Characterization of endogenous nucleic acids that bind to NgAgo in Natronobacterium gregoryi sp2 cells

Lixu Jiang,Bifang He,Chunchao Pu,Zixin Wang,Jian Huang,Lin Ning

doi:10.32604/biocell.2022.016500

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.

Highlights

Eukaryotic Argonautes bind small RNAs and use them as guides for sequence-specific recognition of long RNA targets in a process known as RNA interference (RNAi) (Niaz, 2018)
PIWI expression and purification The pBBR-Tac-Natronobacterium gregoryi Argonaute (NgAgo) plasmid was kindly provided by Chengdu Renhao Biological Technology Co., Ltd., China Recombinant plasmid construction and PIWI domain expression were performed by Hangzhou HuaAn Biotechnology Co., Ltd., China
On SDS–PAGE, PIWI migrated to a position indicative of a mass of approximately 38 kDa (Fig. 2)

Summary

Introduction

Eukaryotic Argonautes (eAgos) bind small RNAs and use them as guides for sequence-specific recognition of long RNA targets in a process known as RNA interference (RNAi) (Niaz, 2018). Many prokaryotic Agos (pAgos) contain divergent variants of conserved nucleic acid interaction domains, as well as extra domains that are absent from eAgos. This suggests that pAgos may have unusual specificities in nucleic acid recognition and transcription inhibition or cleavage (Ryazansky et al, 2018). The N-terminal and PAZ (PIWIArgonaute-Zwille) domains form one lobe, and the MID (middle) and PIWI (P-element Induced Wimpy Testis) domains form the other lobe. The MID and PAZ domains interact with the 5’ and 3’ ends of small nucleic acid guides. The PIWI domain contains an RNase H-like fold with a catalytic tetrad

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