Nicked tRNAs are stable reservoirs of tRNA halves in cells and biofluids.
Nicked tRNAs are stable reservoirs of tRNA halves in cells and biofluids.
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DOI：
10.1073/pnas.2216330120
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发表时间：
2023-01-24
影响因子：
11.1
通讯作者：
Tosar, Juan Pablo
中科院分区：
文献类型：
--
作者：
Costa, Bruno;Li Calzi, Marco;Castellano, Mauricio;Blanco, Valentina;Cuevasanta, Ernesto;Litvan, Irene;Ivanov, Pavel;Witwer, Kenneth;Cayota, Alfonso;Tosar, Juan Pablo
tRNA衍生的小RNA（tDR）在多个水平上调节基因表达。一些tDR在人类生物流体中是丰富的，其中它们主要存在于细胞外囊泡之外。这提出了一个有趣的问题：细胞外tDR如何抵抗细胞外RNA酶的降解。在这项工作中，我们通过发现一些最常检测到的细胞外tDR是全长tRNA的切口形式，因此是dsRNA分子来解决这个谜团。有切口的tRNA不能通过标准分子生物学和/或测序技术进行研究，因为它们是不连续的。然而，我们开发了几种在天然条件下分析有切口的tRNA的方法，并显示了它们在细胞和生物流体中的存在。我们还发现了一个潜在的细胞间通讯途径介导的稳定的非囊泡RNA。非囊泡细胞外RNA（nv-exRNA）构成了细胞外RNA组的大部分，但对其稳定性、功能和作为疾病生物标志物的潜在用途知之甚少。在此，我们测量了几种裸RNA在人血清、尿液和脑脊液（CSF）中孵育时的稳定性。我们鉴定了细胞外产生的tRNA衍生的小RNA（tDR），其在CSF中的半衰期为数小时。与广泛的假设相反，这些内在稳定的小RNA是含有断裂的磷酸二酯键的全长tRNA（即，有切口的tRNA）。标准分子生物学方案，包括基于苯酚的RNA提取和加热，诱导有切口的tRNA的人工变性和随后的tDR的体外产生。断裂的键是逆转录酶的障碍，阻止了在其天然状态下的带切口的tRNA的扩增和/或测序。为了解决这个问题，我们利用噬菌体和细菌tRNA修复系统的内在活性，对在天然条件下纯化的切口tRNA进行酶修复。酶修复在北方印迹中再生了RNA酶R抗性tRNA大小的条带，并使全长tRNA的RT-PCR扩增成为可能。我们还在天然条件下通过色谱方法从tDR中分离出有切口的tRNA，鉴定了应激细胞内和囊泡耗尽的人类生物流体中的有切口的tRNA。有切口的tRNA的解离产生单链tDR，其可以被人上皮细胞自发地摄取，将稳定的nv-exRNA定位为细胞间通讯途径中的潜在相关参与者。
tRNA-derived small RNAs (tDRs) regulate gene expression at multiple levels. Some tDRs are abundant in human biofluids, where they are mostly present outside extracellular vesicles. This poses the intriguing question about how extracellular tDRs resist degradation by extracellular RNases. In this work, we solved this mystery by finding out that some of the most frequently detected extracellular tDRs are nicked forms of full-length tRNAs and are therefore dsRNA molecules. Nicked tRNAs cannot be studied by standard molecular biology and/or sequencing techniques because they are discontinuous. However, we developed several methods for the analysis of nicked tRNAs under native conditions and showed their presence in cells and biofluids. We also uncovered a potential intercellular communication pathway mediated by stable nonvesicular RNAs. Nonvesicular extracellular RNAs (nv-exRNAs) constitute the majority of the extracellular RNAome, but little is known about their stability, function, and potential use as disease biomarkers. Herein, we measured the stability of several naked RNAs when incubated in human serum, urine, and cerebrospinal fluid (CSF). We identified extracellularly produced tRNA-derived small RNAs (tDRs) with half-lives of several hours in CSF. Contrary to widespread assumptions, these intrinsically stable small RNAs are full-length tRNAs containing broken phosphodiester bonds (i.e., nicked tRNAs). Standard molecular biology protocols, including phenol-based RNA extraction and heat, induce the artifactual denaturation of nicked tRNAs and the consequent in vitro production of tDRs. Broken bonds are roadblocks for reverse transcriptases, preventing amplification and/or sequencing of nicked tRNAs in their native state. To solve this, we performed enzymatic repair of nicked tRNAs purified under native conditions, harnessing the intrinsic activity of phage and bacterial tRNA repair systems. Enzymatic repair regenerated an RNase R-resistant tRNA-sized band in northern blot and enabled RT-PCR amplification of full-length tRNAs. We also separated nicked tRNAs from tDRs by chromatographic methods under native conditions, identifying nicked tRNAs inside stressed cells and in vesicle-depleted human biofluids. Dissociation of nicked tRNAs produces single-stranded tDRs that can be spontaneously taken up by human epithelial cells, positioning stable nv-exRNAs as potentially relevant players in intercellular communication pathways.
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影响因子：
16
作者：
Mateescu B;Kowal EJ;van Balkom BW;Bartel S;Bhattacharyya SN;Buzás EI;Buck AH;de Candia P;Chow FW;Das S;Driedonks TA;Fernández-Messina L;Haderk F;Hill AF;Jones JC;Van Keuren-Jensen KR;Lai CP;Lässer C;Liegro ID;Lunavat TR;Lorenowicz MJ;Maas SL;Mäger I;Mittelbrunn M;Momma S;Mukherjee K;Nawaz M;Pegtel DM;Pfaffl MW;Schiffelers RM;Tahara H;Théry C;Tosar JP;Wauben MH;Witwer KW;Nolte-'t Hoen EN
通讯作者：
Nolte-'t Hoen EN
影响因子：
14.9
作者：
Chen C;Ridzon DA;Broomer AJ;Zhou Z;Lee DH;Nguyen JT;Barbisin M;Xu NL;Mahuvakar VR;Andersen MR;Lao KQ;Livak KJ;Guegler KJ
通讯作者：
Guegler KJ
应激诱导的 tRNA 衍生 RNA：原始真核生物兰氏贾第鞭毛虫中的一类新型小 RNA。
影响因子：
14.9
作者：
Li Y;Luo J;Zhou H;Liao JY;Ma LM;Chen YQ;Qu LH
通讯作者：
Qu LH
DOI：
10.1093/plcell/koac043
发表时间：
2022-04-26
期刊：
The Plant cell
影响因子：
--
作者：
通讯作者：
--
影响因子：
5
作者：
Akiyama Y;Lyons SM;Fay MM;Tomioka Y;Abe T;Anderson PJ;Ivanov P
通讯作者：
Ivanov P