCDC88C, localize to centrosome most strongly in prophase. The detection of translation factors together with mRNAs at centrosomes likely reflects where translation occurs. Taken together, these data demonstrate a fine tuning of spatio-temporal dynamics for the centrosomal localization of ASPM, NUMA1, and HMMR mRNAs, with each mRNA localizing at a specific stage and place during cell division.
The localization of the 8 centrosomal mRNAs is inhibited by puromycin but not cycloheximide Next, we analyzed the localization mechanism of these mRNAs and first questioned whether localization requires translation. To this end, we used a HeLa cell line expressing Centrin1-GFP to label centrosomes and treated it for 20 minutes with either cycloheximide, which blocks ribosome elongation, or puromycin, which induces premature chain termination. We first analyzed the mRNAs localizing during interphase (NIN, BICD2, CCDC88C, CEP350, HMMR and PCNT). Remarkably, these six mRNAs became delocalized after puromycin treatment while cycloheximide had no effect ( Figure 3A and 3B). Long puromycin treatments prevent entry into mitosis. However, a 5-minute incubation was sufficient to inhibit the centrosomal localization of ASPM, NUMA1 and HMMR mRNAs at all the mitotic phases in which they normally localize ( Figures S4, S5, and S6), while cycloheximide still had no effect. Since cycloheximide inhibits translation but leaves the nascent peptide chain on ribosomes, while puromycin removes it, our data suggest that mRNA localization to centrosomes requires the nascent peptide. RNA localization is thus expected to occur co-translationally for all the 8 mRNAs, pointing toward a common localization mechanism.

Translation of ASPM coding sequence is necessary and sufficient for localizing its mRNA at centrosomes
To investigate how mRNAs localize to centrosomes in more detail, we focused on ASPM and first asked whether the 5' and 3' UTRs were necessary for its localization. To this end, a full-length ASPM mouse coding sequence (CDS) was fused to the C-terminal of GFP and expressed via transient transfection in HeLa Kyoto cells. To detect mRNAs produced from this reporter only, we performed smFISH with probes directed against the GFP RNA sequence. Mitotic cells expressing the plasmid could be identified by the accumulation of GFP-ASPM, which localized on centrosomes and the mitotic spindle. Interestingly, we could detect ASPM-GFP mRNAs on mitotic centrosomes in most of the transfected cells ( Figure 4A). This demonstrated that the 5' and 3' UTRs of ASPM mRNA are not required for its centrosomal enrichment.
Next, we explored how the same GFP-ASPM mRNA would localize if the nascent ASPM protein was not translated. To test this, we introduced a stop codon between the GFP and ASPM coding sequences, generating a GFP-stop-ASPM construct. Transient transfection showed a diffuse GFP signal as expected. Interestingly, mRNAs translating this reporter failed to localize to mitotic centrosomes labeled by an immunofluorescence (IF) against gamma tubulin ( Figure 4B and 4C). Taken together, this demonstrated that the nascent ASPM polypeptide is required for trafficking its own transcript to mitotic centrosomes.

ASPM mRNAs are actively transported toward centrosomes and anchored on the mitotic spindle
To gain more insights into the localization mechanism, we imaged the endogenous ASPM mRNAs in living mitotic cells. To this end, we inserted 24 MS2 repeats in the 3' UTR of the endogenous gene, using CRISPR/Cas9 mediated homology-directed repair in HeLa Kyoto cells ( Figure 5A). Heterozygous clones were confirmed by genotyping ( Figure S7A).
Moreover, two-color smFISH performed with MS2 and ASPM probes showed that the tagged mRNA accumulated at centrosomes in mitosis ( Figure 5B), indicating that the MS2 13 sequences did not interfere with localization. We then stably expressed low levels of the MS2-coat protein (MCP) fused to GFP and a nuclear localization signal (MCP-GFP-NLS).
This fusion protein binds the MS2 repeat and allows to visualize the tagged RNA in living cells (Bertrand et al., 1998). Indeed, mitotic cells expressing MCP-GFP-NLS displayed diffraction limited fluorescent spots that localized near the centrosomes. Moreover, these spots co-localized with single RNA molecules revealed with probes against either endogenous ASPM mRNA or the MS2 tag, indicating that binding of MCP-GFP-NLS to the tagged mRNA did not abolish RNA localization to the centrosome ( Figure S7B).
Next, we performed live cell experiments. We labeled DNA using SiR-DNA to identify the mitotic phase and we imaged cells in 3D at a rate of 2-4 fps using spinning disk microscopy. During prometaphase, three populations of ASPM mRNA molecules were observed: (i) mRNAs diffusing in the cytosolic space, (ii) immobile molecules that corresponded to mRNAs localizing at the centrosome, and (iii) mRNAs undergoing directed movements towards the centrosome ( Figure 5C). The thickness of mitotic cells yielded low signal-to-noise ratios which, combined with the rapid movements of the mRNAs, made single particle tracking with automated software difficult. We thus manually tracked mRNA molecules undergoing directed movements and observed that they moved at speeds ranging from 0.5 to 1 µm/sec ( Figure 5E), which is compatible with motor-mediated transport (Schiavo et al., 2013).
In metaphase and anaphase where the mRNA is at centrosomes, we expected several possibilities for the movement of mRNA molecules: (i) stable anchoring to the centrosome; (ii) diffusion within a confined space around the centrosome; or (iii) diffusion away from centrosomes and re-localization by a motor-dependent mechanism.
Live imaging revealed that ASPM mRNA localizing at mitotic centrosome did not diffuse and were immobile ( Figure 5D, Movie 1). In addition, directed movements toward Safieddine et al.
14 centrosomes were also observed in mid-mitosis albeit to a lesser extent than in prometaphase ( Figure 5D arrows, Movie 1). Interestingly, we also observed that some ASPM mRNAs were attached on the spindle fibers rather than on the spindle poles (Movie 2). Taken together, these live imaging experiments demonstrated that ASPM mRNAs are actively transported to the mitotic centrosomes at the onset of mitosis and are then anchored on the spindle poles and fibers.

ASPM polysomes are actively transported towards centrosomes during prophase and prometaphase
Since the localization of ASPM transcripts required in-cis translation and might thus involve the transport of translated mRNAs, we next imaged ASPM polysomes. To this end, we used the SunTag system that allows to image nascent polypeptide chains (Morisaki et al., 2016;Pichon et al., 2016;Wang et al., 2016;Wu et al., 2016;Yan et al., 2016). The SunTag is composed of a repeated epitope inserted in the protein of interest and a singlechain antibody fused to GFP. Binding of the fluorescent antibody to the epitope occurs when it emerges from the ribosome, and this allows visualizing nascent protein chains and polysomes in live cells. We engineered a HeLa Kyoto cell line with 32 SunTag repeats fused to the 5' end of the ASPM gene, using CRISPR/Cas9 mediated homology-directed repair ( Figure 6A). Heterozygous clones were confirmed by genotyping ( Figure S8A). The cells were then transduced with a lentivirus expressing the scFv monochain antibody fused to GFP (scFv-sfGFP). Bright GFP foci were observed and confirmed to be polysomes based on both their sensitivity to puromycin ( Figure S8B) and co-localization with endogenous ASPM mRNA by smiFISH ( Figure 6B). Moreover, ASPM mRNAs having the SunTag accumulated on mitotic centrosomes ( Figure S8C), indicating that the tagging process did not abolish centrosomal RNA targeting.
We first imaged SunTag-ASPM polysomes and mRNA together in fixed cells ( Figure   6B). In early prophase, most mRNAs and polysomes did not localize to centrosomes, in agreement with the smFISH data ( Figure S2). In prometaphase, metaphase and anaphase, the accumulation of the SunTag-ASPM mature protein at the spindle poles prevented visualizing ASPM polysomes at this location. However, some ASPM polysomes were observed outside the spindle area indicating that translation was pursued during the entirety of mitosis ( Figure 6B). At the end of telophase, ASPM mRNAs could be seen translated on the nuclear envelope as previously reported for cells in interphase (Chouaib et al., 2019).
We then performed live imaging in 3D at acquisition rates of 1-1.3 stacks per second, using spinning disk microscopy. We first imaged cells in prophase (Movie 3).
Remarkably, while many ASPM polysomes were dispersed in the cytoplasm at the beginning of prophase, they displayed rapid directed motions towards the centrosome, leading to their accumulation at this location at the end of the movie ( Figure 6C, Movie 3).
Single particle tracking showed that an average of 51% of ASPM polysomes displayed such directed movements in early prophase ( Figure 6D, E). Directed movements were also detected in prometaphase but less frequently (Movie 4). Calculating the average velocity of polysomes undergoing directed movements showed that their speed ranged from around 0.25 to 2 µm/s, which is compatible with the velocities of both ASPM mRNAs and motor-dependent transport ( Figure 6F). Taken together, this data directly proved that ASPM polysomes were actively transported to the centrosome at the onset of mitosis.

ASPM mRNAs are translated on MTs during interphase
Similar to MS2-tagged ASPM mRNAs, some SunTag-ASPM polysomes did not co-localize with the spindle poles but rather with spindle fibers that were weakly labeled by the mature SunTag-ASPM protein ( Figure 6B, arrows). This prompted us to investigate in Safieddine et al.

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more details the role of microtubules in the metabolism of ASPM mRNAs. We labeled MTs in living cells using a far-red dye (SiR-Tubulin) and performed sequential two-color 3D live imaging using a spinning disk microscope. We first imaged interphase cells and remarkably, we observed that many ASPM polysomes remained stably anchored to MTs during the course of the movies (66%; Figure 7A-C, Figure S9A, and Movie 5). In addition, we also observed directed motion of single polysomes, albeit at a low frequency (around 4%). We then characterized in more details the movements of ASPM polysomes and for this we classified them in four categories: (i) polysomes localizing on MTs; (ii) localizing at the nuclear envelope (as previously reported; Chouaib et al., 2019); (iii) niether localizing on MTs nor at the nuclear envelope, and thus free in the cytosol; and (iv) showing directed transport ( Figure 7C, Figure S9A). The histogram of displacements between consecutive frames revealed a diffusion coefficient of 0.011µm 2 /s for polysomes on MTs, 0.004 µm 2 /s for the ones on the nuclear envelope and 0.041 µm 2 /s for those not on MT or the envelope (i.e. freely diffusing; Figure S9 B-D). We also calculated the mean square displacement (MSD) as a function of time ( Figure 7D). This confirmed that the ASPM polysomes that were free in the cytosol diffused several folds faster than those bound to MTs or the nuclear envelope. As a control, we depolymerized MTs with a 10minute nocodazole treatment before starting imaging. SiR-tubulin labeling confirmed the absence of MTs in treated cells, and we then tracked ASPM polysomes, excluding the ones attached to nuclear envelope ( Figure S10A-C, Movie 6). The histogram of displacements and MSD curve revealed a single population with a diffusion coefficient of 0.035µm 2 /s, similar to polysomes not on MT or the nuclear envelope in untreated cells ( Figure 7D and Figure S9E). Overall, this showed that a large fraction of ASPM mRNAs are locally translated on MTs during interphase, and that these polysomes are stably anchored to MTs, thereby limiting their diffusion.

ASPM polysomes are transported to mitotic centrosomes by either sliding on MTs or being pulled with entire MTs
To assess whether MTs are necessary for ASPM mRNA localization to centrosomes, we combined a brief nocodazole treatment (10 minutes) with smFISH, using the ASPM SunTag clone. A 10-minute treatment depolymerized MTs whereas centrosomes were still visible ( Figure S10A, white arrowheads). ASPM mRNAs and polysomes no longer accumulated at mitotic centrosomes after MT depolymerization, despite the fact that the mRNAs were still translated (Figure S10 D-F). This indicated that intact MTs are required for ASPM mRNA localization.
We then performed dual color imaging of MTs and ASPM polysomes during mitosis. Tracks were shorter than in the previous mono-color movies because maintaining high framerates required the recording of only 3 Z planes in two-color experiments, as opposed to 15-20 in the single-color movies. Nevertheless, this allowed us to distinguish two types of movements towards centrosomes. In the first, ASPM polysomes rapidly slided along an immobile MT ( Figure 7E, Movie 7). This likely corresponded to motor-driven movements of polysomes along MT cables. In the second type of movements, an ASPM polysome is stably attached to a MT and both were pulled together towards the centrosome ( Figure 7F, Movie 8). In this case, the MT appears being hauled towards the centrosome and drags a tethered polysome with it. Indeed, MTs pulling and sliding has been previously described during mitosis (Forth and Kapoor, 2017;Enos et al., 2018). This demonstrated that ASPM polysomes are transported to the mitotic centrosomes via two mechanisms: sliding on MT, and tethering to MT coupled to MT remodeling.

NUMA1 mRNAs and polysomes also display directed transport towards the centrosome in early mitosis
To assess the generality of the mechanism found with ASPM, we tagged another centrosomal mRNA, NUMA1. Using CRISPR/Cas9 mediated homology-directed repair in HeLa Kyoto cells, we generated a clone with an MS2x24 tag in the NUMA1 3'UTR, and another clone with a SunTag fused to the N-terminus of the protein. Proper recombination was verified by genotyping ( Figure S11 A, B). In the NUMA1 MS2x24 clone, two-color smFISH performed against either the MS2 tag or the endogenous NUMA1 mRNAs revealed that MS2 tagging did not prevent NUMA1 mRNA from localizing to mitotic centrosomes ( Figure 8A). Likewise, the SunTagged NUMA1 mRNA also localized to mitotic centrosomes ( Figure S11C). Furthermore, in the SunTag NUMA1 clone, smiFISH against the endogenous NUMA1 mRNA revealed that the mRNA colocalized with bright SunTag foci in both interphase and mitosis ( Figure 8B and S11D). A puromycin treatment removed these bright cytoplasmic SunTag foci, confirming that they were NUMA1 polysomes ( Figure S11E). Therefore, tagging the endogenous NUMA1 mRNA with MS2 and SunTag repeats did not abolish its centrosomal localization.
Following stable expression of MCP-GFP-NLS or scFv-sfGFP, we imaged single NUMA1 mRNAs and polysomes, respectively, in living interphase and mitotic cells. In interphase, we could observe some NUMA1 polysomes undergoing rapid rectilinear movements ( Figure S11F). Remarkably, in prometaphase, we observed rapid directed motion of both mRNAs and polysomes toward the centrosome, indicating that NUMA1 polysomes are actively transported towards centrosomes at the onset of mitosis, similar to what we observed for ASPM ( Figure 8C-D, Movies 9 and 10). We manually calculated the mean particle velocity. Both NUMA1 mRNAs and polysomes had average speeds of around 0.9 µm/s, which is compatible with motor directed movements and also similar to the velocities measured for ASPM ( Figure 8E). These live imaging experiments of endogenous transcripts and polysomes show that active polysome transport is a localization mechanism shared by several centrosomal mRNAs.
Drosophila orthologs of the human centrosomal mRNAs also localize to centrosomes and also require the nascent protein We next examined whether centrosomal mRNA localization is evolutionary conserved. To this end, we investigated the localization of the Drosophila orthologs of the human centrosomal mRNAs. Out of the 8 human mRNAs, 5 had clear orthologs: ASPM (Asp), NUMA1 (Mud), BICD2 (BicD), CCDC88C (Girdin), and PCNT (Plp). We used S2R+ cells as a model and co-labeled these mRNAs with centrosomes, using smFISH coupled to IF against gamma-tubulin. Remarkably, we observed centrosomal enrichment for 4 of these 5 Drosophila mRNAs (Figure 9, Table 1). Moreover, while we could not obtain clear signals for the fifth mRNA in S2R+ cells (Plp), it was annotated as centrosomal in a previous largescale screen (see Table 1). Interestingly, we also observed a cell-cycle dependent localization for Asp, Mud, and Girdin mRNAs (Figure S12). A large fraction of cells in interphase did not localize Mud mRNAs whereas it localized to centrosomes during mitosis (metaphase, anaphase, and telophase; Figure 9B and S12). Asp mRNA showed a similar dynamic, with the exception that it was not localized in telophase. In contrast, Girdin mRNAs only accumulated on mitotic centrosomes during telophase ( Figure S12).
These observations indicate that a conserved cell-cycle regulated centrosomal translational program occurs in both human and Drosophila cells.
Finally, we tested whether mRNAs in Drosophila depend on their nascent peptide to localize to centrosomes. We performed puromycin and cycloheximide treatments and co-labeled the mRNAs and centrosomes in S2R+ cells. As in human cells, puromycin Safieddine et al. 20 treatment abolished centrosomal accumulation of all 4 mRNAs while cycloheximide did not ( Figure 9). This indicated that not only the identity of centrosomal mRNAs is conserved from human to Drosophila, but also the localization mechanism, which requires the nascent polypeptide in both cases.

Discussion
Here, we studied centrosomal mRNA localization in human cells. We uncovered a complex choreography of mRNA trafficking at centrosomes, particularly during mitosis, and we provide definitive evidence for a nascent chain-dependent transport of polysomes by motors and MTs. Remarkably, both the identity of localized mRNAs and the localization mechanism appear conserved from Drosophila to humans.

A targeted smFISH screen reveals that centrosomal mRNAs are conserved from human to Drosophila
We used high-throughput smFISH to screen 602 genes encoding nearly the entire centrosomal proteome and we identified four new mRNAs localized at the centrosome (Table 1; Table S1). In total, eight human mRNAs now belong to this class: NIN, CEP350, PCNT, BICD2, CCDC88C, ASPM, NUMA1 and HMMR. All the corresponding proteins localize to the centrosome suggesting that their mRNAs are locally translated. Most of them also perform important centrosomal functions. NIN is localized to the sub-distal appendage of mother centrosomes and it functions in microtubule nucleation as well as centrosome maturation (Ou et al., 2002;Stillwell et al., 2004). CEP350 is also localized to sub-distal appendages and it is important for centriole assembly and MT anchoring to centrosomes (Yan et al., 2006;Le Clech, 2008). Pericentrin (PCNT) is a major component of the pericentriolar material (PCM) and it plays a structural role by bridging the centrioles to the PCM (Lee and Rhee, 2011). BICD2 contributes to centrosomal positioning and to centrosomal separation at the onset of mitosis (Raaijmakers et al., 2012;Splinter et al., 2010). ASPM and NUMA1 are two MT minus end binding proteins that accumulate at centrosomes during mitosis, and they control several aspects of spindle assembly and function (Jiang et al., 2017;Seldin et al., 2016). Finally, HMMR acts to separate Safieddine et al.

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centrosomes and to nucleate MT during spindle assembly. It also modulate the cortical localization of NUMA-dynein complexes to correct mispositioned spindles (Connell et al., 2017). The diversity of functions performed by these proteins suggests that RNA localization and local translation play an important role for the centrosome.
Remarkably, we found that 5 of the human centrosomal mRNAs had orthologs in Drosophila, and 4 of these localized to centrosomes in S2R+ cells. Moreover, the fifth Drosophila mRNA, Plp, was reported to localize to centrosomes in Drosophila embryos (Table 1; Lécuyer et al., 2007;Wilk et al., 2016). These data show a striking and unprecedented degree of evolutionary conservation in RNA localization, where the same family of mRNAs is localized to the same subcellular site, from Drosophila to humans. This likely underlies conserved features in mRNA localization mechanism and/or function.

A cell-cycle dependent translational program operates at centrosomes
Our data reveal that centrosomal mRNA localization varies with phases of the cell cycle.
Moreover, the two mitotic mRNAs localized with different kinetics: ASPM localized during all mitotic phases while NUMA1 only during prophase and prometaphase. Finally, HMMR was the only transcript that localized at the cytokinetic bridge at the end of cell division, together with its protein. Interestingly, the Drosophila centrosomal mRNAs also localize in a cell cycle-dependent way, indicating that this feature is also conserved during evolution. This shows the variety, complexity and precision of centrosomal mRNA localization, as well as its potential role during the centrosome cycle. These data 23 demonstrate the existence of a unique and conserved translational program at centrosomes, which is cell cycle regulated.
It is interesting to speculate why these eight proteins and not others are locally translated. Since they function in centrosome/spindle maturation and that this occurs over short time periods, having optimal amounts at centrosomes at the right time point of the cell cycle is crucial. Interestingly, most of these proteins have relatively large sizes (more than 2000 aa, with the exception of HMMR and BICD2), and it would thus take some time to synthesize them. A local translational regulation may thus provide an efficient and rapid method for targeting them to the centrosome when needed. This might be particularly important in prophase where most mRNAs localize, because it is a relatively short phase and also the site of important changes in centrosome composition and function.
Another possibility is that mRNA accumulation at centrosomes plays a structural role. An emerging model is that phase separation helps the formation of centrosomes (Woodruff et al., 2018). Since RNAs are often critical components of phase separated condensates (Navarro et al., 2019), their accumulation at centrosomes could contribute to their formation. Finally, a likely and not exclusive possibility is that these 8 proteins need to be assembled co-translationally with their partners at the centrosome. Cotranslational folding occurs for many proteins and this can be facilitated by the presence of a protein's partner. This may further provide an elegant mechanism for RNA localization (see below).

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For all the eight centrosomal transcripts studied here, premature ribosome termination delocalized the mRNAs while freezing the ribosome and the nascent protein chain on the mRNA had no effect. In the case of a GFP-tagged ASPM mRNA, we further observed that preventing translation of the nascent ASPM protein via a stop codon abolished centrosomal RNA localization, indicating that translation is required in cis. Most importantly, polysomes coding for ASPM and NUMA1 were actively transported to centrosomes at rates compatible with motor-driven transport (0.5-1 m/s). Together, this shows that centrosomal mRNA localization relies on an active mechanism driven by the nascent peptide. While RNA localization is often conceptualized as an RNA-driven process that transports silenced mRNAs, our data contradict this dogma and show that for centrosomal mRNAs, polysome transport mediated by the nascent protein is the rule.
These observations suggest that the nascent polypeptide contains a localization signal that would drive the polysome toward centrosomes. Interestingly, we found that ASPM polysomes are actively transported via two mechanisms. The first is motorized transport whereby a polysome slides on MTs. The second involves the pulling of an entire MT with an ASPM polysome attached to it. The N-terminal part of ASPM contains two calponin-homology domains that can bind MTs. It is possible that once these domains are translated, they bind MTs and cause the entire ASPM polysome to attach. In agreement with this possibility, local translation of ASPM at MTs can also be seen during interphase.

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The paradigm for translation-dependent RNA localization is that of secreted proteins. In this case, translation initially leads to the synthesis of the signal peptide, which is recognized by SRP. This halts the ribosome until the entire complex docks on the SRP receptor on the ER, where translation resumes. It is thus tempting to envision a scenario where ribosomes translating centrosomal mRNAs enter a pause and only resume translation after reaching the centrosome. It has been shown that unfolded domains can halt ribosomes (Liu et al., 2013;Shalgi et al., 2013). Moreover, in a recent case of cotranslational assembly, the ribosome enters a pause at a specific location, which is relieved upon interaction with the partner of the nascent protein (Panasenko et al., 2019).
If indeed the proteins encoded by the centrosomal mRNAs need to be co-translationally assembled, it is possible that the domain responsible for this would remain unfolded before the polysome reaches the centrosome. It could thus halt the ribosomes while the nascent polypeptide located upstream of this unfolded domain could connect the polysome to transport systems and drive it to the centrosome. This could be an elegant and general mechanism that ensures RNA localization and local translation, as it could work at any place in the cell. This could explain why co-translational mRNA targeting appears to be a widespread mechanism in cell lines (Chouaib et al., 2019). 0018-01), the Institut Pasteur, the Ligue Nationale Contre le Cancer and the Labex EpiGenMed, from the framework "Investissements d'avenir".

Author contribution statement
The HT-smFISH methodology was conceived by EB and developed by AMT, CL, EC, EB, FL, MP, TG and VG. The idea of screening the centrosomal proteome was conceived by EB, the gene/probe lists were generated by CL, EB and MP, the screen was conducted by EC and

Conflict of interest disclosure
The authors declare no competing financial interests.
Drugs were used at the following final concentrations: 100 µg/ml for puromycin, 200 µg/ml for cycloheximide, and 5 µg/ml for nocodazole. Treatment of cells with translation inhibitors was for 20 minutes (reduced to 5 min for mitotic cells when indicated).
Treatment of cells with nocodazole was for 10 minutes. Transfection of the GFP-ASPM CDS constructs was done using JetPrime (Polyplus) and 2 µgs of DNA were transfected overnight in a 6 well plate containing 2ml of medium.

Insertion of the MS2 cassette by CRISPR/Cas9
The recombination cassettes contained 500 bases of homology arms flanking a 3xHA tag, a stop codon and 24 MS2 repeats. A start codon was placed after the MS2 repeats followed by an IRES, a neomycin resistance gene, and a stop codon. The IRES-Neo r segment was flanked by two LoxP sites having the same orientation. HeLa Kyoto cells were transfected using JetPrime (Polyplus) and a cocktail of four plasmids, including the recombination cassette and constructs expressing Cas9-nickase and two guide RNAs with an optimized scaffold (Pichon et al. 2016). Insertion was targeted at the stop codon of the ASPM and NUMA1 genes. Cells were selected on 400 µg/ml G418 neomycin for a few weeks.
Individual clones were then picked and analyzed by PCR genotyping, fluorescent microscopy and smFISH/smiFISH with probes against both the endogenous ASPM or NUMA1 mRNA and MS2 sequences. Stable MCP-GFP-NLS expression was then set up via retroviral infection. The sequences targeted by the guide RNAs were (PAM sequences are underlined): TCTCTTCTCAAAACCCAATCTGG for ASPM guide 1, and GCAAGCTATTCAAATGGTGATGG for ASPM guide 2; GAGGTCAGCATCGGGGACACAGG for NUMA1 guide 1, and AGTGCCTTCTCTCAGCTCCCAGG for NUMA1 guide 2.

Insertion of SunTag cassette by CRISPR/Cas9
The recombination cassettes contained 500 bases of homology arms flanking a puromycin resistance gene translated from the endogenous ATG sequence, followed by a P2A

Single molecule fluorescent in situ hybridization
Cells grown on glass coverslips or 96-well glass bottom plates (SensoPlates, Greiner) were fixed for 20 min at RT with 4% paraformaldehyde (Electron Microscopy Sciences) diluted in PBS (Invitrogen), and permeabilized with 70% ethanol overnight at 4°C.
For smFISH performed on BAC cells, we used a set of 44 amino-modified oligonucleotide probes against the GFP-IRES-Neo sequence present in the BAC construction (sequences given in Table S2). Each oligonucleotide probe contained 4 primary amines that were conjugated to Cy3 using the Mono-Reactive Dye Pack  Table S2) was used per 100μl of hybridization mix.
For smiFISH using DNA probes (Tsanov et al., 2016), 24-48 unlabeled primary probes were used (sequences given in Table S2). In addition to hybridizing to their targets, these probes contained a FLAP sequence that was pre-hybridized to a secondary fluorescent oligonucleotide. To this end, 40 pmoles of primary probes were prehybridized to 50 pmoles of secondary probe in 10 µl of 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, pH 7.9. Pre-hybridization was performed on a thermocycler with the following program: 85°C for 3 min, 65°C for 3 min, and 25°C for 5 min. The final hybridization mixture contained the probe duplexes (2 µl per 100 µl of final volume), with 1X SSC, 0.34 mg/ml tRNA, 15% Formamide, 2 mM VRC, 0.2 mg/ml RNAse-free BSA, 10% Dextran sulfate. Slides were then processed as above.
For smiFISH using RNA probes in the high-throughput smFISH screen, a pool of DNA oligonucleotides (GenScript) was used to generate the primary probes. The oligonucleotide design was based on the Oligostan script (Tsanov et al., 2016). A first series of PCR was performed using the oligopool as template and each of the gene-specific barcoding primers. A second series of PCR was achieved using the following primers:

Imaging of fixed cells
Microscopy slides were imaged on a Zeiss Axioimager Z1 wide-field microscope equipped with a motorized stage, a camera scMOS ZYLA 4.2 MP, using a 63x or 100x objective (Plan

Image analysis and quantifications
Mitotic phases were identified based on visual inspection of DNA condensation and cell shape. Early prophase was defined by its low DNA compaction, which increased in late prophase. Early prometaphase was marked by the rupture of the nuclear envelope, while late prometaphase additionally displayed cell rounding. For late mitosis, we subdivided cells into early telophase (without cytokinesis), and late telophase (with cytokinesis marked by the accumulation of HMMR-GFP at cytokinetic bridges). Centrosomal localization was assessed by visual inspection of individual cells.

Single molecule dynamics analysis and single particle tracking
The dynamics of ASPM mRNAs, and both NUMA1 mRNAs and polysomes were assessed as follows: mRNAs anchored to the centrosomes as well as those undergoing directed motion were identified based on visual inspection. The speed of individual mRNA molecules displaying directed motions was measured across at least 3 frames and calculated using ImageJ.
Single particle tracking of ASPM polysomes was performed using the TrackMate plugin in ImageJ. The DoG detector was used. Blob diameter was set to 0.7-0.8 microns and the detection threshold was between 100-120. Median filtering and sub-pixel localization were additionally used. The simple LAP tracker option was used to construct Tracks were imported and analyzed in R. Instant 1D displacements between frames were calculated along the x and y axis, and the resulting histograms were fitted to a Gaussian function, for which variance is directly proportional to the diffusion coefficient (D). We also calculated a mean MSD as a function of time, by aligning all tracks at their start and averaging the resulting 2D displacements.

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Supplementary spreadsheets: Table S1: Summary of all screened mRNAs