Project description:Circular RNAs (circRNAs) are single-stranded RNAs that are joined head to tail with largely unknown functions. Here we show that transfection of purified in vitro generated circRNA into mammalian cells led to potent induction of innate immunity genes and confers protection against viral infection. The nucleic acid sensor RIG-I is necessary to sense foreign circRNA, and RIG-I and foreign circRNA co-aggregate in cytoplasmic foci. CircRNA activation of innate immunity is independent of a 5' triphosphate, double-stranded RNA structure, or the primary sequence of the foreign circRNA. Instead, self-nonself discrimination depends on the intron that programs the circRNA. Use of a human intron to express a foreign circRNA sequence abrogates immune activation, and mature human circRNA is associated with diverse RNA binding proteins reflecting its endogenous splicing and biogenesis. These results reveal innate immune sensing of circRNA and highlight introns-the predominant output of mammalian transcription-as arbiters of self-nonself identity.
Project description:Genome binding/occupancy profiling by high throughput sequencing | Expression profiling by high throughput sequencing | Other Mammalian nuclei contain Pol I, Pol II, and Pol III. However, to what extent and how they are cross-regulated remains elusive. Here, we performed orthogonal multi-omics profiling after acute degradation of the largest subunits of Pol I, Pol II, and Pol III, and showed that they mainly affect specific genes. In contrast, the loss of Pol I or Pol II causes few changes for other RNA polymerases and confirms those known. The changes of Pol II transcription after Pol III depletion are the largest among all the cross-regulatory types. Meta-analyses reveal that Pol III depletion increases nucleosome positioning, reduces the FACT complex occupancy, and perturbs Pol II elongation for nearby mRNA genes. Furthermore, the nucleosome positioning changes also underpinning the Pol II effects on Pol III-mediated tRNA transcription. Our results suggest that Pol III works together with Pol II to coordinate their transcription activities by maintaining local chromatin architecture.
Project description:Genome binding/occupancy profiling by high throughput sequencing | Expression profiling by high throughput sequencing | Other Mammalian nuclei contain Pol I, Pol II, and Pol III. However, to what extent and how they are cross-regulated remains elusive. Here, we performed orthogonal multi-omics profiling after acute degradation of the largest subunits of Pol I, Pol II, and Pol III, and showed that they mainly affect specific genes. In contrast, the loss of Pol I or Pol II causes few changes for other RNA polymerases and confirms those known. The changes of Pol II transcription after Pol III depletion are the largest among all the cross-regulatory types. Meta-analyses reveal that Pol III depletion increases nucleosome positioning, reduces the FACT complex occupancy, and perturbs Pol II elongation for nearby mRNA genes. Furthermore, the nucleosome positioning changes also underpinning the Pol II effects on Pol III-mediated tRNA transcription. Our results suggest that Pol III works together with Pol II to coordinate their transcription activities by maintaining local chromatin architecture.
Project description:Genome binding/occupancy profiling by high throughput sequencing | Expression profiling by high throughput sequencing | Other Mammalian nuclei contain Pol I, Pol II, and Pol III. However, to what extent and how they are cross-regulated remains elusive. Here, we performed orthogonal multi-omics profiling after acute degradation of the largest subunits of Pol I, Pol II, and Pol III, and showed that they mainly affect specific genes. In contrast, the loss of Pol I or Pol II causes few changes for other RNA polymerases and confirms those known. The changes of Pol II transcription after Pol III depletion are the largest among all the cross-regulatory types. Meta-analyses reveal that Pol III depletion increases nucleosome positioning, reduces the FACT complex occupancy, and perturbs Pol II elongation for nearby mRNA genes. Furthermore, the nucleosome positioning changes also underpinning the Pol II effects on Pol III-mediated tRNA transcription. Our results suggest that Pol III works together with Pol II to coordinate their transcription activities by maintaining local chromatin architecture.
Project description:Genome binding/occupancy profiling by high throughput sequencing | Expression profiling by high throughput sequencing | Other Mammalian nuclei contain Pol I, Pol II, and Pol III. However, to what extent and how they are cross-regulated remains elusive. Here, we performed orthogonal multi-omics profiling after acute degradation of the largest subunits of Pol I, Pol II, and Pol III, and showed that they mainly affect specific genes. In contrast, the loss of Pol I or Pol II causes few changes for other RNA polymerases and confirms those known. The changes of Pol II transcription after Pol III depletion are the largest among all the cross-regulatory types. Meta-analyses reveal that Pol III depletion increases nucleosome positioning, reduces the FACT complex occupancy, and perturbs Pol II elongation for nearby mRNA genes. Furthermore, the nucleosome positioning changes also underpinning the Pol II effects on Pol III-mediated tRNA transcription. Our results suggest that Pol III works together with Pol II to coordinate their transcription activities by maintaining local chromatin architecture.
Project description:The super elongation complex (SEC) contains the positive transcription elongation factor b (P-TEFb) and a subcomplex, ELL2-EAF1, which stimulates transcription elongation by RNA polymerase II (Pol II). Here we report the cryo-EM structure of ELL2-EAF1 bound to a Pol II elongation complex at 2.8 Å resolution. The ELL2-EAF1 dimerization module directly binds the Pol II lobe, explaining how SEC delivers P-TEFb to Pol II. The same site on the lobe also binds the initiation factor TFIIF, consistent with SEC binding only after the transition from transcription initiation to elongation. Structure-guided functional analysis shows that elongation stimulation requires the dimerization module and an ELL2 protein linker that tethers this module to the Pol II protrusion. Our results show that SEC stimulates elongation allosterically and indicate that this stimulation involves stabilization of a further closed conformation of the Pol II active center cleft.
Project description:Transcription-coupled DNA repair (TCR) removes transcription-blocking DNA lesions through the coordinated assembly of repair factors on arrested RNA polymerase II (Pol II). This process requires the site-specific ubiquitylation of Pol II by the CRL4CSA ubiquitin ligase. Pol II ubiquitylation is facilitated by ELOF1, a recently identified TCR factor. Using cryogenic electron microscopy, biochemical assays, and cell biology, we reveal that ELOF1 functions as an adaptor to correctly dock CRL4CSA onto Pol II and facilitate the recruitment of UVSSA. The ELOF1-CSA-UVSSA interaction positions CRL4CSA on arrested Pol II, leading to its ubiquitylation upon ligase activation by neddylation. ELOF1-mediated repositioning enables a TFIIS-like element in UVSSA to reach the Pol II active site and the pore region to prevent Pol II re-activation. These findings provide the structural and molecular basis underlying the transition of Pol II from a transcriptionally active to an arrested state, primed for DNA repair.
Project description:In plants, the plant-specific RNA polymerase V (Pol V) transcripts non-coding RNA and provides a docking platform for the association of accessory proteins in the RNA-directed DNA methylation (RdDM) pathway. Various components were uncovered in the process of DNA methylation, but it is still not clear how the transcription of Pol V is regulated. Here, we found the conserved elongation factor, SPT6L, bound to thousands of intergenic regions in an RNA polymerase II (Pol II) independent manner. The intergenic enrichment of SPT6L, interestingly, co-occupied with the largest subunit of Pol V (NRPE1) and the mutation of SPT6L led to the reduction of DNA methylation but not Pol V enrichment. Furthermore, the association of SPT6L at Pol V loci was dependent on the Pol V associated factor, SPT5L, rather than the presence of Pol V, and the interaction between SPT6L and NRPE1 was compromised in spt5l. Finally, Pol V RIP-seq revealed that SPT6L is required to maintain the amount and length of Pol V transcripts. Our findings revealed the critical role of a Pol II conserved elongation factor in Pol V mediated DNA methylation and transcription, and shed light on the mutual regulation between Pol V and Pol II in plants.
Project description:The conserved Ccr4-Not complex regulates all aspects of the RNA polymerase II (Pol II) gene expression pathway, while it also controls proteasome assembly and function. The Not4 RING domain ubiquitin ligase is a core subunit that also contains an RNA recognition motif (RRM) and C3H1 domain (collectively referred to as the RRM-C) whose function is unknown. Herein, we demonstrate that the Not4 RRM-C contributes to both Not4-dependent regulation of the proteasome and Pol II. Disruption of both Not4 ligase activity and the RRM-C domain simultaneously replicates the proteasome-dependent deubiquitylation and catalytic activity misregulation found in Not4-deficient cells. Transcriptome analysis reveals that the Not4 RRM-C affects a subset of Pol II-dependent genes involved in specific biological functions, including transcription elongation, cyclin-dependent kinase regulated nutrient responses, and ribosomal biogenesis. At these genes, the Not4 RRM-C negatively regulates Pol II binding. While Not4 RRM-C disruption modestly increases between Ccr4-Not and Pol II, a Not4 ligase mutant decreases Ccr4-Not association with Pol II, thus implicating Not4-dependent ubiquitylation in mediating Ccr4-Not interaction with Pol II. Collectively, our results suggests the cellular RNA environment may integrate control of global proteostasis and Pol II transcription through the Not4 ubiquitin ligase.