Small RNA populations revealed by blocking rRNA fragments in Drosophila melanogaster reproductive tissues.
Small RNA populations revealed by blocking rRNA fragments in Drosophila melanogaster reproductive tissues.
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DOI：
10.1371/journal.pone.0191966
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发表时间：
2018
期刊：
影响因子：
3.7
通讯作者：
Chapman T
中科院分区：
文献类型：
--
作者：
Fowler EK;Mohorianu I;Smith DT;Dalmay T;Chapman T
RNA干扰（RNA interference, RNAi）是一种复杂且高度保守的由小RNA介导的调控机制。最近高通量测序技术的进步使得对特定身体部位和组织的sRNA丰度和谱的分析越来越详细。这使得研究微小rna （miRNAs）和小干扰rna （sirna）的局部作用成为可能。然而，在被比较的样本中，非编码rna比例的变化可能会阻碍这些分析。特定组织中存在的较长非编码RNA（如核糖体RNA或转移RNA）片段的比例可能存在显著差异，这可能反映了组织特异性生物功能的差异。例如，在果蝇中，一些组织含有丰富的30nt rRNA片段（2S rRNA）以及丰富的5 ‘和3 ’端rRNA片段。这些可能会给sRNA文库的构建带来困难，因为它们会淹没测序空间并模糊sRNA丰度。在这里，我们解决了这一问题，并提出了一种改进的“rRNA阻断”协议，用于在黑颊龙生殖组织中构建高清（HD）适配器sRNA库。结果显示，被阻断寡核苷酸靶向的2S rnas的总reads数从80%减少到0.01%。此外，使用多个rRNA阻断寡核苷酸结合最丰富的rRNA片段，使我们能够以更高的分辨率揭示潜在的sRNA群体。对阻断和未阻断样品的测序文库进行对比，结果显示，rRNA阻断并没有改变存在的miRNA群体，而是增强了它们的丰度。我们认为，这种rRNA阻断程序有可能改善对不同组织内和组织间差异表达的sRNAs的深入分析。
RNA interference (RNAi) is a complex and highly conserved regulatory mechanism mediated via small RNAs (sRNAs). Recent technical advances in high throughput sequencing have enabled an increasingly detailed analysis of sRNA abundances and profiles in specific body parts and tissues. This enables investigations of the localized roles of microRNAs (miRNAs) and small interfering RNAs (siRNAs). However, variation in the proportions of non-coding RNAs in the samples being compared can hinder these analyses. Specific tissues may vary significantly in the proportions of fragments of longer non-coding RNAs (such as ribosomal RNA or transfer RNA) present, potentially reflecting tissue-specific differences in biological functions. For example, in Drosophila, some tissues contain a highly abundant 30nt rRNA fragment (the 2S rRNA) as well as abundant 5’ and 3’ terminal rRNA fragments. These can pose difficulties for the construction of sRNA libraries as they can swamp the sequencing space and obscure sRNA abundances. Here we addressed this problem and present a modified “rRNA blocking” protocol for the construction of high-definition (HD) adapter sRNA libraries, in D. melanogaster reproductive tissues. The results showed that 2S rRNAs targeted by blocking oligos were reduced from >80% to < 0.01% total reads. In addition, the use of multiple rRNA blocking oligos to bind the most abundant rRNA fragments allowed us to reveal the underlying sRNA populations at increased resolution. Side-by-side comparisons of sequencing libraries of blocked and non-blocked samples revealed that rRNA blocking did not change the miRNA populations present, but instead enhanced their abundances. We suggest that this rRNA blocking procedure offers the potential to improve the in-depth analysis of differentially expressed sRNAs within and across different tissues.
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影响因子：
3.7
作者：
Blenkiron C;Tsai P;Brown LA;Tintinger V;Askelund KJ;Windsor JA;Phillips AR
通讯作者：
Phillips AR
影响因子：
7.3
作者：
Henras, Anthony K.;Plisson-Chastang, Celia;O'Donohue, Marie-Francoise;Chakraborty, Anirban;Gleizes, Pierre-Emmanuel
通讯作者：
Gleizes, Pierre-Emmanuel
rRNA 衍生的 RNA 片段 (rRF) 的两个特征系列构成了一类新型小 RNA
影响因子：
3.7
作者：
Chen Z;Sun Y;Yang X;Wu Z;Guo K;Niu X;Wang Q;Ruan J;Bu W;Gao S
通讯作者：
Gao S
影响因子：
7
作者：
Stage, Deborah E.;Eickbush, Thomas H.
通讯作者：
Eickbush, Thomas H.
影响因子：
4.5
作者：
Campbell, Joshua D.;Liu, Gang;Lenburg, Marc E.
通讯作者：
Lenburg, Marc E.