Biochemical Requirements for Two Dicer-Like Activities from Wheat Germ

RNA silencing pathways were first discovered in plants. Through genetic analysis, it has been established that the key silencing components called Dicer-like (DCL) genes have been shown to cooperatively process RNA substrates of multiple origin into distinct 21, 22 and 24 nt small RNAs. However, only few detailed biochemical analysis of the corresponding complexes has been carried out in plants, mainly due to the large unstable complexes that are hard to obtain or reconstitute in heterologous systems. Reconstitution of activity needs thorough understanding of all protein partners in the complex, something that is still an ongoing process in plant systems. Here, we use biochemical analysis to uncover properties of two previously identified native dicer-like activities from wheat germ. We find that standard wheat germ extract contains Dicer-like enzymes that convert double-stranded RNA (dsRNA) into two classes of small interfering RNAs of 21 and 24 nt in size. The 21 nt dicing activity, likely an siRNA producing complex known as DCL4, is 950 kDa-1.2 mDa in size and is highly unstable during purification processes but has a rather vast range for activity. On the contrary, the 24 nt dicing complex, likely the DCL3 activity, is relatively stable and comparatively smaller in size, but has stricter conditions for effective processing of dsRNA substrates. While both activities could process completely complementary dsRNA albeit with varying abilities, we show that DCL3-like 24 nt producing activity is equally good in processing incompletely complementary RNAs.

RNA沉默通路（RNA silencing pathways）最初在植物中被发现。经遗传分析证实，核心沉默元件——类Dicer（DCL）基因可协同加工多种来源的RNA底物，生成长度分别为21、22与24 nt的特异性小RNA。然而，目前针对植物来源此类对应复合物的详细生化研究仍较为匮乏，这主要是因为该类复合物分子量庞大且稳定性较差，难以从异源表达系统中获取或重构。要重构该复合物的活性，需完整解析复合物内所有蛋白伴侣的组成，而这一工作在植物研究领域仍在持续推进。
本研究通过生化分析，解析了此前在小麦胚芽中发现的两种天然类Dicer酶活性的特性。我们发现，常规小麦胚芽提取物中含有类Dicer酶，可将双链RNA（dsRNA）切割为两类长度分别为21 nt与24 nt的小干扰RNA（small interfering RNAs, siRNA）。其中，21 nt切割活性对应的复合物（即已知的生成siRNA的DCL4复合物），分子量介于950 kDa至1.2 mDa之间，在纯化过程中稳定性极差，但活性适用范围较广。与之相对，24 nt切割复合物（推测为DCL3活性复合物）稳定性相对更强，分子量也更小，但对双链RNA底物的有效加工条件更为严苛。尽管两类活性均可加工完全互补的双链RNA，只是加工效率存在差异，但本研究证实，类DCL3的24 nt生成活性，在加工不完全互补的RNA时同样表现优异。