An advanced protocol for profiling RNA-binding proteins in Arabidopsis using plant phase extraction DOI Creative Commons
Yong Zhang, Ye Xu, Todd H. Skaggs

et al.

Biology Methods and Protocols, Journal Year: 2023, Volume and Issue: 8(1)

Published: Jan. 1, 2023

Abstract RNA-binding proteins (RBPs) are key players in regulating cell fate and essential developmental processes. Systematic profiling of the proteome (RBPome) is thus indispensable for researchers aiming to understand mechanisms post-transcriptional gene regulation. RBPome identification methods developed humans, mice, bacteria have successfully identified RBPomes these organisms. However, biochemical genetic complexities plant tissues greatly hindered effectiveness plants. Moreover, RBPs been predominantly discovered through oligo d(T) based affinity purification (RNA-interactome capture). Since polyadenylated RNA only accounts less than 5% total population eukaryotic cells, there a pressing need develop comprehensive, yet unbiased, method capture full spectrum Here, we describe detailed protocol Plant Phase Extraction (PPE), recently identify Arabidopsis (Zhang Y, Xu Skaggs TH, et al. phase extraction: enhanced discovery its dynamics Cell 2023; 35: 2750–72.) [1]. The PPE enables efficient enrichment both poly(A) non-poly(A) from various quickly reproducibly. Most importantly, allows unveiling dynamic RBP–RNA interactions under abiotic biotic stress conditions during different stages. This provides much broader more accurate understanding RBPs, marking significant advancement molecular biology.

Language: Английский

An advanced protocol for profiling RNA-binding proteins in Arabidopsis using plant phase extraction DOI Creative Commons
Yong Zhang, Ye Xu, Todd H. Skaggs

et al.

Biology Methods and Protocols, Journal Year: 2023, Volume and Issue: 8(1)

Published: Jan. 1, 2023

Abstract RNA-binding proteins (RBPs) are key players in regulating cell fate and essential developmental processes. Systematic profiling of the proteome (RBPome) is thus indispensable for researchers aiming to understand mechanisms post-transcriptional gene regulation. RBPome identification methods developed humans, mice, bacteria have successfully identified RBPomes these organisms. However, biochemical genetic complexities plant tissues greatly hindered effectiveness plants. Moreover, RBPs been predominantly discovered through oligo d(T) based affinity purification (RNA-interactome capture). Since polyadenylated RNA only accounts less than 5% total population eukaryotic cells, there a pressing need develop comprehensive, yet unbiased, method capture full spectrum Here, we describe detailed protocol Plant Phase Extraction (PPE), recently identify Arabidopsis (Zhang Y, Xu Skaggs TH, et al. phase extraction: enhanced discovery its dynamics Cell 2023; 35: 2750–72.) [1]. The PPE enables efficient enrichment both poly(A) non-poly(A) from various quickly reproducibly. Most importantly, allows unveiling dynamic RBP–RNA interactions under abiotic biotic stress conditions during different stages. This provides much broader more accurate understanding RBPs, marking significant advancement molecular biology.

Language: Английский

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