Characterization of endogenous nucleic acids that bind to NgAgo in Natronobacterium gregoryi sp2 cells
  • LIXU JIANG1, LIN NING2, CHUNCHAO PU1, ZIXIN WANG1, BIFANG HE1,3, JIAN HUANG1,*
1 School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, 611731, China
2 School of Healthcare Technology, Chengdu Neusoft University, Chengdu, 611844, China
3 School of Medicine, Guizhou University, Guiyang, 550025, China
* Address correspondence to: Jian Huang, hj@uestc.edu.cn
Received 11 March 2021; Accepted 16 April 2021 ; Published online 17 June 2021
Abstract
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.
Keywords
NgAgo, PIWI, Immunoprecipitation, ChIP-seq, RIP-seq, Post-transcriptional regulation