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Depletion of tRNA-halves enables effective small RNA sequencing of low-input murine serum samples

ABSTRACT: Depletion of tRNA-halves enables effective small RNA sequencing of low-input murine serum samples

PROVIDER: PRJNA349073 | ENA |

REPOSITORIES: ENA

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Depletion of tRNA-halves enables effective small RNA sequencing of low-input murine serum samples.

Project description: Not available

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Small RNA sequencing of serum-derived RNA for the evaluation of 5' tRNA halves depletion protocols

Project description:5' tRNA halves are abundantly present in small RNA libraries prepared from serum samples, thereby limiting the detection of other small RNA species. In this study, we developed and compared two protocols for the selective depletion of 5' tRNA halves in RNA isolated from murine serum samples. To evaluate the efficacy of both protocols on small RNA sequencing, we performed the two 5' tRNA halves depletion protocols on total RNA isolated from a serum pool collected from healthy mice. Each RNA sample was divided into three and the resulting fractions were either depleted using beads or RNase H, or used as a control. The experiment was performed in triplicate. Upon depletion, libraries were prepared using TruSeq Small RNA Library Preparation kit. For both protocols, very high depletion efficiencies were observed, with more than 99% of the targeted tRNA-types being depleted. This resulted in a more than 6-fold increase in mapped miRNA reads and 60% more detected mature miRNAs when performing small RNA sequencing on murine serum samples. Importantly, we observed no considerable effects on the relative expression values of miRNAs.

2017-02-22 | GSE88913 | GEO

Depletion of tRNA-halves enables effective small RNA sequencing of low-input murine serum samples

Project description:This SuperSeries is composed of the SubSeries listed below.

2017-02-22 | GSE88915 | GEO

A 5’ tRNA-Ala derived small RNA regulates anti-fungal defense in plants

Project description:Apart from their primordial role in protein synthesis, tRNAs can be cleaved to produce tRNA-derived small RNAs (tsRNAs). The biological functions of tsRNAs in plants remain largely unknown. In this study, we developed RtcB-based RNA sequencing, a method that captures and distinguishes between 2’,3’-cyclic-phosphate (cP)-terminated RNAs and 3’-OH-terminated RNAs, and profiled 5’ tsRNAs and 5’ tRNA halves in Arabidopsis thaliana. We found that Arabidopsis 5’ tsRNAs and 5’ tRNA halves predominantly contain a cP at the 3’ end and require S-like RNase 1 (RNS1) and RNS3 for their production. One of the most abundant 5’ tsRNA, 5’ tsR-Ala, likely by associating with AGO1, negatively regulates Cytochrome P450 71A13 (CYP71A13) expression and camalexin biosynthesis to repress anti-fungal defense. 5’ tsR-Ala is downregulated upon fungal infection. Our study provides a global view of 5’ tsRNAs and 5’ tRNA halves in Arabidopsis and unravels an important role of a 5’ tsRNA in regulating anti-fungal defense.

2022-02-16 | GSE183560 | GEO

A chromatin integration labeling method enables lower-input epigenomic profiling

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A growth-essential Tetrahymena Piwi protein carries tRNA fragment cargo

Project description:Argonaute/Piwi proteins associate with small RNAs that typically provide sequence specificity for RNP function in gene and genome regulation. Here we show that Twi12, a Tetrahymena Piwi protein essential for growth, is loaded with mature tRNA fragments. The tightly bound ~18-22 nt tRNA 3M-bM-^@M-^Y fragments are biochemically distinct from the tRNA halves produced transiently in response to stress. Notably, the end positions of Twi12-bound tRNA 3' fragments precisely match RNAs detected in total small RNA of mouse embryonic stem cells and human cancer cells. Our studies demonstrate unanticipated evolutionary conservation of mature tRNA processing to tRNA-fragment small RNAs. Two libraries are analyzed here: sRNAs associated with slightly overexpressed ZZ-tagged Twi12 purified under native conditions (size range 15-34nt), and those associated after formaldehyde crosslinking (15-22nt).

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Evaluation of three small RNA library preparation kits on serum-derived RNA

Project description:We evaluated the effect of the small RNA library preparation method on 5' tRNA-halves and miRNA abundance in libraries prepared from serum RNA using three commercially available small RNA library preparation kits (TruSeq small RNA library preparation kit v2 (Illumina), TailorMix miRNA sample preparation kit v2 (Seqmatic) and the NEBNext Multiplex Small RNA library prep kit (New England Biolabs)). RNA isolated from 100 µl of serum collected from healthy mice was used as input for the preparation of a small RNA library in duplicate and libraries were single end sequenced.

2017-02-22 | GSE88914 | GEO

A growth-essential Tetrahymena Piwi protein carries tRNA fragment cargo

Project description:Argonaute/Piwi proteins associate with small RNAs that typically provide sequence specificity for RNP function in gene and genome regulation. Here we show that Twi12, a Tetrahymena Piwi protein essential for growth, is loaded with mature tRNA fragments. The tightly bound ~18-22 nt tRNA 3’ fragments are biochemically distinct from the tRNA halves produced transiently in response to stress. Notably, the end positions of Twi12-bound tRNA 3' fragments precisely match RNAs detected in total small RNA of mouse embryonic stem cells and human cancer cells. Our studies demonstrate unanticipated evolutionary conservation of mature tRNA processing to tRNA-fragment small RNAs.

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Altered Expression Profiles of tRNA-derived fragments in relapsed/refractory multiple myeloma

Project description:tRNA related fragments(tRF) and tRNA halves(tiRNA) are novel class of short non-coding RNA derived from tRNAs. Using RNA sequencing, we evaluated the tRFs/tiRNAs expression profiles in relapsed/refractory multiple myeloma and multiple myeloma patients. Bioinformatics analyses indicated that tRFs/tiRNAs may be involved in the progression and drug-resistance of multiple myeloma.

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Processing by RNase 1 forms tRNA halves and distinct Y RNA fragments in the extracellular environment

Project description:In this study, we use small RNA sequencing to investigate the effect of a null mutation of RNase 1 on the levels of tRNA halves and Y RNA fragments in the extracellular environment of cultured human cells. Complemented and extended by the use of northern blots, our results demonstrate that tRNAs and Y RNAs in the vesicle-depleted extracellular compartment are released from cells as full-length precursors. Following their release, tRNAs and Y RNAs are processed by RNase 1 into distinct fragments. In addition, our findings show that standard sequencing methods employed to detect tRNA fragments leave many of such fragments undetected and that a combination of end healing, 3’ deacylation and RNA modification removal before library preparation can substantially improve detection of tRNA halves and reduce biases.

2020-06-30 | GSE148516 | GEO

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