Functional Genomics Unit, Institut Pasteur de Montevideo, Montevideo 11400, Uruguay.
Nuclear Research Center, Faculty of Science, Universidad de la República, Montevideo 11400, Uruguay.
Nucleic Acids Res. 2018 Sep 28;46(17):9081-9093. doi: 10.1093/nar/gky495.
We have previously shown that 5' halves from tRNAGlyGCC and tRNAGluCUC are the most enriched small RNAs in the extracellular space of human cell lines, and especially in the non-vesicular fraction. Extracellular RNAs are believed to require protection by either encapsulation in vesicles or ribonucleoprotein complex formation. However, deproteinization of non-vesicular tRNA halves does not affect their retention in size-exclusion chromatography. Thus, we considered alternative explanations for their extracellular stability. In-silico analysis of the sequence of these tRNA-derived fragments showed that tRNAGly 5' halves can form homodimers or heterodimers with tRNAGlu 5' halves. This capacity is virtually unique to glycine tRNAs. By analyzing synthetic oligonucleotides by size exclusion chromatography, we provide evidence that dimerization is possible in vitro. tRNA halves with single point substitutions preventing dimerization are degraded faster both in controlled nuclease digestion assays and after transfection in cells, showing that dimerization can stabilize tRNA halves against the action of cellular nucleases. Finally, we give evidence supporting dimerization of endogenous tRNAGlyGCC 5' halves inside cells. Considering recent reports have shown that 5' tRNA halves from Ala and Cys can form tetramers, our results highlight RNA intermolecular structures as a new layer of complexity in the biology of tRNA-derived fragments.
我们之前已经表明，tRNAGlyGCC 和 tRNAGluCUC 的 5' 片段是人类细胞系细胞外空间中最丰富的小 RNA，尤其是非囊泡部分。细胞外 RNA 被认为需要通过囊泡包裹或核糖核蛋白复合物形成来保护。然而，非囊泡 tRNA 片段的去蛋白处理并不影响它们在排阻层析中的保留。因此，我们考虑了它们在细胞外稳定性的替代解释。对这些 tRNA 衍生片段的序列进行计算机分析表明，tRNAGly 的 5' 片段可以与 tRNAGlu 的 5' 片段形成同源二聚体或异源二聚体。这种能力实际上是甘氨酸 tRNA 所独有的。通过排阻层析分析合成寡核苷酸，我们提供了体外二聚化可能的证据。不能形成二聚体的单点取代 tRNA 片段在受控核酸酶消化测定和转染细胞后更快降解，表明二聚化可以稳定 tRNA 片段免受细胞核酸酶的作用。最后，我们提供了支持细胞内内源性 tRNAGlyGCC 5' 片段二聚化的证据。考虑到最近的报道表明 Ala 和 Cys 的 5' tRNA 片段可以形成四聚体，我们的结果强调了 RNA 分子间结构作为 tRNA 衍生片段生物学的一个新的复杂性层次。
