Dynamics of RNA localization to nuclear speckles are connected to splicing efficiency [ARTR-seq]
Nuclear speckles, a type of membraneless nuclear organelle in higher eukaryotic cells, play a vital role in gene expression regulation. Using the reverse transcription-based RBP binding sites sequencing (ARTR-seq) method, we study human transcripts associated with nuclear speckles. We identify three gene groups whose transcripts demonstrate different speckle localization properties and dynamics – stably enriched in nuclear speckle post-transcriptionally, transiently enriched in speckles at the pre-mRNA stage co-transcriptionally, and not enriched in speckles. We show that nuclear speckles specifically facilitate splicing of speckle-enriched transcripts. We further reveal RNA sequence features contributing to transcript speckle localization, underscoring a tight interplay between genome organization, RNA cis-elements, and transcript speckle enrichment, and connecting transcript speckle localization with splicing logic. Finally, we show that speckles can act as hubs for the regulated retention of introns during cellular stress. Collectively, our data highlight a role of nuclear speckles in both co- and post-transcriptional splicing regulation. Overall design: We employed ARTR-seq, utilizing pAG-RTase, a fusion of Protein A/G and reverse transcriptase, to map the nuclear speckle-enriched transcriptome. This method combines Protein A/G's IgG binding domains to target IgG subclasses, facilitating binding to antibodies against nuclear speckle scaffold proteins (SON or SRRM2). Reverse transcription was initiated with exogenous biotin dNTPs, followed by enrichment of biotinylated cDNAs using streptavidin beads and library preparation for sequencing.