Project description:The RECQL5 helicase is crucial for maintaining genome stability and functions at the intersection of transcription, replication, and DNA repair. RECQL5 associates with RNA15 polymerase II (Pol II) to modulate transcription elongation, yet the underlying mechanism remains unclear. Here, we report cryo-electron microscopy structures of human RECQL5 bound to multiple transcription elongation complexes. Combined with biochemical analysis, we identified an a-helix of RECQL5 responsible for Pol II binding and inhibition of transcription elongation. We further reveal that the transcription-coupled DNA repair (TCR) complex20 reactivates the transcription of RECQL5-inhibited Pol II through concerted actions of its translocase activity and competition with the RECQL5 helix for engaging Pol II. Our results suggest a model in which RECQL5 and TCR coordinately regulate the transcription elongation rate to ensure transcription efficiency while maintaining genome stability.
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:Splice-switching antisense oligonucleotides (ASOs) and engineered U7 small nuclear ribonucleoprotein (U7 Sm OPT) are the most commonly used methods for exon skipping. However, challenges remain, such as limited organ delivery and repeated dosing for ASOs and unknown risks of by-products produced by U7 Sm OPT. Here, we showed that antisense circular RNAs (AS-circRNAs) can effectively mediate exon skipping in both minigene and endogenous transcripts. We also showed a relatively higher exon skipping efficiency at the tested Dmd minigene than U7 Sm OPT. AS-circRNA specifically targets the precursor mRNA splicing without off-target effects. Moreover, AS-circRNAs with adeno-associated virus (AAV) delivery corrected the open reading frame and restored the dystrophin expression in a mouse model of Duchenne muscular dystrophy. In conclusion, we develop an alternative method for regulating RNA splicing, which might be served as a novel tool for genetic disease treatment.
Project description:Rationale: Circular RNA (circRNA) therapeutics hold great promise as an iteration strategy in messenger RNA (mRNA) therapeutics due to their inherent stability and durable protein translation capability. Nevertheless, the efficiency of RNA circularization remains a significant constraint, particularly in establishing large-scale manufacturing processes for producing highly purified circRNAs. Hence, it is imperative to develop a universal and more efficient RNA circularization system when considering synthetic circRNAs as therapeutic agents with prospective clinical applications. Methods: We initially developed a chimeric RNA circularization system based on the original permuted intron-exon (PIE) and subsequently established a high-performance liquid chromatography (HPLC) method to obtain highly purified circRNAs. We then evaluated their translational ability and immunogenicity. The circRNAs expressing human papillomavirus (HPV) E7 peptide (43-62aa) and dimerized receptor binding domain (dRBD) from SARS-CoV-2 were encapsulated within lipid nanoparticles (LNPs) as vaccines, followed by an assessment of the in vivo efficacy through determination of antigen-specific T and B cell responses, respectively. Results: We have successfully developed a universal chimeric permuted intron-exon system (CPIE) through engineering of group I self-splicing introns derived from Anabaena pre-tRNALeu or T4 phage thymidylate (Td) synthase gene. Within CPIE, we have effectively enhanced RNA circularization efficiency. By utilizing size exclusion chromatography, circRNAs were effectively separated, which exhibit low immunogenicity and sustained potent protein expression property. In vivo data demonstrate that the constructed circRNA vaccines can elicit robust immune activation (B cell and/or T cell responses) against tumor or SARS-CoV-2 and its variants in mouse models. Conclusions: Overall, we provide an efficient and universal system to synthesize circRNA in vitro, which has extensive application prospect for circRNA therapeutics.
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.