Abstract
High-throughput sequencing of ribosome footprints precisely maps and quantifies in vivo mRNA translation. The ribosome footprint sequencing has undergone continuing development since its original report. Here we provide a detailed protocol for construction of high-quality ribosome footprint library of rice. Rice total polysomes are isolated with a modified low ionic polysome extraction buffer. After nuclease digestion, rice ribosome footprints are extracted using SDS method followed by column purification. High-quality rice ribosome footprint library with peak reads of approximately 28-nucleotide (nt) length and strong 3-nt periodicity is constructed via key steps including rRNA depletion, end repair, 3’ adapter ligation, reverse transcription, circularization, PCR enrichment and several rounds of purification. Biological significance of rice ribosome footprint library is further revealed by the comparison of transcriptomic and translatomic responses to salt stress and the utilization for novel open reading frame (ORF) identification. This improved protocol for rice ribosome footprint library construction will facilitate the global comprehension and quantitative measurement of dynamic translation in rice.
Highlights
Translation of mRNAs, one of indispensable steps for gene expression, is directly associated with final proteome, which is profoundly involved in all aspects of cellular, physiological and developmental processes such as cell growth and division (Miettinen et al, 2019), organogenesis (Fujii et al, 2017), reproductivity (Sousa Martins et al, 2016), oncogenesis (Robichaud et al, 2019) and acclimation to variable environmental conditions (Merchante et al, 2017) in all kinds of organisms
Total polysomes are first isolated from tissues of interest such as rice seedling shoots, the monosomes that are derived from the nuclease-treated total polysomes are collected for isolation of ribosome footprints, which are subjected to rRNA trimming and library construction, and the final product is obtained for high-throughput sequencing followed by data analysis after PCR enrichment and PAGE purification of ribosome footprint library (Figure 1)
The functional upstream open reading frames (uORFs) have been reported in yeast under starvation condition (Ingolia et al, 2009) and Arabidopsis under phosphate deficiency (Bazin et al, 2017)
Summary
Translation of mRNAs, one of indispensable steps for gene expression, is directly associated with final proteome, which is profoundly involved in all aspects of cellular, physiological and developmental processes such as cell growth and division (Miettinen et al, 2019), organogenesis (Fujii et al, 2017), reproductivity (Sousa Martins et al, 2016), oncogenesis (Robichaud et al, 2019) and acclimation to variable environmental conditions (Merchante et al, 2017) in all kinds of organisms. High-quality rice ribosome footprint library with peak reads of about 28 nt length and strong 3-nt periodicity, two crucial characteristics superior to most ribosome footprint libraries constructed in Arabidopsis previously (Liu et al, 2013; Juntawong et al, 2014; Merchante et al, 2015; Bazin et al, 2017), is prepared by following key steps of rRNA depletion, end repair, 3’ adapter ligation, reverse transcription, circularization, PCR enrichment and several rounds of purification The generality of this protocol is confirmed by evaluating the length, 3-nt periodicity and gene body distribution of ribosome footprints in the libraries constructed with different rice cultivars under normal and salt stress conditions. We propose a series of key points, to which more attentions should be paid during library construction process
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