RNA localization to nuclear speckles follows splicing logic

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 spliceosomal 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 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. Overall design: To ensure that RNA transcribed from our synthetic constructs does not deviate from the intended sequence, we performed Nanopore full-length RNA sequencing after plasmid transfection. We applied unbiased transcriptome-wide sequencing without using any gene-specific primers, ensuring that even transcripts significantly deviating from the intended sequence (e.g., cryptic transcription start site, or cryptic splicing) will be captured.