Genome-Wide Profiling of Uncapped mRNA

Abstract

Gene transcripts are under extensive posttranscriptional regulation, including the regulation of their stability. A major route for mRNA degradation produces uncapped mRNAs, which can be generated by decapping enzymes, endonucleases, and small RNAs. Profiling uncapped mRNA molecules is important for the understanding of the transcriptome, whose composition is determined by a balance between mRNA synthesis and degradation. In this chapter, we describe a method to profile these uncapped mRNAs at the genome scale.

Tudor Staphylococcal Nuclease Links Formation of Stress Granules and Processing Bodies with mRNA Catabolism in Arabidopsis

“…To address this possibility, we analyzed the mRNA degradome of wild-type (Col and Ler) and tsn1 tsn2 seedlings grown under heat stress (39°C for 40 min) and control (23°C) conditions. Uncapped mRNA fractions composed of intermediates of the 59→39 decay pathway were purified and used for measuring the abundance of individual mRNAs by using cDNA arrays (Jiao et al, 2008;Jiao and Riechmann, 2012). Before microarray hybridization, a control experiment confirmed that there was no contamination from intact 59 cap mRNAs in our uncapped mRNA preparations (Jiao et al, 2008) (Supplemental Figure 8).…”

“…So the highest value in all samples became the mean of the highest values, the second highest value became the mean of the second highest values, and so on. Before microarray hybridization, the size distribution of cRNA samples from positive and control experiments, with and without T4 RNA ligase, respectively, was evaluated using an Agilent 2100 bioanalyzer (Jiao and Riechmann, 2012). A method described previously was used to define when a probe was to be considered as a positive signal .…”

“…Two independent sets of biological samples were used for the experiments. Uncapped mRNA purification was performed as described previously, using T4 RNA ligase (Ambion) to add an RNA adaptor to the uncapped mRNA (Jiao et al, 2008;Jiao and Riechmann, 2012). Uncapped mRNA was reverse transcribed, and then second-strand cDNA (double-stranded DNA [dsDNA]-uncapped) was synthesized using a specific primer as described previously (Jiao and Riechmann, 2012).…”

“…Uncapped mRNA purification was performed as described previously, using T4 RNA ligase (Ambion) to add an RNA adaptor to the uncapped mRNA (Jiao et al, 2008;Jiao and Riechmann, 2012). Uncapped mRNA was reverse transcribed, and then second-strand cDNA (double-stranded DNA [dsDNA]-uncapped) was synthesized using a specific primer as described previously (Jiao and Riechmann, 2012). The dsDNA samples along with total mRNA samples were sent to OakLabs, where total mRNA samples were reverse transcribed and the second-strand cDNA (dsDNAtotal) was synthesized using Agilent's LIQA kit.…”

Genome-wide analysis of uncapped mRNAs under heat stress in Arabidopsis

“…Total mRNA was isolated using RNeasy mini kit (Qiagen) following the manufacturer's instructions. Uncapped mRNA purification was performed as previously described, using a method based on the RNA ligase-mediated 5′ rapid amplification of cDNA ends (RLM 5´-RACE) [3] , [4] . Briefly, T4 RNA ligase (Ambion) was used to add an RNA adaptor to poly(a) + RNA having a free 5′ monophosphate (uncapped mRNAs).…”