Tracking Nuclear Poly(A) RNA Movement Within and Among Speckle Nuclear Bodies and the Surrounding Nucleoplasm

The movement of polyadenylated RNA transcripts (poly(A) RNA) through speckles in the nucleus can be detected and studied using fluorescence correlation microscopy (FCM) and photoactivation RNA tracking techniques. Speckles, sometimes called interchromatin granule clusters, are nuclear bodies that contain pre-mRNA splicing factors and poly(A) RNA. In the methods described here, speckles are marked in live cells using monomeric red fluorescent protein fused to SC35, a splicing protein that is a common speckle component. Endogenous poly(A) RNAs are tagged by in vivo hybridization with fluorescein-labeled oligo(dT) and FCM is performed at the marked speckles and in the nucleoplasm to measure the mobility of the tagged poly(A) RNA. The majority of the nuclear poly(A) RNA population diffuses rapidly throughout the nucleoplasm, and thus this method allows one to ask whether poly(A) RNA that is located in speckles at a given time is undergoing a dynamic transit or is, in contrast, a more immobile, perhaps structural, component. To visualize the movement of poly(A) RNA away from speckles, poly(A) RNA is tagged with caged-fluorescein-labeled oligo(dT) and speckle-associated poly(A) RNAs are specifically photoactivated using a laser beam directed through a pinhole in a rapid digital imaging microscopy system. The spatial distribution of the now-fluorescent RNA as it moves from the speckle photoactivation site is then recorded over time. Temperature and/or ATP levels can also be varied to test whether movement or localization of the poly(A) RNA is dependent on metabolic energy.