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 from probe-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.

The nuclear cap-binding complex (CBC) stimulates multiple steps in several RNA maturation pathways, but how it functions in humans is incompletely understood. For small, capped RNAs such as pre-snRNAs, the CBC recruits PHAX. Here, we identify the CBCAP complex, composed of CBC, ARS2 and PHAX, and show that both CBCAP and CBC-ARS2 complexes can be reconstituted from recombinant proteins. ARS2 stimulates PHAX binding to the CBC and snRNA 3'-end processing, thereby coupling maturation with export. In vivo, CBC and ARS2 bind similar capped noncoding and coding RNAs and stimulate their 3'-end processing. The strongest effects are for cap-proximal polyadenylation sites, and this favors premature transcription termination. ARS2 functions partly through the mRNA 3'-end cleavage factor CLP1, which binds RNA Polymerase II through PCF11. ARS2 is thus a major CBC effector that stimulates functional and cryptic 3'-end processing sites.

Local translation allows for a spatial control of gene expression. Here, we use 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 reveal a common translation-dependent localization mechanism requiring the nascent protein. Using ASPM and NUMA1 as models, single mRNA and polysome imaging reveals 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 polysome transport mediated by nascent proteins.

Promoter-proximal pausing of RNA polymerase II is a key process regulating gene expression. In latent HIV-1 cells, it prevents viral transcription and is essential for latency maintenance, while in acutely infected cells the viral factor Tat releases paused polymerase to induce viral expression. Pausing is fundamental for HIV-1, but how it contributes to bursting and stochastic viral reactivation is unclear. Here, we performed single molecule imaging of HIV-1 transcription. We developed a quantitative analysis method that manages multiple time scales from seconds to days and that rapidly fits many models of promoter dynamics. We found that RNA polymerases enter a long-lived pause at latent HIV-1 promoters (>20 minutes), thereby effectively limiting viral transcription. Surprisingly and in contrast to current models, pausing appears stochastic and not obligatory, with only a small fraction of the polymerases undergoing long-lived pausing in absence of Tat. One consequence of stochastic pausing is that HIV-1 transcription occurs in bursts in latent cells, thereby facilitating latency exit and providing a rationale for the stochasticity of viral rebounds.

Spinal muscular atrophy is a neuromuscular disease resulting from mutations in the SMN1 gene, which encodes the survival motor neuron (SMN) protein. SMN is part of a large complex that is essential for the biogenesis of spliceosomal small nuclear RNPs. SMN also colocalizes with mRNAs in granules that are actively transported in neuronal processes, supporting the hypothesis that SMN is involved in axonal trafficking of mRNPs. Here, we have performed a genome-wide analysis of RNAs present in complexes containing the SMN protein and identified more than 200 mRNAs associated with SMN in differentiated NSC-34 motor neuron-like cells. Remarkably, ∼30% are described to localize in axons of different neuron types. In situ hybridization and immuno-fluorescence experiments performed on several candidates indicate that these mRNAs colocalize with the SMN protein in neurites and axons of differentiated NSC-34 cells. Moreover, they localize in cell processes in an SMN-dependent manner. Thus, low SMN levels might result in localization deficiencies of mRNAs required for axonogenesis.

Spinal muscular atrophy results from deletions or mutations in the survival of motor neuron (SMN1) gene. The SMN protein has an essential role in the biogenesis of spliceosomal snRNPs, but the link between a defect in this process and specific splicing inhibition of pre-mRNAs has not been established. In this study, we report the construction of a temperature-degron (td) allele of the Schizosaccharomyces pombe SMN protein and show that its depletion at 371C affects splicing and formation of U1, U2, U4 and U5 snRNPs, but not of U6 and U3 ribonucleoproteins. The function of the tdSMN allele in snRNP assembly is already perturbed at 251C, suggesting a deleterious effect of the tag at this temperature. Using a genome-wide approach, we report that introns react unequally to lower levels of snRNPs in tdSMN cells and that increasing the length of the polypyrimidine tract can improve the splicing efficiency of some, but not all, affected introns. Altogether, our results suggest that the defects observed in tdSMN fission yeast cells mimic splicing deficits observed in SMN-deficient metazoan cells.

BackgroundmicroRNAs (miRNAs) play crucial roles in major biological processes and their deregulations are often associated with human malignancies. As such, they represent appealing candidates as targets of innovative therapies. Another interesting aspect of their biology is that they are present in various biological fluids where, advantageously, they appear to be very stable. A plethora of studies have now reported their potential as biomarkers that can be used in diagnosis, prognosis and/or theranostic issues. However, the application of circulating miRNAs in clinical practices still requires the identification of highly efficient, robust and reproducible methods for their isolation from biological samples.In that context, we performed an independent cross-comparison of three commercially available RNA extraction kits for miRNAs isolation from human blood samples (Qiagen and Norgen kits as well as the new NucleoSpin miRNAs Plasma kit from Macherey-Nagel). miRNAs were further profiled using the Taqman Low Density Array technology.ResultsWe found that, although these 3 kits had equal performances in extracting miRNAs from peripheral blood mononuclear cells, the Macherey-Nagel kit presented several advantages when isolating miRNAs from sera. Besides, our results have indicated that, depending on the quantity of the biological samples used, the extraction procedure directly impacted on the G/C composition of the miRNAs detected.ConclusionOverall, our study contributes to the definition of a reliable framework for profiling circulating miRNAs.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-395) contains supplementary material, which is available to authorized users.

BackgroundDespite its critical role for mammalian gene regulation, the basic structural landscape of chromatin in living cells remains largely unknown within chromosomal territories below the megabase scale.ResultsHere, using the 3C-qPCR method, we investigate contact frequencies at high resolution within interphase chromatin at several mouse loci. We find that, at several gene-rich loci, contact frequencies undergo a periodical modulation (every 90 to 100 kb) that affects chromatin dynamics over large genomic distances (a few hundred kilobases). Interestingly, this modulation appears to be conserved in human cells, and bioinformatic analyses of locus-specific, long-range cis-interactions suggest that it may underlie the dynamics of a significant number of gene-rich domains in mammals, thus contributing to genome evolution. Finally, using an original model derived from polymer physics, we show that this modulation can be understood as a fundamental helix shape that chromatin tends to adopt in gene-rich domains when no significant locus-specific interaction takes place.ConclusionsAltogether, our work unveils a fundamental aspect of chromatin dynamics in mammals and contributes to a better understanding of genome organization within chromosomal territories.