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缺乏功能性CCA尾的全长转运RNA(tRNA)被选择性地分选到细胞外囊泡的腔内。

Full-length tRNAs lacking a functional CCA tail are selectively sorted into the lumen of extracellular vesicles.

作者信息

Scheepbouwer Chantal, Aparicio-Puerta Ernesto, Gómez-Martin Cristina, van Eijndhoven Monique A J, Drees Esther E E, Bosch Leontien, de Jong Daphne, Wurdinger Thomas, Zijlstra Josée M, Hackenberg Michael, Gerber Alan, Pegtel D Michiel

机构信息

Department of Neurosurgery, Cancer Center Amsterdam, Amsterdam University Medical Center, VU University, Amsterdam, Netherlands.

Cancer Center Amsterdam, Cancer Biology, Amsterdam, Netherlands.

出版信息

bioRxiv. 2024 May 12:2024.05.12.593148. doi: 10.1101/2024.05.12.593148.

Abstract

Small extracellular vesicles (sEVs) are heterogenous lipid membrane particles typically less than 200 nm in size and secreted by most cell types either constitutively or upon activation signals. sEVs isolated from biofluids contain RNAs, including small non-coding RNAs (ncRNAs), that can be either encapsulated within the EV lumen or bound to the EV surface. EV-associated microRNAs (miRNAs) are, despite a relatively low abundance, extensively investigated for their selective incorporation and their role in cell-cell communication. In contrast, the sorting of highly-structured ncRNA species is understudied, mainly due to technical limitations of traditional small RNA sequencing protocols. Here, we adapted ALL-tRNAseq to profile the relative abundance of highly structured and potentially methylated small ncRNA species, including transfer RNAs (tRNAs), small nucleolar RNAs (snoRNAs), and Y RNAs in bulk EV preparations. We determined that full-length tRNAs, typically 75 to 90 nucleotides in length, were the dominant small ncRNA species (>60% of all reads in the 18-120 nucleotides size-range) in all cell culture-derived EVs, as well as in human plasma-derived EV samples, vastly outnumbering 21 nucleotides-long miRNAs. Nearly all EV-associated tRNAs were protected from external RNAse treatment, indicating a location within the EV lumen. Strikingly, the vast majority of luminal-sorted, full-length, nucleobase modification-containing EV-tRNA sequences, harbored a dysfunctional 3' CCA tail, 1 to 3 nucleotides truncated, rendering them incompetent for amino acid loading. In contrast, in non-EV associated extracellular particle fractions (NVEPs), tRNAs appeared almost exclusively fragmented or 'nicked' into tRNA-derived small RNAs (tsRNAs) with lengths between 18 to 35 nucleotides. We propose that in mammalian cells, tRNAs that lack a functional 3' CCA tail are selectively sorted into EVs and shuttled out of the producing cell, offering a new perspective into the physiological role of secreted EVs and luminal cargo-selection.

摘要

小细胞外囊泡(sEVs)是异质性的脂质膜颗粒,大小通常小于200nm,由大多数细胞类型组成性分泌或在激活信号作用下分泌。从生物流体中分离出的sEVs含有RNA,包括小非编码RNA(ncRNAs),这些RNA可以包裹在囊泡腔内或与囊泡表面结合。尽管细胞外囊泡相关的微小RNA(miRNAs)丰度相对较低,但因其选择性掺入及其在细胞间通讯中的作用而受到广泛研究。相比之下,高度结构化的ncRNA种类的分选研究较少,主要是由于传统小RNA测序方案的技术限制。在这里,我们采用全tRNA测序来分析高度结构化和潜在甲基化的小ncRNA种类的相对丰度,包括转运RNA(tRNAs)、小核仁RNA(snoRNAs)和Y RNAs在大量囊泡制剂中的情况。我们确定,全长tRNAs通常长度为75至90个核苷酸,是所有细胞培养来源的囊泡以及人血浆来源的囊泡样本中占主导地位的小ncRNA种类(在18 - 120个核苷酸大小范围内的所有读数中>60%),其数量远远超过21个核苷酸长的miRNAs。几乎所有与囊泡相关的tRNAs都能抵御外部RNA酶处理,表明其位于囊泡腔内。令人惊讶的是,绝大多数腔内分选的、全长的、含核碱基修饰的囊泡 - tRNA序列都有一个功能失调的3' CCA尾,截短了1至3个核苷酸,使其无法进行氨基酸加载。相比之下,在非囊泡相关的细胞外颗粒部分(NVEPs)中,tRNAs几乎只出现片段化或“切口”成18至35个核苷酸长度的tRNA衍生小RNA(tsRNAs)。我们提出,在哺乳动物细胞中,缺乏功能性3' CCA尾的tRNAs被选择性地分选到囊泡中并运出产生细胞,这为分泌囊泡和腔内货物选择的生理作用提供了新的视角。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/efab/11100784/f13f2096380b/nihpp-2024.05.12.593148v1-f0001.jpg

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