As nucleic acid-guided endonucleases,some prokaryotic Argonautes have been used as programmable nucleases.Natronobacterium gregoryi Argonaute(NgAgo)has also been proposed for gene editing,but this remains very controv...As nucleic acid-guided endonucleases,some prokaryotic Argonautes have been used as programmable nucleases.Natronobacterium gregoryi Argonaute(NgAgo)has also been proposed for gene editing,but this remains very controversial.Until now,the endogenous nucleic acids that bind to NgAgo in Natronobacterium gregoryi sp2(N.gregoryi sp2)have not been characterized.We expressed the conserved PIWI domain of NgAgo and used it to induce anti-PIWI antibody.We also cultured the N.gregoryi sp2 strain and performed immunoprecipitation,chromatin immunoprecipitation(ChIP),and RNA immunoprecipitation(RIP)assays.The nucleic acids that endogenously bound NgAgo in N.gregoryi sp2 cells were sequenced and analyzed.The results showed that NgAgo endogenously bound RNA rather than DNA.NgAgo-associated RNAs were mainly transcripts of genes that encoded tRNA,transcriptional regulators,RNA polymerases,and RNA-binding proteins.NgAgo mainly binds to the transcripts inside genes or in their upstream sequences.Interestingly,the top enriched motif of peaks was the same as that of miR-1289,suggesting that NgAgo may regulate gene expression post-transcriptionally.GO enrichment analysis showed that the peak-associated genes were enriched in transmembrane transport processes.These results revealed that NgAgo binds RNA and may function in post-transcriptional regulation in vivo.展开更多
Plastid biogenesis and the coordination of plastid and nuclear genome expression through anterograde and retrograde signaling are essential for plant development.GENOMES UNCOUPLED1(GUN1)plays a central role in retrogr...Plastid biogenesis and the coordination of plastid and nuclear genome expression through anterograde and retrograde signaling are essential for plant development.GENOMES UNCOUPLED1(GUN1)plays a central role in retrograde signaling during early plant development.The putative function of GUN1 has been extensively studied,but its molecular function remains controversial.Here,we evaluate published transcriptome data and generate our own data from gun1 mutants grown under signaling-relevant condi-tions to show that editing and splicing are not relevant for GUN1-dependent retrograde signaling.Our study of the plastid(post)transcriptome of gun1 seedlings with white and pale cotyledons demonstrates that GUN1 deficiency significantly alters the entire plastid transcriptome.By combining this result with a penta-tricopeptide repeat code-based prediction and experimental validation by RNA immunoprecipitation ex-periments,we identified several putative targets of GUN1,including tRNAs and RNAs derived from ycf1.2,rpoC1,and rpoC2 and the ndhH–ndhA–ndhI–ndhG–ndhE–psaC–ndhD gene cluster.The absence of plastid rRNAs and the significant reduction of almost all plastid transcripts in white gun1 mutants ac-count for the cotyledon phenotype.Our study provides evidence for RNA binding and maturation as the long-sought molecular function of GUN1 and resolves long-standing controversies.We anticipate that ourfindings will serve as a basis for subsequent studies on mechanisms of plastid gene expression and will help to elucidate the function of GUN1 in retrograde signaling.展开更多
基金The datasets generated during the current study are available in the Sequence Read Archive(SRA)repository under the accession number PRJNA720376(BioProject)GenBank under the accession number PKKI00000000.
文摘As nucleic acid-guided endonucleases,some prokaryotic Argonautes have been used as programmable nucleases.Natronobacterium gregoryi Argonaute(NgAgo)has also been proposed for gene editing,but this remains very controversial.Until now,the endogenous nucleic acids that bind to NgAgo in Natronobacterium gregoryi sp2(N.gregoryi sp2)have not been characterized.We expressed the conserved PIWI domain of NgAgo and used it to induce anti-PIWI antibody.We also cultured the N.gregoryi sp2 strain and performed immunoprecipitation,chromatin immunoprecipitation(ChIP),and RNA immunoprecipitation(RIP)assays.The nucleic acids that endogenously bound NgAgo in N.gregoryi sp2 cells were sequenced and analyzed.The results showed that NgAgo endogenously bound RNA rather than DNA.NgAgo-associated RNAs were mainly transcripts of genes that encoded tRNA,transcriptional regulators,RNA polymerases,and RNA-binding proteins.NgAgo mainly binds to the transcripts inside genes or in their upstream sequences.Interestingly,the top enriched motif of peaks was the same as that of miR-1289,suggesting that NgAgo may regulate gene expression post-transcriptionally.GO enrichment analysis showed that the peak-associated genes were enriched in transmembrane transport processes.These results revealed that NgAgo binds RNA and may function in post-transcriptional regulation in vivo.
基金Deutsche Forschungsgemeinschaft to C.S.-L.,D.L.,and T.K.(TRR175,projects A02,C01,and C05)Research in the Hua laboratory was supported by a US NSF CAREER award(MCB-1750361).
文摘Plastid biogenesis and the coordination of plastid and nuclear genome expression through anterograde and retrograde signaling are essential for plant development.GENOMES UNCOUPLED1(GUN1)plays a central role in retrograde signaling during early plant development.The putative function of GUN1 has been extensively studied,but its molecular function remains controversial.Here,we evaluate published transcriptome data and generate our own data from gun1 mutants grown under signaling-relevant condi-tions to show that editing and splicing are not relevant for GUN1-dependent retrograde signaling.Our study of the plastid(post)transcriptome of gun1 seedlings with white and pale cotyledons demonstrates that GUN1 deficiency significantly alters the entire plastid transcriptome.By combining this result with a penta-tricopeptide repeat code-based prediction and experimental validation by RNA immunoprecipitation ex-periments,we identified several putative targets of GUN1,including tRNAs and RNAs derived from ycf1.2,rpoC1,and rpoC2 and the ndhH–ndhA–ndhI–ndhG–ndhE–psaC–ndhD gene cluster.The absence of plastid rRNAs and the significant reduction of almost all plastid transcripts in white gun1 mutants ac-count for the cotyledon phenotype.Our study provides evidence for RNA binding and maturation as the long-sought molecular function of GUN1 and resolves long-standing controversies.We anticipate that ourfindings will serve as a basis for subsequent studies on mechanisms of plastid gene expression and will help to elucidate the function of GUN1 in retrograde signaling.
