Distinct Stress‐Dependent Signatures of Cellular and Extracellular tRNA‐Derived Small RNAs

Abstract The cellular response to stress is an important determinant of disease pathogenesis. Uncovering the molecular fingerprints of distinct stress responses may identify novel biomarkers and key signaling pathways for different diseases. Emerging evidence shows that transfer RNA‐derived small RNAs (tDRs) play pivotal roles in stress responses. However, RNA modifications present on tDRs are barriers to accurately quantifying tDRs using traditional small RNA sequencing. Here, AlkB‐facilitated methylation sequencing is used to generate a comprehensive landscape of cellular and extracellular tDR abundances in various cell types during different stress responses. Extracellular tDRs are found to have distinct fragmentation signatures from intracellular tDRs and these tDR signatures are better indicators of different stress responses than miRNAs. These distinct extracellular tDR fragmentation patterns and signatures are also observed in plasma from patients on cardiopulmonary bypass. It is additionally demonstrated that angiogenin and RNASE1 are themselves regulated by stressors and contribute to the stress‐modulated abundance of sub‐populations of cellular and extracellular tDRs. Finally, a sub‐population of extracellular tDRs is identified for which AGO2 appears to be required for their expression. Together, these findings provide a detailed profile of stress‐responsive tDRs and provide insight about tDR biogenesis and stability in response to cellular stressors.

collected fractions was measured and the three protein-enriched fractions were pooled and dialyzed successively in Dialysis Buffer and Storage Buffer. The dialyzed protein supernatant containing recombinant His-AlkB protein was validated by Coomassie Bule staining and western blotting, and used for ARM-seq.

Human Cell Line Culture and Stress Treatment
HEK293 cells were purchased from ATCC (ATCC, CRL-1573) and cultured in D10 medium, which consisted of DMEM with high glucose and pyruvate (Thermo Fisher, Cat# 11995073) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher, Cat# 10437028) and 1% Penicillin/Streptomycin (Thermo Fisher, Cat# 15140122). BeWo cells (ATCC, CCL-98), were cultured in F-12K Medium (ATCC, Cat# 30-2004) containing 10% FBS and 1% Penicillin/Streptomycin. For the glucose and serum deprivation (GSD) treatment, HEK293 and BeWo cells were cultured in glucose-free DMEM basal medium for 24 hours. For the hypoxia treatment, HEK293 and BeWo cells were fed with complete medium and maintained in hypoxia chamber with 0.1% oxygen for 24 hours. For the oxidative stress treatment, HEK293 and BeWo cells were treated with 0.8 mM hydrogen peroxide (H2O2) and 1.7 mM H2O2 separately in complete medium for 24 hours. FBS used for control, hypoxia and H2O2 treatments was filtered through 0.2 μm filter and followed by 24 h ultracentrifugation at 120,000 g to deplete extracellular vesicles (EVs) before use.

Neonatal Rat Ventricular Cardiomyocytes (CMs) and Cardiac Fibroblast (CFs) Isolation and Stress Treatment
All animal procedures conformed to the animal welfare regulations of the Massachusetts General Hospital Subcommittee on Research Animal Care (SRAC). Neonatal rat ventricular CMs and CFs were isolated as previously described 2 . Briefly, the ventricular heart tissues were carefully dissected from 1-day old Sprague Dawley rat neonates and minced using a sterile sharp razor blade, followed by 8 rounds of digestion in the ADS buffer (NaCl 116 mM, HEPES 20 mM, NaH2PO4 1 mM, KCl 5.4 mM, MgSO4 0.8 mM, and Glucose 5.5 mM) containing 0.05 mg/ml collagenase type II (Worthington, Cat# LS004177) and 1 mg/ml pancreatin (Sigma, Cat# P3292). Then the single cell suspensions were pooled and plated onto 10 cm tissue culture dishes with CM medium [DMEM medium supplemented with 10% horse serum (Thermo fisher, Cat# 26050-088), 5% FBS and 1% Penicillin/Streptomycin] for 1 hour. The attached cells were considered to be CFs and maintained in D10 medium. The floating cells were then purified via Percoll (GE Healthcare, Cat# 17-0891-01) gradient; the cells in the middle layer, which were considered as CMs, were collected and maintained in CM medium.
For the GSD treatment, CF and CM cells were exposed to glucose-free DMEM basal medium for 5 hours. For the hypoxia treatment, CF and CM cells were fed with glucose free DMEM basal medium and maintained in hypoxia chamber with 0.2% oxygen for 5 hours. For the reoxygenation (ReO2) treatment, CF and CM cells were exposed to hypoxia treatment for 5 hours and then fed with complete medium and cultured in normoxia for an additional 24 hours. FBS and horse serum used for control and ReO2 treatment were also EV-depleted by filtration and ultracentrifugation.

CPB Patient Plasma Sample Collection
Plasma samples were prospectively collected from six patients undergoing elective aortic valve replacement surgery with cardiopulmonary bypass by a single surgeon at a single institution in 2020 (https://clinicaltrials.gov/ct2/show/NCT00985049). Written informed consent approved by the Partners Healthcare Institutional Review Board (Boston, MA) was provided. Baseline plasma samples were obtained from venous blood immediately after commencement of cardiopulmonary bypass, which included intermittent cold blood cardioplegia for myocardial protection. Postischemic samples were obtained immediately before removal of the aortic cross-clamp at the end of the de-airing procedure. Plasma samples were collected using K2EDTA tubes and stored at −80℃ before RNA isolation. Median ischemic time was 73 minutes (Inter-quartile range [IQR] 68-121).
Of the six patients included in this analysis, 3 (50%) were female and the median age was 73 years (IQR 66-75).

Quantitative Polymerase Chain Reaction (qPCR)
qPCR was performed as previously described 3 . Briefly, 1 μg of cellular total RNA was reverse transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher, Cat# 4368813). qPCR was then performed using a QuantStudio 6 Flex Real-Time PCR Systems (Thermo Fisher) with SsoAdvanced™ Universal SYBR® Green Supermix (BioRad, Cat# 1725275). The sequences of qPCR primers used are listed in Table S8.

Western Blot
Western blotting was performed as previously described 3 . Briefly, cell pellets were resuspended in RIPA buffer (Thermo Fisher, Cat# 89900) containing Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher, Cat# 78441), and PMSF, and lysed via sonication. The protein concentration of clarified cell lysate was then measured using the BCA protein assay kit (Thermo

Standardized tDR naming system
tDRnamer (http://trna.ucsc.edu/tDRnamer/) was used for the standardized tDR naming in this study, which consists of five parts as shown in the example below: The instruction manual for tDRnamer can be found at http://trna.ucsc.edu/tDRnamer/docs/naming/.
Briefly, ①. Prefix -"tDR" stands for "tRNA Derived RNA". ②. Position -This includes the start and end positions of the tDR relative to the source tRNA. If the position is located at the leader or trailer sequence of the precursor tRNA gene, the numbering will be preceded with a letter "L" or "T" respectively. ③. Source tRNA -This is the name of the tRNA from which the tDR is derived.
tRNA names from Genomic tRNA Database are used. ④. Matching tRNA transcripts -If a tDR is mapped to multiple tRNA genes, an optional component with prefix "M" and the number of matching tRNAs will be added to the tDR name. ⑤. Variations -For a substitution, the annotation will include the base in tDR, the position of the substitution relative to the tDR, and the substituted base in the source tRNA. For an insertion, the annotation will include a prefix "I", followed by the position of the insertion relative to the tDR and the inserted base. For a deletion, the annotation will include a prefix "D", followed by the position before the deletion relative to the tDR and the deleted base from the source tRNA.             A. Genomic DNA PCR validation of the ANG-knockout monoclonal HEK cells.

B.
Genomic DNA PCR validation of the RNASE1-knockout monoclonal HEK cells.