Table_2_The in vivo dsRNA Cleavage Has Sequence Preference in Insects.XLSX

Exogenous dsRNA enters the insect body and can induce the RNAi effect only when it is cleaved into siRNA. However, what kinds of base composition are easier to cut and what kinds of siRNA will be produced in vivo is largely unknown. In this study, we found that dsRNA processing into siRNA has sequence preference and regularity in insects. We injected 0.04 mg/g dsRNA into Asian corn borers or cotton bollworms according to their body weight, and then the siRNAs produced in vivo were analyzed by RNA-Seq. We discovered that a large number of siRNAs were produced with GGU nucleotide residues at the 5′- and 3′-ends and produced a siRNA peak on the sequence. Once the GGU site is mutated, the number of siRNAs will decrease significantly and the siRNA peak will also lost. However, in the red flour beetle, a member of Coleoptera, dsRNA was cut at more diverse sites, such as AAG, GUG, and GUU; more importantly, these enzyme restriction sites have a high conservation base of A/U. Our discovery regarding dsRNA in vivo cleavage preference and regularity will help us understand the RNAi mechanism and its application.

外源性双链RNA（dsRNA）进入昆虫体内后，仅当被切割为小干扰RNA（siRNA）时，方可诱导RNA干扰（RNAi）效应。然而，究竟何种碱基组成的序列更易被切割，以及体内会生成何种类型的siRNA，目前仍未明确。本研究发现，昆虫体内dsRNA加工生成siRNA的过程存在序列偏好性与规律性。我们根据亚洲玉米螟与棉铃虫的体重，向其体内注射剂量为0.04 mg/g的dsRNA，随后通过RNA测序（RNA-Seq）对体内生成的siRNA进行分析。我们发现，大量生成的siRNA在5'端与3'端均带有GGU核苷酸残基，且在对应序列上呈现siRNA特征峰。一旦GGU位点发生突变，siRNA的生成量将显著下降，其特征峰也会消失。但在鞘翅目昆虫赤拟谷盗中，dsRNA的切割位点更为多样，涵盖AAG、GUG与GUU等序列；更为关键的是，这类酶切位点的碱基组成高度偏好A/U。本研究关于dsRNA体内切割偏好性与规律性的发现，将有助于我们深入理解RNAi的作用机制，并推动其相关应用。