Single-molecule Live-cell Imaging of a Non-coding Rna

RNA is a recent target for bioimaging studies. For example, subcellular localization of particular mRNAs has been investigated and the dynamics of them have been partially revealed [1, 2]. On the other hand, few studies have been reported on live cell imaging of non-coding RNAs (ncRNAs) in spite of the importance of ncRNAs that are implicated in controlling biological events especially in the nucleus. We herein describe development of an RNA probe for live-cell imaging of a non-coding RNA, telomeric-repeat containing RNA (TERRA). This probe consists of a pair of split EGFP fragments and a mutated RNA-binding domain PUM-HD of human pumilio1. The mutated PUM-HD were designed to binds specifically to a particular 8-base RNA sequence in the telomeric-repeat region of TERRA. Upon multiple probe molecules bind to the repeat region, the EGFP fragments occur intermolecular reconstitution, recovering fluorescence (Figure 1). Using this probe, we performed simultaneous fluorescence observation of TERRA, telomere, and a telomere-related protein hnRNPA1 in living cells. A telomere probe (TRF1-iRFP) and hnRNPA1-SNAPf were expressed in living cells in addition to the present TERRA probe, and subjected to total internal reflection fluorescence (TIRF) microscope observation. The TIRF microscope equipped triple excitation lasers (488, 561, and 640 nm) and three cameras, being applicable to simultaneous observation of TERRA, telomere, and hnRNPA1. In the TIRF imaging, we observed diffusing single molecule TERRA and hnRNPA1, and stable telomeres in identical cells. In the merged images of TERRA and hnRNPA1, we detected colocalization events of TERRA and hnRNPA1 continuing 0.3 sec or more, which indicates complex formation of these molecules. Then we analyzed spatial distribution of the complex formation. The complexes were intensively formed in regions of 1 μm from individual telomeres. Moreover, we found that the complex formed between static TERRA and diffusing hnRNPA1. Based on these results, we discuss the possibility of the present probe and microscopic methodology to establish a mechanistic models of TERRA.