tRNA-derived fragments (tRFs): establishing their turf in post-transcriptional gene regulation

Krishna, Srikar; Raghavan, Srikala; DasGupta, Ramanuj; Palakodeti, Dasaradhi

doi:10.1007/s00018-020-03720-7

Cited by 58 publications

(46 citation statements)

References 78 publications

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“…Many categories of sRNA, including miRNA, piwi-interacting RNA, transfer RNA (tRNA)/tRNA fragments (tRFs), and Y RNAs, play important roles in the regulation of gene expression ( 44 , 45 ). The profile of these RNAs in the bloodstream and other biofluids holds promise as an approach for diagnostic monitoring of human disease ( 46 , 47 ).…”

Section: Resultsmentioning

confidence: 99%

Low-bias ncRNA libraries using ordered two-template relay: Serial template jumping by a modified retroelement reverse transcriptase

Upton

Ferguson

Temoche-Diaz

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Selfish, non-long terminal repeat (non-LTR) retroelements and mobile group II introns encode reverse transcriptases (RTs) that can initiate DNA synthesis without substantial base pairing of primer and template. Biochemical characterization of these enzymes has been limited by recombinant expression challenges, hampering understanding of their properties and the possible exploitation of their properties for research and biotechnology. We investigated the activities of representative RTs using a modified non-LTR RT from Bombyx mori and a group II intron RT from Eubacterium rectale. Only the non-LTR RT supported robust and serial template jumping, producing one complementary DNA (cDNA) from several templates each copied end to end. We also discovered an unexpected terminal deoxynucleotidyl transferase activity of the RTs that adds nucleotide(s) of choice to 3′ ends of single- and/or double-stranded RNA or DNA. Combining these two types of activity with additional insights about nontemplated nucleotide additions to duplexed cDNA product, we developed a streamlined protocol for fusion of next-generation sequencing adaptors to both cDNA ends in a single RT reaction. When benchmarked using a reference pool of microRNAs (miRNAs), library production by Ordered Two-Template Relay (OTTR) using recombinant non-LTR retroelement RT outperformed all commercially available kits and rivaled the low bias of technically demanding home-brew protocols. We applied OTTR to inventory RNAs purified from extracellular vesicles, identifying miRNAs as well as myriad other noncoding RNAs (ncRNAs) and ncRNA fragments. Our results establish the utility of OTTR for automation-friendly, low-bias, end-to-end RNA sequence inventories of complex ncRNA samples.

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Section: Resultsmentioning

confidence: 99%

Low-bias ncRNA libraries using ordered two-template relay: Serial template jumping by a modified retroelement reverse transcriptase

Upton

Ferguson

Temoche-Diaz

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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“…As such it might represent a novel pathway that supports host-parasite interactions and sustains infection. In this context, complementing the pool of tRNAs transcribed by the parasite with the import of specific exogenous/host tRNAs could play a punctual role during the developmental cycle, either by promoting protein synthesis at specific stages or by enabling unrelated signaling processes such as those reviewed in ( 47 , 48 , 49 ). The absence of other examples of import of exogenous tRNAs beyond the Plasmodium genus complicates its characterization but also offers new opportunities.…”

Section: Discussionmentioning

confidence: 99%

Identification of host tRNAs preferentially recognized by the Plasmodium surface protein tRip

Cela

Théobald-Dietrich

Rudinger-Thirion

et al. 2021

Nucleic Acids Research

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Malaria is a life-threatening and devastating parasitic disease. Our previous work showed that parasite development requires the import of exogenous transfer RNAs (tRNAs), which represents a novel and unique form of host–pathogen interaction, as well as a potentially druggable target. This import is mediated by tRip (tRNA import protein), a membrane protein located on the parasite surface. tRip displays an extracellular domain homologous to the well-characterized OB-fold tRNA-binding domain, a structural motif known to indiscriminately interact with tRNAs. We used MIST (Microarray Identification of Shifted tRNAs), a previously established in vitro approach, to systematically assess the specificity of complexes between native Homo sapiens tRNAs and recombinant Plasmodium falciparum tRip. We demonstrate that tRip unexpectedly binds to host tRNAs with a wide range of affinities, suggesting that only a small subset of human tRNAs is preferentially imported into the parasite. In particular, we show with in vitro transcribed constructs that tRip does not bind specific tRNAs solely based on their primary sequence, hinting that post-transcriptional modifications modulate the formation of our host/parasite molecular complex. Finally, we discuss the potential utilization of the most efficient tRip ligands for the translation of the parasite's genetic information.

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“…Many categories of sRNA, including miRNA, piwi-interacting RNA (piRNA), tRNA/tRNA fragments (tRFs), and Y RNAs, play important roles in the regulation of gene expression (44,45). The profile of these RNAs in the bloodstream and other biofluids holds promise as an approach for diagnostic monitoring of human disease (46,47).…”

Section: Ottr For Ev Rna Sequencingmentioning

confidence: 99%

Low-bias ncRNA Libraries using Ordered Two-Template Relay: Serial Template Jumping by a Modified Retroelement Reverse Transcriptase

Upton

Ferguson

Temoche-Diaz

et al. 2021

Preprint

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Non-long terminal repeat (non-LTR) and group II intron retroelements encode reverse transcriptases (RTs) that copy the retroelement transcript directly into host cell DNA, often at specific target sites. Biochemical characterization of these enzymes has been limited by recombinant expression and purification challenges, hampering understanding of their transposition mechanism and their exploitation for research and biotechnology. Properties of retroelement RTs substantiate their application for end-to-end RNA sequence capture. To investigate this utility, we first compared a non-LTR RT from Bombyx mori and a group II intron RT from Eubacterium rectale. Only the non-LTR RT showed processive template jumping, producing one cDNA from discontinuous templates each copied end-to-end. We also discovered an unexpected terminal deoxynucleotidyl transferase activity of the RTs that adds nucleotide(s) of choice to 3’ ends of single-stranded RNA or DNA. Combining these two types of activity with additional insights about non-templated nucleotide additions to duplexed cDNA product, we developed a streamlined protocol for linking Next Generation Sequencing (NGS) adaptors to both cDNA ends in a single RT reaction. When benchmarked using a reference pool of microRNAs (miRNAs), library production using modified non-LTR retroelment RT for Ordered Two-Template Relay (OTTR) outperformed all commercially available kits and rivaled the low bias of technically demanding home-brew protocols. We applied OTTR to inventory RNAs purified from extracellular vesicles (EVs), identifying miRNAs as well as myriad other non-coding (nc) RNAs and ncRNA fragments. Our results establish the utility of OTTR for automation-friendly, low-bias, end-to-end RNA sequence inventories of complex ncRNA samples.SignificanceRetrotransposons are non-infectious mobile genetic elements that proliferate in host genomes via an RNA intermediate that is copied into DNA by a reverse transcriptase (RT) enzyme. RTs are important for biotechnological applications involving information capture from RNA, since RNA is first converted into complementary DNA for detection or sequencing. Here, we biochemically characterize RTs from two retroelements and uncover several activities that allowed us to design a streamlined, efficient workflow for determining the inventory of RNA sequences in processed RNA pools. The unique properties of non-retroviral RT activities obviate many technical issues associated with current methods of RNA sequence analysis, with wide applications in research, biotechnology, and diagnostics.

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tRNA-derived fragments (tRFs): establishing their turf in post-transcriptional gene regulation

Cited by 58 publications

References 78 publications

Low-bias ncRNA libraries using ordered two-template relay: Serial template jumping by a modified retroelement reverse transcriptase

Low-bias ncRNA libraries using ordered two-template relay: Serial template jumping by a modified retroelement reverse transcriptase

Identification of host tRNAs preferentially recognized by the Plasmodium surface protein tRip

Low-bias ncRNA Libraries using Ordered Two-Template Relay: Serial Template Jumping by a Modified Retroelement Reverse Transcriptase

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