Phosphorylation controls mRNA retention via nuclear speckle cohesion - cellular fractionation data
Nuclear speckles are membraneless organelles that associate with active transcription sites and participate in post-transcriptional mRNA processing. During mitosis, nuclear speckles dissolve following phosphorylation of their protein components. Here, we identify PP1 phosphatases as responsible for counteracting kinase-mediated dissolution. Their overexpression increases speckle cohesion and leads to retention of polyadenylated RNA within speckles and the nucleus. By performing APEX2 proximity labeling combined with RNA-sequencing, we characterized the association of specific RNAs with nuclear speckles depending on their cohesion state. We find that many transcripts are preferentially enriched within nuclear speckles compared to the nucleoplasm, particularly chromatin- and nucleus-associated transcripts. While total polyadenylated RNA retention increased with greater nuclear speckle cohesion, the ratios of most mRNA species to each other were constant, indicating non-selective, or proportional, retention. We then explored whether nuclear speckle cohesion changes in response to environmental perturbations associated with changes in kinase or phosphatase activity. We found that cellular responses to heat shock, oxidative stress, and hypoxia include changes to the cohesion of nuclear speckles and mRNA retention. Our results demonstrate that tuning the material properties of nuclear speckles provides a mechanism for the acute control of mRNA localization. Overall design: Human induced pluripotent stem cells were fractionated into chromatin, nuclear and cytoplasmic fractions using a protocol adapted from Mayer & Churchman, 2017. Five replicates were collected.