Rapid depletion and super-resolution microscopy reveal an unexpected role of the nuclear-speckle protein SRSF5 in paraspeckle assembly and dynamics during cellular stress

Nuclear speckles (NS) and paraspeckles (PS) are adjacent condensates with distinct protein composition, with serine-arginine-rich splicing factors (SRSFs) concentrated in NS. Surprisingly, we find that SRSF5 is present in both. Combining super-resolution imaging, proximity proteomics and iCLIP, we show that SRSF5 binds with PS core proteins to the PS-scaffold RNA NEAT1 and locates between PS spheres. Acute SRSF5 depletion results in reduced PS with differently packaged NEAT1. Under stress, SRSF5's association with PS increases, and without SRSF5, PS cluster assembly is impaired. Interfering with binding to purine-rich RNAs even causes PS-NS fusion. In an intriguing over-compensation, longer SRSF5 depletion reduces TDP-43 levels via premature polyadenylation, leading to NEAT1 isoform switching and more PS. We propose that SRSF5 forms a stress-specific PS shell and acts as a glue for PS clusters. Additionally, we uncover SRSF5 as a novel regulator of TDP-43 and demonstrate how acute depletion distinguishes direct from compensatory effects.