Profiling transcriptome composition and dynamics within nuclear compartments using SLAM-RT&Tag

Abstract

Nuclear compartments are membrane-less regions 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 and tagment (RT&Tag) to human cell lines to identify the transcript populations of Polycomb domains and nuclear speckles. We also developed SLAM-RT&Tag, which combines RNA metabolic labeling with RT&Tag, to quantify transcript dynamics within nuclear compartments. We observed unique transcript populations with differing structures and dynamics within each compartment. Intriguingly, exceptionally long genes are transcribed adjacent to Polycomb domains and are transiently associated with chromatin. By contrast, nuclear speckles act as quality control checkpoints that transiently confine incompletely spliced polyadenylated transcripts and facilitate their post-transcriptional splicing. In summary, we demonstrate that transcripts at Polycomb domains and nuclear speckles undergo distinct RNA processing mechanisms, highlighting the pivotal role of compartmentalization in RNA maturation. • Developed SLAM-RT&Tag to profile RNA dynamics within nuclear compartments • RNAs persistently associated with compartments, like XIST and MALAT1, are rare • Exceptionally long genes are transcribed adjacent to Polycomb domains • Incompletely spliced mature mRNAs transiently pass through nuclear speckles Khyzha et al. developed SLAM-RT&Tag, a kinetic labeling strategy for profiling RNA dynamics within nuclear compartments. Applied to Polycomb domains and nuclear speckles, they identified compartment-specific transcript populations with distinct structural features and demonstrated that most transcripts exhibit transient associations with these compartments.

Topics & Concepts