CoPixie, a novel algorithm for single-particle track colocalization, enables efficient quantification of telomerase dynamics at telomeres.

Single-particle imaging and tracking can be combined with colocalization analysis to study the dynamic interactions between macromolecules in living cells. Indeed, single-particle tracking has been extensively used to study protein-DNA interactions and dynamics. Still, unbiased identification and quantification of binding events at specific genomic loci remains challenging.

TERRA transcripts localize at long telomeres to regulate telomerase access to chromosome ends.

The function of TERRA in the regulation of telomerase in human cells is still debated. While TERRA interacts with telomerase, how it regulates telomerase function remains unknown. Here, we show that TERRA colocalizes with the telomerase RNA subunit hTR in the nucleoplasm and at telomeres during different phases of the cell cycle.

Phosphorylation controls the oligomeric state of She2 and mRNA localization in yeast.

Messenger RNA (mRNA) localization is an important mechanism controlling local protein synthesis. In budding yeast, asymmetric localization of transcripts such as mRNA to the bud tip depends on the She2 RNA-binding protein. She2 assembles as a tetramer to bind RNA, but the regulation of this process as part of the mRNA locasome is still unclear.

Quantitative Imaging of MS2-Tagged hTR in Cajal Bodies: Photobleaching and Photoactivation.

Advances in imaging technologies, gene editing, and fluorescent molecule development have made real-time imaging of nucleic acids practical. Here, we detail methods for imaging the human telomerase RNA template, hTR via the use of three inserted MS2 stem loops and cognate MS2 coat protein (MCP) tagged with superfolder GFP or photoactivatable GFP. These technologies enable tracking of the dynamics of RNA species through Cajal bodies and offer insight into their residence time in Cajal bodies through photobleaching and photoactivation experiments.

Imaging of Telomerase RNA by Single-Molecule Inexpensive FISH Combined with Immunofluorescence.

Fluorescent hybridization (FISH) on the RNA moiety of human telomerase (hTR) with 50-mer probes detects hTR RNA accumulated in Cajal bodies. Using both live-cell imaging and single-molecule inexpensive FISH, our published work revealed that only a fraction of hTR localizes to Cajal bodies, with the majority of hTR molecules distributed throughout the nucleoplasm. This protocol is an application guide to the smiFISH method for the dual detection of hTR RNA and telomeres or Cajal bodies by immunofluorescence.