Double stranded RNA formation leads to preferential nuclear export and gene expression

mRNAs are transcribed and processed in the nucleus before they are exported into the cytoplasm for translation. Export is mediated by the export receptor heterodimer Mex67-Mtr2 in yeast (TAP-p15 in humans). Interestingly, also many lncRNAs leave the nucleus but it is currently unclear why they travel into the cytoplasm. Here we show that antisense (as)RNAs accelerate mRNA export by annealing with their sense counterparts through the helicase Dbp2. These double-stranded (ds)RNAs dominate export compared to single-stranded (ss)RNA, as they have a higher capacity and affinity for the export receptor Mex67. In this way, asRNAs boost gene expression, which is beneficial for cells. This is particularly important upon expression program changes. Consequently, the degradation or prevention of the formation of dsRNA is toxic for cells. This mechanism illuminates the general cellular occurrence of asRNAs and explains their nuclear export. Overall design: Total RNA was isolated from yeast with Trizol. From the total RNA, 90µg RNA was then incubated with 3µg J2 antibody and G-sepharose beads for 120 min at 4°C. The resulting supernatant was clarified a second time with the J2 antibody. The supernatant from the second round was sequenced as the unbound fraction. The J2 eluate from the first precipitation was, after repeated washing, purified with Trizol and sequenced as the J2-Eluate. 3 replicates are included.

信使RNA（mRNA）需在细胞核内完成转录与加工，随后被转运至细胞质进行翻译。酵母的核输出过程由输出受体异二聚体Mex67-Mtr2介导，人类中对应的同源受体为TAP-p15。值得关注的是，诸多长链非编码RNA（lncRNAs）同样可从细胞核转运至细胞质，但目前学界尚未明确其跨核膜运输的具体调控机制。
本研究证实，反义RNA（asRNAs）可通过解旋酶Dbp2介导与对应正义RNA链退火结合，进而加速mRNA的核输出过程。相较于单链RNA（ssRNA），双链RNA（dsRNA）与输出受体Mex67具有更高的结合容量与亲和力，因此在核输出过程中占据主导优势。通过这一途径，反义RNA可增强基因表达水平，对细胞存活具有积极意义，这一效应在细胞基因表达程序发生改变时尤为关键。反之，降解双链RNA或阻碍其形成则会对细胞产生毒性作用。该机制不仅阐明了反义RNA在细胞内的普遍存在性，同时解释了其核输出的分子机理。
整体实验设计：采用Trizol试剂从酵母细胞中提取总RNA。取90μg总RNA，与3μg J2抗体及琼脂糖凝胶G微珠（G-sepharose beads）于4℃下共孵育120分钟。将所得上清液再次使用J2抗体进行纯化以去除杂质，第二轮纯化后的上清液作为未结合组分进行高通量测序。首次免疫沉淀所得的J2洗脱液经反复洗涤后，采用Trizol试剂纯化，作为J2洗脱组分进行高通量测序。本实验共设置3次生物学重复。