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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. 

Inflammation and oxidative stress in pancreatic islets amplify the appearance of various posttranslational modifications to self-proteins. In this study, we identified a select group of carbonylated islet proteins arising before the onset of hyperglycemia in NOD mice. Of interest, we identified carbonyl modification of the prolyl-4-hydroxylase β subunit (P4Hb) that is responsible for proinsulin folding and trafficking as an autoantigen in both human and murine type 1 diabetes. We found that carbonylated P4Hb is amplified in stressed islets coincident with decreased glucose-stimulated insulin secretion and altered proinsulin-to-insulin ratios. Autoantibodies against P4Hb were detected in prediabetic NOD mice and in early human type 1 diabetes prior to the onset of anti-insulin autoimmunity. Moreover, we identify autoreactive CD4+ T-cell responses toward carbonyl-P4Hb epitopes in the circulation of patients with type 1 diabetes. Our studies provide mechanistic insight into the pathways of proinsulin metabolism and in creating autoantigenic forms of insulin in type 1 diabetes. 

Abstract Rapid permafrost degradation and peatland expansion occurred in Eurasia during the Early Holocene and may be analogous to the region’s response to anthropogenic warming. Here we present a 230 Th-dated, multiproxy speleothem record with subdecadal sampling resolution from Kyok-Tash Cave, at the modern permafrost margin in the northern Altai Mountains, southwestern Siberia. Stalagmite K4, covering the period 11,400 to 8,900 years before present, indicates an absence of stable permafrost within three centuries of the Younger Dryas termination. Between 11,400 and 10,400 years ago, speleothem δ 18 O is antiphased between the Altai and Ural ranges, suggesting a reorganization of the westerly wind systems that led to warmer and wetter winters over West Siberia and Altai, relative to the zonally adjacent regions of Northern Eurasia. At the same time, there is evidence of peak permafrost degradation and peatland expansion in West Siberia, consistent with the interpreted climate anomaly. Based on these findings, we suggest that modern permafrost in Eurasia is sensitive to feedbacks in the ocean-cryosphere system, which are projected to alter circulation regimes over the continent. 

Abstract Background Genotype independent transformation and whole plant regeneration through somatic embryogenesis relies heavily on the intrinsic ability of a genotype to regenerate. The critical genetic architecture of non-embryogenic callus (NEC) cells and embryogenic callus (EC) cells in a highly regenerable cotton genotype is unknown. Results In this study, gene expression profiles of a highly regenerable Gossypium hirsutum L. cultivar, Jin668, were analyzed at two critical developmental stages during somatic embryogenesis, non-embryogenic callus (NEC) cells and embryogenic callus (EC) cells. The rate of EC formation in Jin668 is 96%. Differential gene expression analysis revealed a total of 5333 differentially expressed genes (DEG) with 2534 genes upregulated and 2799 genes downregulated in EC. A total of 144 genes were unique to NEC cells and 174 genes were unique to EC. Clustering and enrichment analysis identified genes upregulated in EC that function as transcription factors/DNA binding, phytohormone response, oxidative reduction, and regulators of transcription; while genes categorized in methylation pathways were downregulated. Four key transcription factors were identified based on their sharp upregulation in EC tissue; LEAFY COTYLEDON 1 (LEC1), BABY BOOM (BBM), FUSCA (FUS3) and AGAMOUS-LIKE15 with distinguishable subgenome expression bias. Conclusions This comparative analysis of NEC and EC transcriptomes gives new insights into the genes involved in somatic embryogenesis in cotton.