RNA-Seq of mRNA derived from fourth larval stage larvae from C. elegans strain PE1328 treated for 16 hours at 20 degrees Celsius with 5-Ph-IAA compared to untreated controls

The objective of the experiment was to determine the impact of cap-adjacent 2'-O-ribose methylation on steady-state mRNA levels in a background lacking the decapping exonculease EOL-1. We depleted CMTR-1 protein, which is the main enzyme responsible for of cap-adjacent 2'-O-ribose methylation in C. elegans. This was achieved using a strain that is homozygous for an auxin-degron allele of the endogenous cmtr-1 gene in genetic background that constitutively expressed TIR1(F74G) protein (cshIs140 allele derived from HML1012), and also for an eol-1(fe172[E197K]) mutation. The depletion was achieved using the auxin analogue 5-Ph-IAA. Experimental data consists of six replicates, and depletion of CMTR-1 was confirmed by Western blotting.

本实验旨在探究在缺失脱帽外切酶（decapping exonuclease）EOL-1的遗传背景中，帽邻近2'-O-核糖甲基化（cap-adjacent 2'-O-ribose methylation）对稳态mRNA水平的影响。我们耗竭了CMTR-1蛋白——该蛋白是秀丽隐杆线虫（C. elegans）中负责催化帽邻近2'-O-核糖甲基化的主要酶。本次实验所用的品系为双重纯合菌株：内源cmtr-1基因携带生长素降解标签等位基因，同时携带eol-1(fe172[E197K])突变；其遗传背景组成型表达TIR1(F74G)蛋白，且带有源自HML1012的cshIs140等位基因。我们通过生长素类似物5-Ph-IAA实现了CMTR-1的蛋白耗竭。本实验数据包含6次重复，且已通过蛋白质印迹法（Western blotting）验证了CMTR-1的耗竭效果。