The Biogenesis And Function Of CircRNAs

Circular RNAs(circRNAs)are covalently closed,single-stranded transcripts,produced from back-splicing of exons of precursor RNA.With the improvement of next-generation RNA sequencing and development of algorithms for circRNA discovery,over ten thousand different circRNAs have been identified among a variety of species,such as fruit fly,mouse and human.However,it remains largely unknown about the regulation and functions of the majority of circRNAs.Back-splicing can be modulated by intronic complementary sequences flanking circularized exons.Although having the same cis-elements,expression levels of circRNAs from the same loci are diverse in different cell lines and tissues,suggesting the involvement of trans-factors in circRNA expression regulation.To identify proteins that could potentially affect circRNA expression,we applied a genome-wide si RNA screening that targets all human unique genes with an efficient circRNA expression reporter.And we identified 103 RNA binding proteins(RBPs)potentially involved in regulation of circRNA expression,including multiple factors related with host immune responses,such as the human interleukin enhancer binding factor 3(ILF3).The ILF3 gene produces several isoforms of nuclear factor 90(NF90/NF110)due to alternative splicing and alternative polyadenylation.Furthermore,we demonstrated that NF90 and NF110,which both contain two double-stranded RNA-binding domains(ds RBDs),could promote circRNA production by stabilizing intronicRNA pairs in the nucleus and that they were also components of circRNPs in the cytoplasm.Upon viral infection,the production of circRNAs decreased due to the export of NF90 and NF110 to the cytoplasm,which was important for the NF90 and NF110 to be released from circRNP complexes,allowing their binding to viral m RNAs for prompt immune response.How circRNAs are degraded? Their covalently closed conformation prevents linear RNA decay machinery from accessible for circRNAs.We found that circRNAs were stable in normal situation,but they underwent rapid turnover by endonuclease RNase L upon poly(I:C)stimulation or virus infection.In normal cells,circRNAs preferred to bind nucleic acid receptors with direct antiviral activity,especially the double-stranded RNA(ds RNA)-activated protein kinase R(PKR).SHAPE-Ma P assays revealed that endogenous circRNAs tended to form 16-26 bp imperfect RNA duplexes,which allowed the structure-dependent binding of circRNAs to PKR.Furthermore,we found that rapid turnover of circRNAs by RNase L facilitated PKR activation upon virus infection.In addition,global reduction of circRNA expression and abnormal activation of PKR were observed in systemic lupus erythematosus(SLE)-derived primary cells,suggesting that mis-regulation of circRNA-PKR is linked to SLE.In sum,by studying circRNA regulation and functions,these results together reveal a previously unknown regulation and function of the mysterious circRNAs in viral infection and autoimmune diseases.