Conserved regulation of ZC3H14/Nab2 in circRNA biogenesis

Circular RNAs (circRNAs) are natural outputs of eukaryotic transcription and RNA processing and have emerged as critical regulators in physiology and human diseases. Although a series of cis-elements and trans-factors are reported to globally regulate circRNA biogenesis, most of these regulations are unconserved or at most mammalian conserved. Here, using a genome-wide CRISPR knockout screen, we have identified an evolutionarily conserved polyadenosine RNA-binding protein ZC3H14 to regulate circRNA biogenesis in eukaryotes. Mechanistically, ZC3H14 associates with spliceosome to bind to 3' and 5' exon-intron boundaries of circularized exons, and forms a dimer to promote circRNA biogenesis. ZC3H14 knockout mice exhibit disrupted spermatogenesis and reduced testicular circRNA levels. Additionally, human ZC3H14 is associated with male infertility. Our findings reveal a conserved requirement for ZC3H14/Nab2 in the modulation of eukaryotic backsplicing and link ZC3H14 along with circRNAs to mammalian testicular disorders. Overall design: To investigate direct and specific regulators of backsplicing, we set out to establish a circReporter system with an unbiased genome-wide CRISPR-Cas9 knockout screen, and identified an evolutionarily conserved polyadenosine-binding protein ZC3H14 to regulate circRNA biogenesis. Then, we performed circRNA profiles in human HEK293 cells or mouse testes when ZC3H14 was knocked down or knockout. We also performed the individual-nucleotide resolution crosslink immunoprecipitation (iCLIP) followed by RNA-seq of the endogenous ZC3H14 in HEK293 cells in duplicates and in mouse germ cells Please note that each processed data file was generated from 2-3 samples together as indicated in the corresponding sample description field.