G-quadruplex is involved in the regulation of BmSGF1 expression in the Silkworm, Bombyx mori

Advanced DNA structures,such as the G-quadruplex(G4)and the i-motif,are widely but not randomly present in the genomes of many organisms.A G4 structure was identified in the promoter of the silk gland factor-1 gene(SGFI),which is the main regulatory gene for silk production in Bombyx mori.In this study,a BmSGF1 G4-/-ho-mozygous mutant was generated with the G4 sequence knocked out.The promoter activity of BmSGF1 was lowered in the BmSGF1 G4-/-mutant.Pyridostatin(PDS)stabilized the G4 structure and increased the promoter activity of BmSGF1,whereas anti-sense oligonu-cleotide(ASO)complementary to the G4 sequence suppressed the promoter activity of BmSGF1.Compared with wild-type larvae,the deletion of the BmSGF1 G4 structure de-creased both the expression of BmSGF1 and the fibroin heavy chain gene BmFib-H in the posterior silk gland and the weight of the cocoons.Overall,these results suggest that the promoter G4 structure of BmSGFI participates in the transcription regulation of the BmSGFl gene in the silkworm.

Identification of binding domains and key amino acids involved in the interaction between BmLARK and G4 structure in the BmPOUM2 promoter in Bombyx mori

It has been found that the non-B form DNA structures,like G-quadruplex(G4)and i-motif,are involved in many important biological processes.Our previous study showed that the silkworm transcription factor BmLARK binds to the G4 structure in the promoter of the transcription factor BmPOUM2 and regulates its promoter activity.However,the binding mechanism between BmLARK and BmPOUM2 G4 structure remains unclear.In this study,binding domains and key amino acid residues involved in the interaction between BmLARK and BmPOUM2 G4 were studied.The electrophoretic mobility shift assay results indicated that the two RNA-recognition motifs(RRM)of BmLARK are simultaneously required for the binding with the G4 structure.Either RRM1 or RRM2 alone could not bind with the G4 structure.The zinc-finger motif was not involved in the binding.A series of mutant proteins with specific amino acid mutations were expressed and used to identify the key amino acid residues involving the interaction.The results indicated thatβsheets,especially theβ1 andβ3 sheets,in the RRM domains of BmLARK played critical roles in the binding with the G4 structure.Several amino acid mutations of RRM1/2 in ribonucleoprotein domain 1(RNP1)(motif inβ3 strand)and RNP2(motif inβ1 strand)caused loss of binding ability,indicating that these amino acids are the key sites for the binding.All the results suggest that RRM domains,particularly their the RNP1 and RNP2 motifs,play important roles not only in RNA recognition,but also in the G4 structure binding.