The SID-1 double-stranded RNA transporter is not selective for dsRNA length
The SID-1 double-stranded RNA transporter is not selective for dsRNA length
复制标题
DOI：
10.1261/rna.1286409
复制
发表时间：
2009-03-01
期刊：
影响因子：
4.5
通讯作者：
Hunter, Craig P.
中科院分区：
文献类型：
--
作者：
Shih, Joseph D.;Fitzgerald, Michael C.;Hunter, Craig P.
双链RNA（dsRNA）转运蛋白SID-1能够在秀丽隐杆线虫中实现系统性RNA干扰（RNAi），由此由局部暴露于dsRNA引发的沉默在整个动物及其后代中传播。以前，我们发现在表达C. elegans SID-1有效地触发RNAi。在这些实验中，长dsRNA被证明在沉默靶基因方面比短dsRNA显著更有效。在这里，我们表明这些细胞中积累了同等质量的长或短双链RNA，这表明大小依赖性沉默并不是由于通过SID-1的大小选择性转运造成的。此外，使用脉冲追踪dsRNA摄取实验，我们表明短dsRNA比长dsRNA积累得更快。我们发现，输入速率依赖于dsRNA浓度，与通过SID-1通道的能量独立的、扩散限制的运输一致。异源表达SID-1的果蝇S2细胞和原代培养的C. elegans细胞显示出相似的dsRNA浓度和大小依赖性，表明C.线虫调节蛋白不能显著增强或限制dsRNA通过SID-1的转运。最后，我们发现突变型SID-1与野生型SID-1在S2细胞中共表达干扰SID-1的功能，表明SID-1可能作为一个多聚体。
The double-stranded RNA (dsRNA) transport protein SID-1 enables systemic RNA interference (RNAi) in Caenorhabditis elegans, whereby silencing initiated by local exposure to dsRNA spreads throughout the animal and to its progeny. Previously, we showed that providing dsRNA in the growth medium of Drosophila S2 cells that express C. elegans SID-1 efficiently triggers RNAi. In these experiments long dsRNA proved to be significantly more effective than short dsRNA in silencing the target gene. Here, we show that equivalent masses of long or short dsRNA accumulate in these cells, indicating that size-dependent silencing is not due to size-selective transport through SID-1. Furthermore, using pulse-chase dsRNA uptake experiments, we show that short dsRNA accumulates more rapidly than long dsRNA. We found that import rates are dependent on dsRNA concentration, consistent with energy-independent, diffusion-limited transport through the SID-1 channel. Comparison of silencing efficiencies between Drosophila S2 cells heterologously expressing SID-1 and primary-cultured C. elegans cells shows similar dsRNA concentration and size dependencies, suggesting that C. elegans regulatory proteins do not measurably enhance or restrict dsRNA transport through SID-1. Finally, we find that coexpressing mutant SID-1 with wild-type SID-1 in S2 cells interferes with SID-1 function, indicating that SID-1 may function as a multimer.