Abstract
High-throughput sequencing has had an enormous impact on small RNA research during the past decade. However, sequencing only offers a one-dimensional view of the transcriptome and is often highly biased. Additionally, the ‘sequence, map and annotate’ approach, used widely in small RNA research, can lead to flawed interpretations of the data, lacking biological plausibility, due in part to database issues. Even in the absence of technical biases, the loss of three-dimensional information is a major limitation to understanding RNA stability, turnover and function. For example, noncoding RNA-derived fragments seem to exist mainly as dimers, tetramers or as nicked forms of their parental RNAs, contrary to widespread assumptions. In this perspective, we will discuss main sources of bias during small RNA-sequencing, present several useful bias-reducing strategies and provide guidance on the interpretation of small RNA-sequencing results, with emphasis on RNA fragmentomics. As sequencing offers a one-dimensional projection of a four-dimensional reality, prior structure-level knowledge is often needed to make sense of the data. Consequently, while less-biased sequencing methods are welcomed, integration of orthologous experimental techniques is also strongly recommended.
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References
Bartel, D. P. Metazoan microRNAs. Cell 173, 20–51 (2018).
Wang, X., Ramat, A., Simonelig, M. & Liu, M. F. Emerging roles and functional mechanisms of PIWI-interacting RNAs. Nat. Rev. Mol. Cell Biol. 24, 123–141 (2023).
Shi, J., Zhou, T. & Chen, Q. Exploring the expanding universe of small RNAs. Nat. Cell Biol. 24, 415–423 (2022).
Chen, Q., Zhang, X., Shi, J., Yan, M. & Zhou, T. Origins and evolving functionalities of tRNA-derived small RNAs. Trends Biochem. Sci. 46, 790–804 (2021).
Costa, B. et al. Nicked tRNAs are stable reservoirs of tRNA halves in cells and biofluids. Proc. Natl Acad. Sci. USA 120, e2216330120 (2023).
Chen, X. & Wolin, S. L. Transfer RNA halves are found as nicked tRNAs in cells: evidence that nicked tRNAs regulate expression of an RNA repair operon. RNA https://doi.org/10.1261/rna.079575.122 (2023).
Drino, A. et al. Identification of RNA helicases with unwinding activity on angiogenin-processed tRNAs. Nucleic Acids Res. 51, 1326–1352 (2023).
Akiyama, Y. et al. RTCB complex regulates stress-induced tRNA cleavage. Int. J. Mol. Sci. 23, 13100 (2022).
Spitale, R. C. & Incarnato, D. Probing the dynamic RNA structurome and its functions. Nat. Rev. Genet. 24, 178–196 (2023).
Giraldez, M. D. et al. Comprehensive multi-center assessment of small RNA-seq methods for quantitative miRNA profiling. Nat. Biotechnol. 36, 746–757 (2018).
Hafner, M. et al. RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries. RNA 17, 1697–1712 (2011).
Hu, J. F. et al. Quantitative mapping of the cellular small RNA landscape with AQRNA-seq. Nat. Biotechnol. 39, 978–988 (2021).
Song, Y., Liu, K. J. & Wang, T. H. Elimination of ligation dependent artifacts in T4 RNA ligase to achieve high efficiency and low bias microRNA capture. PLoS ONE 9, e94619 (2014).
Zhang, Z., Lee, J. E., Riemondy, K., Anderson, E. M. & Yi, R. High-efficiency RNA cloning enables accurate quantification of miRNA expression by deep sequencing. Genome Biol. 14, R109 (2013).
Turchinovich, A. et al. Capture and amplification by tailing and switching (CATS): an ultrasensitive ligation-independent method for generation of DNA libraries for deep sequencing from picogram amounts of DNA and RNA. RNA Biol. 11, 817–828 (2014).
Zhu, Y. Y., Machleder, E. M., Chenchik, A., Li, R. & Siebert, P. D. Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction. Biotechniques 30, 892–897 (2001).
Upton, H. E. et al. Low-bias ncRNA libraries using ordered two-template relay: serial template jumping by a modified retroelement reverse transcriptase. Proc. Natl Acad. Sci. USA 118, e2107900118 (2021).
Wulf, M. G. et al. Non-templated addition and template switching by Moloney murine leukemia virus (MMLV)-based reverse transcriptases co-occur and compete with each other. J. Biol. Chem. 294, 18220–18231 (2019).
Dard-Dascot, C. et al. Systematic comparison of small RNA library preparation protocols for next-generation sequencing. BMC Genomics 19, 118 (2018).
Mohr, S. et al. Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing. RNA 19, 958–970 (2013).
Zheng, G. et al. Efficient and quantitative high-throughput tRNA sequencing. Nat. Methods 12, 835–837 (2015).
Nottingham, R. M. et al. RNA-seq of human reference RNA samples using a thermostable group II intron reverse transcriptase. RNA 22, 597–613 (2016).
Xu, H., Yao, J., Wu, D. C. & Lambowitz, A. M. Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction. Sci. Rep. 9, 7953 (2019).
Behrens, A., Rodschinka, G. & Nedialkova, D. D. High-resolution quantitative profiling of tRNA abundance and modification status in eukaryotes by mim-tRNAseq. Mol. Cell 81, 1802–1815.e7 (2021).
Heyer, E. E., Ozadam, H., Ricci, E. P., Cenik, C. & Moore, M. J. An optimized kit-free method for making strand-specific deep sequencing libraries from RNA fragments. Nucleic Acids Res. 43, (2015).
Cozen, A. E. et al. ARM-seq: AlkB-facilitated RNA methylation sequencing reveals a complex landscape of modified tRNA fragments. Nat. Methods 12, 879–884 (2015).
Wang, H. et al. CPA-seq reveals small ncRNAs with methylated nucleosides and diverse termini. Cell Discov. 7, 25 (2021).
Shi, J. et al. PANDORA-seq expands the repertoire of regulatory small RNAs by overcoming RNA modifications. Nat. Cell Biol. 23, 424–436 (2021).
Pinkard, O., McFarland, S., Sweet, T. & Coller, J. Quantitative tRNA-sequencing uncovers metazoan tissue-specific tRNA regulation. Nat. Commun. 11, 4104 (2020).
Shigematsu, M. et al. YAMAT-seq: an efficient method for high-throughput sequencing of mature transfer RNAs. Nucleic Acids Res. 45, e70 (2017).
Nolte’T Hoen, E. N. M. et al. Deep sequencing of RNA from immune cell-derived vesicles uncovers the selective incorporation of small non-coding RNA biotypes with potential regulatory functions. Nucleic Acids Res. 40, 9272–9285 (2012).
Tosar, J. P. et al. Assessment of small RNA sorting into different extracellular fractions revealed by high-throughput sequencing of breast cell lines. Nucleic Acids Res. 43, 5601–5616 (2015).
Sharma, U. et al. Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals. Science 351, 391–396 (2016).
Shurtleff, M. J. et al. Broad role for YBX1 in defining the small noncoding RNA composition of exosomes. Proc. Natl Acad. Sci. USA 114, E8987–E8995 (2017).
Tosar, J. P. et al. Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome. Nucleic Acids Res. 48, 12874–12888 (2020).
Gogakos, T. et al. Characterizing expression and processing of precursor and mature human tRNAs by hydro-tRNAseq and PAR-CLIP. Cell Rep. 20, 1463–1475 (2017).
Choi, J. H. & Sullivan, C. S. DUSP11 and triphosphate RNA balance during virus infection. PLoS Pathog. 17, (2021).
Honda, S. et al. Sex hormone-dependent tRNA halves enhance cell proliferation in breast and prostate cancers. Proc. Natl Acad. Sci. USA 112, E3816–E3825 (2015).
Akat, K. M. et al. Detection of circulating extracellular mRNAs by modified small-RNA-sequencing analysis. JCI Insight 4, 127317 (2019).
Giraldez, M. D. et al. Phospho‐RNA‐seq: a modified small RNA‐seq method that reveals circulating mRNA and lncRNA fragments as potential biomarkers in human plasma. EMBO J. 38, (2019).
Lentzsch, A. M., Yao, J., Russell, R. & Lambowitz, A. M. Template-switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-seq. J. Biol. Chem. 294, 19764–19784 (2019).
Tosar, J. P., Cayota, A., Eitan, E., Halushka, M. K. & Witwer, K. W. Ribonucleic artefacts: are some extracellular RNA discoveries driven by cell culture medium components? J. Extracell. Vesicles 6, 1272832 (2017).
Auber, M., Fröhlich, D., Drechsel, O., Karaulanov, E. & Krämer-Albers, E.-M. Serum-free media supplements carry miRNAs that co-purify with extracellular vesicles. J. Extracell. Vesicles 8, 1656042 (2019).
Fromm, B. et al. A uniform system for the annotation of vertebrate microRNA genes and the evolution of the human microRNAome. Annu. Rev. Genet. 49, 213–242 (2015).
Wei, Z., Batagov, A. O., Carter, D. R. F. & Krichevsky, A. M. Fetal bovine serum RNA interferes with the cell culture derived extracellular RNA. Sci. Rep. 6, 31175 (2016).
Tosar, J. P., Cayota, A. & Witwer, K. Exomeres and supermeres: monolithic or diverse? J. Extracell. Biol. 1, e45 (2022).
Tosar, J. P., Rovira, C. & Cayota, A. Non-coding RNA fragments account for the majority of annotated piRNAs expressed in somatic non-gonadal tissues. Commun. Biol. 1, 2 (2018).
Tosar, J. P., García-Silva, M. R. & Cayota, A. Circulating SNORD57 rather than piR-54265 is a promising biomarker for colorectal cancer: Common pitfalls in the study of somatic piRNAs in cancer. RNA 27, 403–410 (2021).
Tosar, J. P. et al. Dimerization confers increased stability to nucleases in 5′ halves from glycine and glutamic acid tRNAs. Nucleic Acids Res. 46, 9081–9093 (2018).
Lyons, S. M., Gudanis, D., Coyne, S. M., Gdaniec, Z. & Ivanov, P. Identification of functional tetramolecular RNA G-quadruplexes derived from transfer RNAs. Nat. Commun. 8, 1127 (2017).
Lyons, S. M. et al. eIF4G has intrinsic G-quadruplex binding activity that is required for tiRNA function. Nucleic Acids Res. 48, 6223–6233 (2021).
Loughrey, D., Watters, K. E., Settle, A. H. & Lucks, J. B. SHAPE-Seq 2.0: systematic optimization and extension of high-throughput chemical probing of RNA secondary structure with next generation sequencing. Nucleic Acids Res. 42, (2014).
Zubradt, M. et al. DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo. Nat. Methods 14, 75–82 (2017).
Tosar, J. P., Gámbaro, F., Castellano, M. & Cayota, A. RI-SEC-seq: comprehensive profiling of nonvesicular extracellular RNAs with different stabilities. Bio Protoc. 11, e3918 (2021).
Kharel, P. et al. Stress promotes RNA G-quadruplex folding in human cells. Nat. Commun. 14, 205 (2023).
Kaymak, E. & Rando, O. J. Staying together after the breakup: tRNA halves in extracellular fluids. Proc. Natl Acad. Sci. USA 120, e2300300120 (2023).
Munafò, M. R. & Smith, Davey G. Robust research needs many lines of evidence. Nature 553, 399–401 (2018).
Lentzsch, A. M., Yao, J., Russell, R. & Lambowitz, A. M. Template-switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq. J. Biol. Chem. 294, (2019).
Almeida, M. V., de Jesus Domingues, A. M., Lukas, H., Mendez-Lago, M. & Ketting, R. F. RppH can faithfully replace TAP to allow cloning of 5′-triphosphate carrying small RNAs. MethodsX 6, (2019).
Tosar, J. P., Rovira, C., Naya, H. & Cayota, A. Mining of public sequencing databases supports a non-dietary origin for putative foreign miRNAs: Underestimated effects of contamination in NGS. RNA 20, 754–757 (2014).
Tosar, J. P., Witwer, K. & Cayota, A. Revisiting extracellular RNA release, processing, and function. Trends Biochem. Sci. 46, 438–445 (2021).
Chen, Q. et al. Sperm tsRNAs contribute to intergenerational inheritance of an acquired metabolic disorder. Science 351, 397–400 (2016).
Zhang, Y. et al. Dnmt2 mediates intergenerational transmission of paternally acquired metabolic disorders through sperm small non-coding RNAs. Nat. Cell Biol. 20, (2018).
Sarker, G. et al. Maternal overnutrition programs hedonic and metabolic phenotypes across generations through sperm tsRNAs. Proc. Natl Acad. Sci. USA 116, (2019).
Jimenez, N. A. et al. Paternal methotrexate exposure affects sperm small RNA content and causes craniofacial defects in the offspring. Nat. Commun. 14, 1617 (2023).
Boskovic, A., Bing, X. Y., Kaymak, E. & Rando, O. J. Control of noncoding RNA production and histone levels by a 5′ tRNA fragment. Genes Dev. 34, 118–131 (2020).
Gustafsson, H. T. et al. Deep sequencing of yeast and mouse tRNAs and tRNA fragments using OTTR. Preprint at bioRxiv https://doi.org/10.1101/2022.02.04.479139(2022).
Acknowledgements
This work was partially funded by the National Institutes of Health Office of the Director (UG3/UH3CA241694) and Universidad de la República, Uruguay (CSIC I+D_2020_433).
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J.P.T., B.C. and A.C. have filed a patent in the United States on a method for RNA repair and sequencing.
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Tosar, J.P., Castellano, M., Costa, B. et al. Small RNA structural biochemistry in a post-sequencing era. Nat Protoc 19, 595–602 (2024). https://doi.org/10.1038/s41596-023-00936-2
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DOI: https://doi.org/10.1038/s41596-023-00936-2
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