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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 

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. 

RNA molecules often play critical roles in assisting the formation of membraneless organelles in eukaryotic cells. Yet, little is known about the organization of RNAs within membraneless organelles. Here, using super-resolution imaging and nuclear speckles as a model system, we demonstrate that different sequence domains of RNA transcripts exhibit differential spatial distributions within speckles. Specifically, we image transcripts containing a region enriched in binding motifs of serine/arginine-rich (SR) proteins and another region enriched in binding motifs of heterogeneous nuclear ribonucleoproteins (hnRNPs). We show that these transcripts localize to the outer shell of speckles, with the SR motif-rich region localizing closer to the speckle center relative to the hnRNP motif-rich region. Further, we identify that this intra-speckle RNA organization is driven by the strength of RNA-protein interactions inside and outside speckles. Our results hint at novel functional roles of nuclear speckles and likely other membraneless organelles in organizing RNA substrates for biochemical reactions. 

Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current ‘state of the art’ from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of ‘soft recommendations’ about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage ‘open science’ practices.