Effects of local mRNA structure on posttranscriptional gene silencing
Effects of local mRNA structure on posttranscriptional gene silencing
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DOI：
10.1073/pnas.0805781105
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发表时间：
2008-09-16
影响因子：
11.1
通讯作者：
Gewirtz, Alan M.
中科院分区：
文献类型：
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作者：
Rudnick, Stephen I.;Swaminathan, Jyothishmathi;Gewirtz, Alan M.
反义寡脱氧核苷酸（AON）和短干扰RNA（siRNA）通过与mRNA杂交然后指导其切割来实现转录后基因沉默（PTGS）。为了理解mRNA结构对AON-与siRNA-介导的PTGS的约束，在果蝇胚胎全细胞裂解物中监测AON-和siRNA-介导的限定mRNA结构的切割。我们观察到，AON引导的切割比用siRNA引导相同靶位点的切割快约3倍。此外，并且出乎意料地，发现AON介导的切割对于靶序列可及性（如通过未配对核苷酸的存在所测量的）比相应的siRNA少得多的挑剔。尽管如此，在体内，siRNA沉默其mRNA靶标的效率至少是相应AON的2倍。这些看似矛盾的结果表明，额外的，尚未确定的因素在体内调节PTGS效率发挥重要作用。我们使用了一种定义明确的RNA结合蛋白α CP及其相应的高亲和力RNA结合位点来探索这一假设。我们发现，预结合的α CP有效地阻止AON介导的切割的RNA结合位点相比，在α CP的情况下，该网站的切割。我们的结论是，由RNA和结合蛋白形成的高阶结构在确定AON指导的PTGS的效率中起着重要作用。我们推测，旨在去除RNA结合蛋白的策略可能会显着改善AON介导的PTGS在体内。
Antisense oligodeoxynucleotides (AONs) and short interfering RNAs (siRNAs) effect posttranscriptional gene silencing (PTGS) by hybridizing to an mRNA and then directing its cleavage. To understand the constraints that mRNA structure imposes on AON- vs. siRNA-mediated PTGS, AON- and siRNA-mediated cleavage of defined mRNA structures was monitored in Drosophila embryo whole-cell lysates. We observed that AON-directed cleavage was approximate to 3-fold faster than cleavage with a siRNA directed to the same target site. Furthermore, and unexpectedly, AON-mediated cleavage was found to be much less fastidious with respect to target sequence accessibility, as measured by the presence of unpaired nueleotides, than a corresponding siRNA. Nonetheless, in vivo, siRNAs silenced their mRNA target at least 2-fold more efficiently than the corresponding AON. These seemingly contradictory results suggested that additional, as yet undefined factors play an important role in regulating PTGS efficiency in vivo. We used a well defined RNA-binding protein, alpha CP, and its corresponding high-affinity RNA-binding site to explore this hypothesis. We found that prebound alpha CP effectively blocked AON-mediated cleavage of the RNA-binding site compared with cleavage of the site in the absence of alpha CP. We conclude that higher-order structures formed by RNA and bound proteins play an important role in determining the efficiency of AON-directed PTGS. We hypothesize that strategies aimed at removing RNA-binding proteins might significantly improve AON-mediated PTGS in vivo.