A systematic analysis of circRNAs in subnuclear compartments.

CircRNAs are an important class of RNAs with diverse cellular functions in human physiology and disease. A thorough knowledge of circRNAs including their biogenesis and subcellular distribution is important to understand their roles in a wide variety of processes. However, the analysis of circRNAs from total RNA sequencing data remains challenging. Therefore, we developed Calcifer, a versatile workflow for circRNA annotation. Using Calcifer, we analysed APEX-Seq data to compare circRNA occurrence between whole cells, nucleus and subnuclear compartments. We generally find that circRNAs show higher abundance in whole cells compared to nuclear samples, consistent with their accumulation in the cytoplasm. The notable exception is the single-exon circRNA circCANX(9), which is unexpectedly enriched in the nucleus. In addition, we observe that circFIRRE prevails over the linear lncRNA FIRRE in both the cytoplasm and the nucleus. Zooming in on the subnuclear compartments, we show that circRNAs are strongly depleted from nuclear speckles, indicating that excess splicing factors in this compartment counteract back-splicing. Our results thereby provide valuable insights into the subnuclear distribution of circRNAs. Regarding circRNA function, we surprisingly find that the majority of all detected circRNAs possess complete open reading frames with potential for cap-independent translation. Overall, we show that Calcifer is an easy-to-use, versatile and sustainable workflow for the annotation of circRNAs which expands the repertoire of circRNA tools and allows to gain new insights into circRNA distribution and function.

Circular RNA circPLOD2 regulates pericyte function by targeting the transcription factor KLF4.

Circular RNAs are generated by backsplicing and control cellular signaling and phenotypes. Pericytes stabilize capillary structures and play important roles in the formation and maintenance of blood vessels. Here, we characterize hypoxia-regulated circular RNAs (circRNAs) in human pericytes and show that the circular RNA of procollagen-lysine,2-oxoglutarate 5-dioxygenase-2 (circPLOD2) is induced by hypoxia and regulates pericyte functions. Silencing of circPLOD2 affects pericytes and increases proliferation, migration, and secretion of soluble angiogenic proteins, thereby enhancing endothelial migration and network capability. Transcriptional and epigenomic profiling of circPLOD2-depleted cells reveals widespread changes in gene expression and identifies the transcription factor krüppel-like factor 4 (KLF4) as a key effector of the circPLOD2-mediated changes. KLF4 depletion mimics circPLOD2 silencing, whereas KLF4 overexpression reverses the effects of circPLOD2 depletion on proliferation and endothelial-pericyte interactions. Together, these data reveal an important function of circPLOD2 in controlling pericyte proliferation and capillary formation and show that the circPLOD2-mediated regulation of KLF4 significantly contributes to the transcriptional response to hypoxia.

Poison cassette exon splicing of SRSF6 regulates nuclear speckle dispersal and the response to hypoxia.

Hypoxia induces massive changes in alternative splicing (AS) to adapt cells to the lack of oxygen. Here, we identify the splicing factor SRSF6 as a key factor in the AS response to hypoxia. The SRSF6 level is strongly reduced in acute hypoxia, which serves a dual purpose: it allows for exon skipping and triggers the dispersal of nuclear speckles. Our data suggest that cells use dispersal of nuclear speckles to reprogram their gene expression during hypoxic adaptation and that SRSF6 plays an important role in cohesion of nuclear speckles. Down-regulation of SRSF6 is achieved through inclusion of a poison cassette exon (PCE) promoted by SRSF4. Removing the PCE 3' splice site using CRISPR/Cas9 abolishes SRSF6 reduction in hypoxia. Aberrantly high SRSF6 levels in hypoxia attenuate hypoxia-mediated AS and impair dispersal of nuclear speckles. As a consequence, proliferation and genomic instability are increased, while the stress response is suppressed. The SRSF4-PCE-SRSF6 hypoxia axis is active in different cancer types, and high SRSF6 expression in hypoxic tumors correlates with a poor prognosis. We propose that the ultra-conserved PCE of SRSF6 acts as a tumor suppressor and that its inclusion in hypoxia is crucial to reduce SRSF6 levels. This may prevent tumor cells from entering the metastatic route of hypoxia adaptation.