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 t...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.展开更多
Insect cuticle is an apical extracellular matrix produced by the epidermis,tracheal,hind-and foregut epithelia during embryogenesis and renewed during molting and metamorphosis.However,the underlying regulatory mechan...Insect cuticle is an apical extracellular matrix produced by the epidermis,tracheal,hind-and foregut epithelia during embryogenesis and renewed during molting and metamorphosis.However,the underlying regulatory mechanism for embryonic cuticle formation remains largely unclear.Here,we investigate the function of the transcription factor POUM2 in the embryonic cuticular formation in Bombyx mori,a model lepidopteran insect.Clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein-9-mediated knockout of POUM2 resulted in the defect of cuticular deposition,pigmentation,and sclerotization in the embryos.Differentially expressed transcripts analysis of 7-d-old embryos identified 174 up-or downregulated cuticular protein transcripts,8 upregulated chitin degradation transcripts,2 downregulated chitin synthesis transcripts and 48 up-or downregulated transcription factor transcripts in the POUM2−/−embryos.The expression levels of the key factors of the tyrosine metabolic pathway,such as tyrosine hydroxylase(Th),Dopa decarboxylase(DDC),and arylalkylamine N-acetyltransferase(aaNAT),were significantly decreased in the POUM2−/−embryos.POUM2 isoform POUM2-L specifically bound the POU cis-regulatory element(CRE)in the Th promoter and increased the transcription of Th,whereas POUM2-S could not bind the POU CRE,although it also increased the transcription of Th.Heterogeneous nuclear ribonucleoprotein Squid-1 directly bound the POUM2 pre-mRNA(messenger RNA)and inhibited the alternative splicing of POUM2-L to POUM2-S mRNA.These results suggest that POUM2 participates in the cuticular formation by regulating the chitin and cuticular protein synthesis and metabolism,and the cuticular pigmentation and sclerotization by regulating tyrosine metabolism during embryogenesis.This study provides new insights into novel function of POUM2 in embryogenesis.展开更多
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(SG...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.展开更多
Endogenous retroviruses(ERVs),once thought to be mere remnants of ancient viral integrations in the mammalian genome,are now recognized for their critical roles in various physiological processes,including embryonic d...Endogenous retroviruses(ERVs),once thought to be mere remnants of ancient viral integrations in the mammalian genome,are now recognized for their critical roles in various physiological processes,including embryonic devel-opment,innate immunity,and tumorigenesis.Their impact on host organisms is significant driver of evolutionary changes,offering insight into evolutionary mechanisms.In our study,we explored the functionality of ERVs by exam-ining single-cell transcriptomic profiles from human embryonic stem cells and urine cells.This led to the discovery of a unique ERVH48-1 expression pattern between these cell types.Additionally,somatic cell reprogramming efficacy was enhanced when ERVH48-1 was overexpressed in a urine cell-reprogramming system.Induced pluripotent stem cells(iPSCs)generated with ERVH48-1 overexpression recapitulated the traits of those produced by traditional repro-gramming approaches,and the resulting iPSCs demonstrated the capability to differentiate into all three germ layers in vitro.Our research elucidated the role of ERVs in somatic cell reprogramming.展开更多
基金This work was supported by the grants of the Chinese National Natural Science Foundation(Grant no.:31672494,31720103916,31930102)。
文摘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.
基金supported by grants from the National Natural Science Foundation of China(No.31872969,No.31930102).
文摘Insect cuticle is an apical extracellular matrix produced by the epidermis,tracheal,hind-and foregut epithelia during embryogenesis and renewed during molting and metamorphosis.However,the underlying regulatory mechanism for embryonic cuticle formation remains largely unclear.Here,we investigate the function of the transcription factor POUM2 in the embryonic cuticular formation in Bombyx mori,a model lepidopteran insect.Clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein-9-mediated knockout of POUM2 resulted in the defect of cuticular deposition,pigmentation,and sclerotization in the embryos.Differentially expressed transcripts analysis of 7-d-old embryos identified 174 up-or downregulated cuticular protein transcripts,8 upregulated chitin degradation transcripts,2 downregulated chitin synthesis transcripts and 48 up-or downregulated transcription factor transcripts in the POUM2−/−embryos.The expression levels of the key factors of the tyrosine metabolic pathway,such as tyrosine hydroxylase(Th),Dopa decarboxylase(DDC),and arylalkylamine N-acetyltransferase(aaNAT),were significantly decreased in the POUM2−/−embryos.POUM2 isoform POUM2-L specifically bound the POU cis-regulatory element(CRE)in the Th promoter and increased the transcription of Th,whereas POUM2-S could not bind the POU CRE,although it also increased the transcription of Th.Heterogeneous nuclear ribonucleoprotein Squid-1 directly bound the POUM2 pre-mRNA(messenger RNA)and inhibited the alternative splicing of POUM2-L to POUM2-S mRNA.These results suggest that POUM2 participates in the cuticular formation by regulating the chitin and cuticular protein synthesis and metabolism,and the cuticular pigmentation and sclerotization by regulating tyrosine metabolism during embryogenesis.This study provides new insights into novel function of POUM2 in embryogenesis.
基金This research was supported by the National Natural Science Foundation of China(31930102,32250710148,32000337,32100383)Shaoguan University high-level talent research start-up funding project(432/9900064607)Research Projects of Shaoguan University(SY2023KJ03).
文摘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.
基金National Natural Science Foundation of China(32270574,92068201)Science and Technology Projects in Guangzhou(2023A03J0045)+1 种基金Guangzhou Key Laboratory of Biological Targeting Diagnosis and Therapy(202201020379)Key Laboratory of Guangdong Higher Education Institutes(2021KSYS009).
文摘Endogenous retroviruses(ERVs),once thought to be mere remnants of ancient viral integrations in the mammalian genome,are now recognized for their critical roles in various physiological processes,including embryonic devel-opment,innate immunity,and tumorigenesis.Their impact on host organisms is significant driver of evolutionary changes,offering insight into evolutionary mechanisms.In our study,we explored the functionality of ERVs by exam-ining single-cell transcriptomic profiles from human embryonic stem cells and urine cells.This led to the discovery of a unique ERVH48-1 expression pattern between these cell types.Additionally,somatic cell reprogramming efficacy was enhanced when ERVH48-1 was overexpressed in a urine cell-reprogramming system.Induced pluripotent stem cells(iPSCs)generated with ERVH48-1 overexpression recapitulated the traits of those produced by traditional repro-gramming approaches,and the resulting iPSCs demonstrated the capability to differentiate into all three germ layers in vitro.Our research elucidated the role of ERVs in somatic cell reprogramming.
