LTR-Retrotransposon Control by tRNA-Derived Small RNAs
Authors: Andrea J Schorn
Chantal LeBlanc
Michael J Gutbrod
Rob Martienssen
Journal: Cell (2017)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: LTR-retrotransposon mobility; epigenetic reprogramming; mouse endogenous retroviruses; small RNA; tRNA fragments
Affiliations: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA; Watson School of Biological Sciences, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA; Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA; Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA; Watson School of Biological Sciences, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA. Electronic address: .

Abstract:  Transposon reactivation is an inherent danger in cells that lose epigenetic silencing during developmental reprogramming. In the mouse, long terminal repeat (LTR)-retrotransposons, or endogenous retroviruses (ERV), account for most novel insertions and are expressed in the absence of histone H3 lysine 9 trimethylation in preimplantation stem cells. We found abundant 18 nt tRNA-derived small RNA (tRF) in these cells and ubiquitously expressed 22 nt tRFs that include the 3' terminal CCA of mature tRNAs and target the tRNA primer binding site (PBS) essential for ERV reverse transcription. We show that the two most active ERV families, IAP and MusD/ETn, are major targets and are strongly inhibited by tRFs in retrotransposition assays. 22 nt tRFs post-transcriptionally silence coding-competent ERVs, while 18 nt tRFs specifically interfere with reverse transcription and retrotransposon mobility. The PBS offers a unique target to specifically inhibit LTR-retrotransposons, and tRF-targeting is a potentially highly conserved mechanism of small RNA-mediated transposon control. 

Retracted: No

PubPeer comments:
 Just read the new review by Cullen and Schorn in Nutrients (PMID: 32718022) concerning the activity of tRNA fragments on retrovirus activity. It is kind of curious that nobody has ever reproduced the findings reported by Schorn et al., 2017, especially in light of the great interest in the biological function of these small RNAs that are derived from tRNAs. Reading through the review it is striking that Cullen and Schorn re-reference the paper from 2017 over and over again while building great hypotheses based on their experimental evidence that transfection of tRNA fragments have a potent effect on the retrotransposition of active endogenous retroviruses (ERVs) from the mouse when expressed from plasmids in HeLa cells. What is curious, too, is that the amount of tRNA fragments in any cell type is quite low when compared to parental tRNA from which they (somehow) originate. How would a few tRNA fragments be able to block reverse transcription of millions of virus-derived RNAs? I went back to the material and methods in Schorn et al., 2017 in order to look a little closer to how they had derived at the notion that tRNA fragments block retrotransposition in their assays. And, I understand now that their assay is greatly artificial since they use a huge amount of small RNAs to interfere with reverse transcription and subsequent transposition of a plasmid DNA-derived viral RNA. Here is what I found: Retrotransposition Assays 2.5x 10^5 HeLa cells were seeded in 6-well plates the day before transfection and transfected in triplicates with plasmid DNA and 200 pmol tRFs (unless otherwise mentioned) using Lipofectamine 2000 (Thermo Fisher Scientific) resulting in a final concentration of 100 nM tRFs. Let s calculate: 100 nM of tRFs (18 nt species) corresponds to 580 micrograms of small RNAs per liter. That is quite a lot. 200 pmol tRFs hit 2.5x 10^5 cells. That is 1.161 ug of tRFs hit 2.5x 10^5 cells. That means 1.205e+14 tRF molecules hit 2.5x 10^5 cells. That would be 4.82 x10^8 tRF molecules that would hit one cell. That would be 4.65 picograms of one tRF species hitting one cell. Of course, transfection efficiency is never 100%. Well, with a transfection efficiency of 50%, 2,41 x10^8 tRF molecules, or 2.3 picograms of tRFs that would hit one cell. According to molecular biology textbooks, a mammalian cell contains 10-20 pg of RNA ([24] Ogura et al., RNA chip: quality assessment of RNA by microchannel linear gel electrophoresis in injection-molded plastic chips. Clin Chem. 1998 Nov44(11):2249-55. [44] Kawasaki, E. S. Wang, A. M. In PCR Technology: Principles and Applications for DNA Amplification Erlich, H. A., Ed. Stockton Press: New York, 1989 p 92.PubMed ID9799750). About 90% of that RNA is ribosomal RNA, followed by tRNA (5 %), followed by anything else, and certainly among the lowest ranking members will be tRNA fragments of a specific kind and sequence identity (according to the tRNA fragment literature, specific tRFs correspond to 1-5% of the particular parental tRNA mass. In conclusion, the authors loaded a given HeLa cell instead of 25 femtograms of a given tRF (2.5% of 20 picograms = 1 picogram tRNA per cell, 5% of 1 picogram might be tRNA fragments) with a 100-fold excess of this tRF. Interestingly, the supplemental data show that the authors used synthetic tRFs that carried stabilizing 2 -O-methylation at every nucleotide thereby ensuring that the transfected tRFs remain stable and long-lived. That seems like a clever trick but no tRNA nor tRF carries 2 -O-methylation at every nucleotide. Hence, the system used and manipulated by experimentation is highly artificial in nature and while the experiments hint towards the potential for tRFs in blocking reverse transcription and retrotransposition of these retroviruses, the absence of follow-up studies indicate that it is likely harder to prove that something similar is really happening in mammalian cells. ,