In compartmentalized cells, the genetic information must travel from the transcription sites in the nucleus toward the translation sites in the cytoplasm. In addition, RNA molecules play enzymatic or structural roles in a myriad of non-coding RNP particles. Thus, the intracellular transport of RNA molecules is a fundamental problem in biology. To tackle this question, we use cutting edge imaging techniques such as tracking of single molecule, FRAP and photo-activation, and FRET.
We first study RNA transport within the nucleus. For this, we use the model of box C/D snoRNAs which concentrate in nucleoli where they are involved in rRNA maturation. We have shown an important role of Cajal bodies in the early biogenesis of snoRNAs and telomerase. We also observed that PHAX, a protein previously characterized as an snRNA export factor, is required for the transport of snoRNAs toward Cajal bodies. This suggests that PHAX is a specialized transport factor for small RNA molecules across the nucleoplas. More recently, we have shown that the chaperone HSP90 and a conserved co-chaperone complex play an essential role in the assembly of snoRNP and telomerase. This indicates that protein chaperones are directly involved in RNP assembly, possibly to assist protein folding during this process. In addition, this suggests that HSP90 is a master regulator of cell proliferation, by controlling simultaneously cell signalling and cell growth.
Second, we study early steps of mRNA biogenesis. Using HIV-1 as a model system, we have developped a technique that allows us to visualize mRNA synthesis and processing in real-time, by FRAP analysis of a specific transcription site. With this tool, we aim to provide : (i) a detailled kinetic analysis of transcription, splicing, and 3’-end formation in live cells; (ii) a view of spliceosome assembly and turn-over.
Finally, we are also interested in mRNA transport in the cytoplasm. Two model system are used. Using retroviruses, we have analyzed the transport of genomic RNAs toward the viral budding sites at the plasma membrane, and we have observed that they are transported on endosomal vesicles. Using miRNA and mRNAs controlled by miRNA, we have shown that P-bodies are involved in storage of translationally repressed mRNAs. Our goal is to understand how RNA are transported toward this compartment.
This picture shows a yeast cell that expresses an artificial reporter mRNA (red). The nucleus is in blue. In the left image, red spots correspond to individual mRNA molecules detected after in situ hybridization. In the right image, these spots are automatically recognized by an appropriate software. This experiment is part of a european project, aimed at developing system biology of RNA (visit RIBOSYS at http://www.ribosys.org/). We perform systematic quantification of the number and localization individual mRNA molecules, in wild-type cells and a variety of mutant background. |