Nuclear speckles, membraneless organelles in higher eukaryotic cells, play a crucial role in gene expression. Using ARTR-seq, a reverse transcription-based sequencing method, the authors studied the transcriptome associated with nuclear speckles. They identified three groups of genes based on their transcript localization properties: those stably enriched in nuclear speckles, those transiently enriched in speckles at the pre-mRNA stage, and those not enriched in speckles. The study found that stably enriched transcripts contain inefficiently excised introns, and disruption of nuclear speckles specifically affects the splicing of these transcripts. The authors also revealed RNA sequence features contributing to transcript speckle localization, suggesting a tight interplay between transcript speckle enrichment, genome organization, and splicing efficiency. Additionally, they showed that genes with stably enriched transcripts are over-represented among those with heat shock-upregulated intron retention, indicating a connection between speckle localization and cellular stress response. The findings highlight the role of nuclear speckles in both co- and posttranscriptional splicing regulation.Nuclear speckles, membraneless organelles in higher eukaryotic cells, play a crucial role in gene expression. Using ARTR-seq, a reverse transcription-based sequencing method, the authors studied the transcriptome associated with nuclear speckles. They identified three groups of genes based on their transcript localization properties: those stably enriched in nuclear speckles, those transiently enriched in speckles at the pre-mRNA stage, and those not enriched in speckles. The study found that stably enriched transcripts contain inefficiently excised introns, and disruption of nuclear speckles specifically affects the splicing of these transcripts. The authors also revealed RNA sequence features contributing to transcript speckle localization, suggesting a tight interplay between transcript speckle enrichment, genome organization, and splicing efficiency. Additionally, they showed that genes with stably enriched transcripts are over-represented among those with heat shock-upregulated intron retention, indicating a connection between speckle localization and cellular stress response. The findings highlight the role of nuclear speckles in both co- and posttranscriptional splicing regulation.