Single molecule-based screening at medium throughtput of mRNA localization in human cells

La localisation d’ARNm a été découverte en 1983 dans les ovocytes et les embryons des ascidies. Depuis, plusieurs exemples d'ARN localisés ont été trouvés dans de nombreux organismes, y compris les plantes, les levures, les champignons, les insectes, les poissons et les mammifères. Les ARNm localisés contribuent à de nombreuses fonctions biologiques, telles que le développement embryonnaire, la division cellulaire asymétrique, la migration cellulaire, la signalisation, la plasticité neuronale et plein d’autres ...Jusqu'à présent, quelques études ont analysé la localisation d’ARNm de manière systématique. Trois d'entre eux ont été effectués chez la drosophile pendant l'embryogenèse, l'oogenèse ou le stade larvaire et ont analysé environ 16000 ARN au total. Les deux autres études ont été réalisées dans des cellules de mammifères et ont analysé près de 1000 ARNm chacune. Ces études ont montré l'importance de la localisation d’ARNm dans les cellules humaines et son implication dans différents processus biologiques. L'objectif de ma thèse était donc d'augmenter le débit des techniques FISH à l’échelle de molécule unique (smFISH) et d'étudier la localisation d’ARNm dans les cellules HeLa de manière systématique.Une limitation de smFISH est le coût de sondes fluorescentes, qui limite le nombre d'ARNm qui peut être analysé. Par conséquent, j'ai développé un protocole alternatif dans lequel des sondes pour de nombreux gènes ont été synthétisées comme un pool d'oligonucléotides (40 par gène en moyenne, plus de 12000 au total). Les sondes spécifiques d’un ARNm donné ont ensuite été amplifiées par PCR et converties en simple brin par transcription in vitro. J'ai généré un protocole complet, à partir de la conception de la sonde et jusqu'à l'acquisition de l'image. Je me suis intéressé à l’étude des ARNm du cycle cellulaire. En effet, les gènes du cycle cellulaire ont été largement étudiés au niveau de la protéine, mais on sait peu de choses sur la localisation de leurs ARNm. Pendant la mitose, les cellules subissent d'importantes modifications morphologiques et la traduction locale pourrait être un moyen d'atteindre la localisation des protéines. Le screening sur ces ARNm est en cours.Parallèlement à ces expériences, j'ai réalisé des expériences de smFISH sur 100 gènes choisis au hasard et 50 régulateurs de la transition G2/M du cycle cellulaire, en utilisant un protocole de smFISH classique. Dans cette configuration, on disposait d'une collection de lignées cellulaires HeLa, dans laquelle chaque cellule contient un chromosome artificiel bactérien avec le gène d'intérêt marqué au GFP. Par conséquent, en utilisant des sondes qui s'hybridaient à la séquence GFP, je pourrais utiliser le même ensemble de sondes marquées pour étudier la localisation de tous les ARNm. Un autre avantage est que la localisation des protéines pourrait être évaluée simultanément. Mes résultats indiquent que 4 ARNm ont montré une localisation spécifique lors du screening de 100 gènes choisis d’une manière aléatoire et 15 ARNm parmi les 54 régulateurs de la transition G2 / M. Ces ARNm appartiennent à cinq classes de localisation: "blobs", qui sont des agrégats d'ARNm cytoplasmiques; «clusters», qui sont des zones de concentration locale élevée d'ARNm, mais où une molécule unique d’ARNm peut encore être résolu; «nuclear membrane », où les ARNm se concentrent autour de l'enveloppe nucléaire; "spindle", qui sont des ARNm accumulés sur l'appareil de division mitotique, “spots" qui sont des agrégats d'ARNm cytoplasmiques où une molécule unique d’ARNm ne peut pas être résolu, et qui sont plus grands que les blobs. La colocalisation entre l'ARNm et la GFP, qui suggère une traduction locale, n'a été trouvée que pour 1 ARNm.Ces screenings aléatoires et ciblés effectués à petite échelle montrent une fréquence et une diversité inattendues dans les modèles de localisation d’ARNm. Cela ouvre la voie pour effectuer des screenings à plus grande échelle.MRNA localization was discovered in 1983 in ascidian oocytes and early embryos. Since then many examples of localized RNAs have been found in many organisms, including plants, yeast, fungi, insects, fish and mammals. Localized mRNAs contribute to many biological functions, such as embryonic patterning, asymmetric cell division, cell migration, signaling, neuronal plasticity and others…Until now, only few studies analyzed RNA localization in a systematic manner. Three of them were done in Drosophila, during embryogenesis, oogenesis or larval stage and analyzed around 16000 mRNAs in total. The two other studies were done in mammalian cells and analyzed nearly 1000 mRNAs each. These studies opened a door and raised questions regarding the importance of mRNA localization in human cells and its implication in different biological processes. The goal of my thesis was thus to increase the throughput of single molecule FISH techniques (smFISH) and to study mRNA localization in HeLa cells in a systematic manner.One limitation in smFISH is the cost of the fluorescent oligonucleotide probes, which limits the number of mRNAs that can be analyzed. Therefore, I developed an alternative protocol in which probes for many genes were synthesized as a pool of oligonucleotides (40 per gene in average, more than 12000 in total). Gene-specific probes were then amplified by PCR and converted into single strand by in vitro transcription. I generated a complete protocol, starting from probe design and up to image acquisition. I was interested in studying cell cycle genes. Indeed, cell cycle genes have been extensively studied at the protein level but little is known concerning the localization of their mRNAs. During mitosis, cells go through important morphological modifications and local translation could be a mean of achieving protein localization. This screen is ongoing.In parallel to these experiments, I performed a smFISH based screen on 100 randomly chosen genes and 50 regulators of the G2/M transition of the cell cycle, using a traditional smFISH protocol. In this set-up, I took advantage of a library of HeLa cell lines, in which each cell line contains a bacterial artificial chromosome with the gene of interest tagged with GFP. Therefore, using oligonucleotides hybridizing to the GFP sequence, I could use the same probe set to study the localization of all the tagged mRNAs. A further advantage is that protein localization could be assessed simultaneously. My results indicate that two mRNAs showed a specific localization when screening 100 random genes, and 16 mRNAs among the 50 regulators of the G2/M transition. These mRNAs belong to five localization classes: "blobs", which are cytoplasmic mRNA aggregates; "clusters", which are areas of high local mRNA concentration but where individual mRNA can still be resolved; "nuclear envelope", where mRNAs concentrate around the nuclear envelope; "spindle", which are mRNAs accumulating on the cell division apparatus during mitosis, “spots" which are cytoplasmic mRNA aggregates where individual mRNA can’t be resolved and are bigger than blobs. Interestingly, colocalization between mRNA and GFP, which suggests local translation, was only found for 1 mRNA.These random and targeted screens performed at small-scale show an unexpected frequency and diversity in mRNA localization patterns, therefore pointing to new functions related to this process. This will stimulate future studies aiming at performing screenings at a higher scale

A computational framework to study sub-cellular RNA localization

Automated analysis of RNA localisation in smFISH data has been elusive. Here, the authors simulate and use a large dataset of images to design and validate a framework for highly accurate classification of sub-cellular RNA localisation patterns from smFISH experiments

Smifish and fish-quant – a flexible single rna detection approach with super-resolution capability

Single molecule FISH (smFISH) allows studying transcription and RNA localization by imaging individual mRNAs in single cells. We present smiFISH (single molecule inexpensive FISH), an easy to use and flexible RNA visualization and quantification approach that uses unlabelled primary probes and a fluorescently labelled secondary detector oligonucleotide. The gene-specific probes are unlabelled and can therefore be synthesized at low cost, thus allowing to use more probes per mRNA resulting in a substantial increase in detection efficiency. smiFISH is also flexible since differently labelled secondary detector probes can be used with the same primary probes. We demonstrate that this flexibility allows multicolor labelling without the need to synthesize new probe sets. We further demonstrate that the use of a specific acrydite detector oligonucleotide allows smiFISH to be combined with expansion microscopy, enabling the resolution of transcripts in 3D below the diffraction limit on a standard microscope. Lastly, we provide improved, fully automated software tools fromprobe-design to quantitative analysis of smFISH images. In short, we provide a complete workflow to obtain automatically counts of individual RNA molecules in single cells

Smifish and fish-quant – a flexible single rna detection approach with super-resolution capability

Single molecule FISH (smFISH) allows studying transcription and RNA localization by imaging individual mRNAs in single cells. We present smiFISH (single molecule inexpensive FISH), an easy to use and flexible RNA visualization and quantification approach that uses unlabelled primary probes and a fluorescently labelled secondary detector oligonucleotide. The gene-specific probes are unlabelled and can therefore be synthesized at low cost, thus allowing to use more probes per mRNA resulting in a substantial increase in detection efficiency. smiFISH is also flexible since differently labelled secondary detector probes can be used with the same primary probes. We demonstrate that this flexibility allows multicolor labelling without the need to synthesize new probe sets. We further demonstrate that the use of a specific acrydite detector oligonucleotide allows smiFISH to be combined with expansion microscopy, enabling the resolution of transcripts in 3D below the diffraction limit on a standard microscope. Lastly, we provide improved, fully automated software tools fromprobe-design to quantitative analysis of smFISH images. In short, we provide a complete workflow to obtain automatically counts of individual RNA molecules in single cells

HT-smFISH: a cost effective and flexible workflow for high-throughput single molecule RNA imaging

International audienceThe ability to visualize RNA in its native subcellular environment by using single-molecule fluorescence in situ hybridization (smFISH) has reshaped our understanding of gene expression and cellular functions. A major hindrance of smFISH is the difficulty to perform systematic experiments in medium- or high-throughput formats, principally because of the high cost of generating the individual fluorescent probe sets. Here, we present high-throughput smFISH (HT-smFISH), a simple and cost-efficient method for imaging hundreds to thousands of single endogenous RNA molecules in 96-well plates. HT-smFISH uses RNA probes transcribed in vitro from a large pool of unlabeled oligonucleotides. This allows the generation of individual probes for many RNA species, replacing commercial DNA probe sets. HT-smFISH thus reduces costs per targeted RNA compared with many smFISH methods and is easily scalable and flexible in design. We provide a protocol that combines oligo pool design, probe set generation, optimized hybridization conditions and guidelines for image acquisition and analysis. The pipeline requires knowledge of standard molecular biology tools, cell culture and fluorescence microscopy. It is achievable in ~20 d. In brief, HT-smFISH is tailored for medium- to high-throughput screens that image RNAs at single-molecule sensitivity

HT-smFISH: a cost effective and flexible workflow for high-throughput single molecule RNA imaging

International audienceThe ability to visualize RNA in its native subcellular environment by using single-molecule fluorescence in situ hybridization (smFISH) has reshaped our understanding of gene expression and cellular functions. A major hindrance of smFISH is the difficulty to perform systematic experiments in medium- or high-throughput formats, principally because of the high cost of generating the individual fluorescent probe sets. Here, we present high-throughput smFISH (HT-smFISH), a simple and cost-efficient method for imaging hundreds to thousands of single endogenous RNA molecules in 96-well plates. HT-smFISH uses RNA probes transcribed in vitro from a large pool of unlabeled oligonucleotides. This allows the generation of individual probes for many RNA species, replacing commercial DNA probe sets. HT-smFISH thus reduces costs per targeted RNA compared with many smFISH methods and is easily scalable and flexible in design. We provide a protocol that combines oligo pool design, probe set generation, optimized hybridization conditions and guidelines for image acquisition and analysis. The pipeline requires knowledge of standard molecular biology tools, cell culture and fluorescence microscopy. It is achievable in ~20 d. In brief, HT-smFISH is tailored for medium- to high-throughput screens that image RNAs at single-molecule sensitivity

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.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 localization screen reveals translation factories and widespread co-translational RNA targeting

Publié sur BioRxiv le 21 mai 2020 : https://www.biorxiv.org/content/10.1101/2020.05.20.106989v1Local translation allows a spatial control of gene expression. Here, we performed a dual protein/mRNA localization screen, using smFISH on 523 human cell lines expressing GFP-tagged genes. A total of 32 mRNAs displayed specific cytoplasmic localizations, and we observed local translation at unexpected locations, including cytoplasmic protrusions, cell edges, endosomes, Golgi, the nuclear envelope and centrosomes, the latter being cell cycle dependent. Quantitation of mRNA distribution and automatic pattern classification revealed a high degree of localization heterogeneity between cells. Surprisingly, mRNA localization frequently required ongoing translation, indicating widespread co-translational RNA targeting. Interestingly, while P-body accumulation was frequent (15 mRNAs), four mRNAs accumulated in foci that were distinct structures. These foci lacked the mature protein, but nascent polypeptide imaging showed that they were specialized translation factories. For β-catenin, foci formation was regulated by Wnt, relied on APC-dependent polysome aggregation, and led to nascent protein degradation. Thus, translation factories uniquely regulate nascent protein metabolism and create a fine granular compartmentalization of translation

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

International audienceLocal 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