Profiling transcriptome composition and dynamics within nuclear compartments using SLAM-RT&Tag
Nuclear compartments are membrane-less nuclear regions that are enriched in functionally related molecules. RNA is a major component of many nuclear compartments, but the identity and dynamics of transcripts within nuclear compartments are poorly understood. Here, we applied Reverse Transcribe & Tagment (RT&Tag) to identify the transcript populations of Polycomb domains and of nuclear speckles within the nucleus. We also developed SLAM-RT&Tag, which combines RNA metabolic labeling with RT&Tag, to quantify transcript dynamics within nuclear compartments. Intriguingly, exceptionally long genes are transcribed adjacent to Polycomb domains, and are transiently associated with chromatin. In contrast, diverse incompletely spliced mature transcripts migrate to nuclear speckles, and are generally transient within speckles. Speckle transcripts are retained longer when splicing is inhibited, suggesting post-transcriptional processing is coupled to speckle dynamics. Our findings argue that nuclear speckles act as quality control checkpoints that transiently confine incompletely spliced transcripts and facilitate their post-transcriptional splicing. Overall design: We developed SLAM-RT&Tag, which combines RNA metabolic labeling with RT&Tag, to quantify transcript dynamics within nuclear compartments.