RNA degradation in proliferating and differentiated C2C12 muscle precursor cells analyzed on Affymetrix exon arrays

Steady-state RNA levels are a result of RNA synthesis and degradation. The importance of transcription-factor mediated induction or repression of mRNA synthesis is well established, but the role and mechanisms of RNA degradation are less well understood. We globally evaluated the RNA decay rates in proliferating and differentiated mouse myoblasts on whole-genome Affymetrix exon arrays, allowing for the assessment of directionality of RNA degradation and the detection of splice variant-specific differences in RNA decay rates. We found large differences in decay rates. mRNAs coding for proteins involved in signal transduction and transcriptional regulation have shortest half lives, whereas mRNAs coding for DNA replication enzymes and muscle contraction proteins are among the most stable. Many genes differentially expressed between proliferating and differentiated myoblasts demonstrate major differences in RNA decay rates. Quantitative PCR experiments confirmed the higher stability of transcripts with increased expression levels. RNA degradation has no apparent preferential directionality. RNA degradation appears to affect the ratio of different splice variants. For example, Itga7 isoforms with higher abundance in differentiated than in proliferating cells are more stable in differentiated cells, despite the sharing of a common 3’ untranslated region. Thus, where it was previously thought that the abundance of different splice isoforms was mainly controlled by tissue-specific splicing factors, we now demonstrate that isoforms may be produced at comparable levels but degraded with different efficiencies, depending on the differentiation status of the cells. Our results indicate that control of RNA degradation rates contributes significantly to the differentiation stage-dependent differences in abundance of transcripts and splice variants. Keywords: total RNA expression profiling; cultured cells We analyzed proliferating C2C12 cells and C2C12 cells differentiated into myotubes by serum starvation (8 days after induction of differentiation). We analyzed 7 time points after addition of actionmycin D (0, 10, 20, 30, 60, 150, 480 minutes). There were two biological replicates per condition.

稳态RNA水平是RNA合成与降解共同作用的结果。转录因子介导的mRNA合成诱导或抑制的重要性已得到充分证实，但RNA降解的作用与机制仍有待深入阐明。我们利用全基因组Affymetrix外显子芯片（whole-genome Affymetrix exon arrays），对增殖与分化状态的小鼠成肌细胞的RNA降解速率进行了全局分析，借此可评估RNA降解的方向性，并检测不同剪接变体间的RNA降解速率差异。本研究发现降解速率存在显著差异：编码信号转导与转录调控相关蛋白的mRNA半衰期最短，而编码DNA复制酶及肌肉收缩蛋白的mRNA则是稳定性最高的一类。大量在增殖与分化成肌细胞间差异表达的基因，其RNA降解速率存在显著差异。定量聚合酶链反应（qPCR，Quantitative PCR）实验证实，表达水平上调的转录本稳定性更高。RNA降解未表现出明显的方向性偏好，且似乎会影响不同剪接变体的比例。例如，相较于增殖状态的细胞，在分化细胞中丰度更高的整合素α7（Itga7）剪接变体，尽管共享同一3’非翻译区（3’ untranslated region），但在分化细胞中的稳定性更强。因此，此前学界普遍认为不同剪接异构体的丰度主要由组织特异性剪接因子调控，而本研究证实，异构体的转录水平可能相近，但会根据细胞的分化状态以不同效率被降解。本研究结果表明，RNA降解速率的调控，对转录本与剪接变体丰度随分化阶段的差异变化具有重要贡献。关键词：总RNA表达谱；培养细胞
我们分析了增殖状态的C2C12细胞，以及通过血清饥饿诱导分化8天后形成肌管的C2C12细胞。我们在添加放线菌素D（actionmycin D）后的7个时间点（0、10、20、30、60、150、480分钟）进行了样本检测，每组实验设置2次生物学重复。