5′ tRNA halves are present as abundant complexes in serum, concentrated in blood cells, and modulated by aging and calorie restriction

Background Small RNAs complex with proteins to mediate a variety of functions in animals and plants. Some small RNAs, particularly miRNAs, circulate in mammalian blood and may carry out a signaling function by entering target cells and modulating gene expression. The subject of this study is a set of circulating 30–33 nt RNAs that are processed derivatives of the 5′ ends of a small subset of tRNA genes, and closely resemble cellular tRNA derivatives (tRFs, tiRNAs, half-tRNAs, 5′ tRNA halves) previously shown to inhibit translation initiation in response to stress in cultured cells. Results In sequencing small RNAs extracted from mouse serum, we identified abundant 5′ tRNA halves derived from a small subset of tRNAs, implying that they are produced by tRNA type-specific biogenesis and/or release. The 5′ tRNA halves are not in exosomes or microvesicles, but circulate as particles of 100–300 kDa. The size of these particles suggest that the 5′ tRNA halves are a component of a macromolecular complex; this is supported by the loss of 5′ tRNA halves from serum or plasma treated with EDTA, a chelating agent, but their retention in plasma anticoagulated with heparin or citrate. A survey of somatic tissues reveals that 5′ tRNA halves are concentrated within blood cells and hematopoietic tissues, but scant in other tissues, suggesting that they may be produced by blood cells. Serum levels of specific subtypes of 5′ tRNA halves change markedly with age, either up or down, and these changes can be prevented by calorie restriction. Conclusions We demonstrate that 5′ tRNA halves circulate in the blood in a stable form, most likely as part of a nucleoprotein complex, and their serum levels are subject to regulation by age and calorie restriction. They may be produced by blood cells, but their cellular targets are not yet known. The characteristics of these circulating molecules, and their known function in suppression of translation initiation, suggest that they are a novel form of signaling molecule.

control diet. Tissues were flash frozen in liquid nitrogen. Serum samples were centrifuged at 110,000 g for 2 hrs, and supernatant and pellet fractions were separated. Samples of 0.2 ml serum mixed with 1.8 ml PBS were subjected to ultrafiltration through Vivaspin 2 columns (GE Healthcare) with 30, 100, or 300 kDa MW cut-off, and concentrate and filtrate fractions were collected. All samples were stored at -80 °C before RNA extraction. For plasma preparation, mouse blood samples were mixed with 0.5M EDTA (10 µl/ml) or sodium heparin (5.5 mg/ml) and centrifuged at 10,000 g for 10 min. The plasma supernatant was transferred to new tubes, centrifuged at 16,000 g for 15 min to remove any residual cells and cell-debris, and stored at -80 °C before use. Total RNA including small RNA was isolated from tissue samples, cell pellets or serum fractions with miRNeasy kit (Qiagen).
Collection of human blood and RNA extraction from serum and plasma. Human blood samples were collected with Institutional Review Board approval after obtaining informed consent. Blood was collected from one young adult male in BD Vacutainer Venous Blood Collection Tubes (BD Diagnostics): K2 EDTA Spray-Dried (BD-366643) or Spray-Coated Sodium Heparin (BD367874). Blood was transferred to Leucosep Centrifuge Tubes (Grenier Bio One #227290P) and centrifuged at 800 g for 15 min at room temperature. The plasma supernatant was transferred to fresh tubes, centrifuged at 16,000 g for 15 min to remove any residual cells and cell-debris, and stored at -80 °C before use. Total RNA including small RNA was isolated from plasma or serum with miRNeasy kit (Qiagen).

Preparation of leukocytes from mouse and human blood and RNA extraction.
Blood was collected on EDTA, centrifuged at 1000 g for 15 minutes to separate the plasma and blood cells. The buffy coat was collected, incubated in erythrocyte lysis buffer (Qiagen), and washed with PBS. Leukocyte pellets were flash frozen in liquid nitrogen, and stored at -80 °C before use. Total RNA including small RNA was isolated from leukocytes pellets with miRNeasy kit (Qiagen).
Real time quantitative PCR (qPCR). For qPCR assays, 10 fmoles of the synthetic C. elegans cel-miR-39 (Qiagen #MSY0000010) were spiked into 0.2 ml of serum or plasma before RNA extraction to account for variations during RNA extraction, cDNA synthesis, and real-time PCR. One fourth of total RNA extracted from 0.2 ml serum or plasma was reverse transcribed using the miScript Reverse Transcription Kit (Qiagen) according to the manufacturer's protocol. The obtained reverse transcription product was amplified using the following Qiagen reagents: SYBR Green PCR Master Mix, Universal Primer, and miScript Primer Assays for miR-16, miR-24, and miR-Cel-39. Real-time qPCR was carried out on a Bio-Rad CFX96 thermocycler.

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Pall GS & Hamilton AJ (2008) Improved northern blot method for enhanced detection of small RNA. Nature protocols 3(6):1077--1084. Figure S1 -Length distribution of sequencing reads that mapped to the RepeatMasker classes of DNA, LINE, LTR, Low_complexity, RC, SINE, Satellite, and Simple_repeat. Read length distribution is displayed by abundance of sequencing. Figure S2 -Scarcity of 5' tRNA-Asn halves in mouse serum. Northern blot analysis of RNA extracted from U2OS cells cultured in the absence (-) or presence (+) of sodium arsenite (AS), or from 0.4 ml of mouse serum, or from the supernatant (Sup) after ultracentrifugation of 0.4 ml of mouse serum at 110000g. The blot was hybridized to a 32 P-end-labeled oligonucleotide probe complementary to the 5' end of tRNA-Gly-GCC (A) or the 5' end of tRNA-Asn-GTT (B). The blot hybridized to the 5' end of tRNA-Gly-GCC was exposed to an X-ray film for 25 minutes, while the blot hybridized to the 5' end of tRNA-Asn was exposed for 5 days. The positions of full length tRNAs and tRNA halves are indicated on the right. DM: decade markers.