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Nuclear speckles are nuclear membraneless organelles in higher eukaryotic cells playing a vital role in gene expression. Using an in situ reverse transcription–based sequencing method, we study nuclear speckle–associated human transcripts. Our data indicate the existence of three gene groups whose transcripts demonstrate different speckle localization properties: stably enriched in nuclear speckles, transiently enriched in speckles at the pre–messenger RNA stage, and not enriched. We find that stably enriched transcripts contain inefficiently excised introns and that disruption of nuclear speckles specifically affects splicing of speckle-enriched transcripts. We further reveal RNA sequence features contributing to transcript speckle localization, indicating a tight interplay between transcript speckle enrichment, genome organization, and splicing efficiency. Collectively, our data highlight a role of nuclear speckles in both co- and posttranscriptional splicing regulation. Last, we show that genes with stably enriched transcripts are over-represented among genes with heat shock–up-regulated intron retention, hinting at a connection between speckle localization and cellular stress response.
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This content will become publicly available on May 28, 2026
RNA localization to nuclear speckles follows splicing logic
Summary Nuclear speckles are membraneless organelles implicated in multiple RNA processing steps. In this work, we systematically characterize the sequence logic determining RNA localization to nuclear speckles. We find extensive similarities between the speckle localization code and the RNA splicing code, even for transcripts that do not undergo splicing. Specifically, speckle localization is enhanced by the presence of unspliced exon-like or intron-like sequence features. We demonstrate that interactions required for early splicesomal complex assembly contribute to speckle localization. We also show that speckle localization of isolated endogenous exons is reduced by disease-associated single nucleotide variants. Finally, we find that speckle localization strongly correlates with splicing kinetics of splicing-competent constructs and is tightly linked to the decision between exon inclusion and skipping. Together, these results suggest a model in which RNA speckle localization is associated with the formation of the early spliceosomal complex and enhances the efficiency of splicing reactions. HighlightsSequences containing hallmarks of pre-mRNA dictate speckle localizationRNA speckle localization is coupled to early spliceosome assemblyDisease-associated single nucleotide variants reduce localization of isolated exonsRNA speckle localization strongly correlates with splicing kineticsGraphical Abstract
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- Award ID(s):
- 2246530
- PAR ID:
- 10597627
- Publisher / Repository:
- bioRxiv
- Date Published:
- Format(s):
- Medium: X
- Institution:
- bioRxiv
- Sponsoring Org:
- National Science Foundation
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