A hypoxia-responsive tRNA-derived small RNA confers renal protection via RNA autophagy

Abstract

Transfer RNA-derived small RNAs (tsRNAs, or tDRs) perform a range of cellular functions. Here, we showed that a hypoxia-induced tDR, derived from the 3’ end of tRNA-Asp-GTC (tRNA-Asp-GTC-3’tDR), activated autophagic flux in kidney cells, while its silencing blocked autophagic flux. Functional gain/loss-of-function studies in murine kidney disease models demonstrated a significant reno-protective function of tRNA-Asp-GTC-3’tDR. Mechanistically, tRNA-Asp-GTC-3’tDR assembled stable G-quadruplex structures and sequestered pseudouridine synthase PUS7, preventing catalytic pseudouridylation of histone mRNAs. The resulting pseudouridylation deficiency directed histone mRNAs to the autophagosome-lysosome pathway, triggering RNA autophagy. This tDR-induced RNA autophagy pathway was activated during murine and human kidney diseases, suggesting clinical relevance. Thus, tRNA-Asp-GTC-3’tDR plays a role in regulating RNA autophagy, which helps to maintain homeostasis in kidney cells and protects against kidney injury.

Transfer RNAs (tRNAs) act as amino acid carriers and mRNA decoders during protein synthesis (1). The human genome contains more than 500 tRNA genes, with about 50% actively transcribing tRNAs, making them the most abundant RNA species within cells (2, 3). Certain tRNAs can be cleaved into fragments of various lengths, termed tRNA-derived small RNAs (tsRNAs or tDRs), particularly as a response to stress conditions (4). The development of specialized RNA sequencing methodologies has accelerated the recognition of a plethora of tDRs (5–8). We now understand that tRNA cleavage represents an evolutionarily conserved process that produces new RNA species in all domains of life (9). The biogenesis of tDR is orchestrated by various ribonucleases, such as DICER, RNaseL, and Angiogenin, together with RNA-binding proteins (RBPs) and RNA modification enzymes, rendering it sensitive to fluctuating cellular dynamics (10).

The intricate regulation of tDR biogenesis, along with the unique linear sequences and the complex three-dimensional structures of tDRs, underscore their multifaceted roles as an emerging class of regulatory small non-coding RNAs. tDR species have been implicated in a myriad of biological processes (11), including regulation of translation and transcription (12–17), RNAi-like functions (18), and regulation of mRNA stability through competitive binding of RBPs (19).

From a comprehensive tDR atlas generated using ARM-seq to characterize stress-regulated tDRs across multiple cell types, we identified over 3000 significantly regulated hypoxia-responsive tDRs (20). In this work, we focused on tRNA-Asp-GTC-3’tDR (derived from the 3’ end of tRNA-Asp-GTC), as the most upregulated tDR in response to hypoxia, and studied its biogenesis and regulation. Using silencing and overexpression tools, we explored the functional role of tRNA-Asp-GTC-3’tDR in regulating cellular stress responses in kidney cells. We next investigated the role of tRNA-Asp-GTC-3’tDR in the pathogenesis of kidney injury in two different kidney disease models using complementary gain-of-function (GOF) and loss-of-function (LOF) approaches. Lastly, we characterized the key structural motifs and binding partners of tRNA-Asp-GTC-3’tDR that were the critical determinants of its function in kidney cells.

Hypoxia-induced tRNA-Asp-GTC-3’tDR enhances autophagic flux

In a comprehensive stress-specific cellular and extracellular tDR atlas (20), analysis of multiple cell types identified numerous tDRs highly regulated by different stressors. To characterize their functions, we focused on tDRs demonstrating the most significant stress-induced changes in HEK cells. ARM-seq data showed that hypoxia induced the most substantial changes in tDR levels in HEK cells (Fig. S1A). The top 2 tDRs upregulated by hypoxia originated from the tRNA-Asp-GTC-2 gene, with one from the 5’end (named by tDRnamer (21) as tDR-1:32-Asp-GTC-2; hereafter tRNA-Asp-GTC-5’tDR), and another one from the 3’ end (named by tDRnamer as tDR-39:72-Asp-GTC-2-M2; hereafter tRNA-Asp-GTC-3’tDR) (Fig. 1A, Table S1). Northern blotting validated their upregulation, demonstrating additionally that tDR isoforms comprise <3% of the full-length tRNA-Asp-GTC (Fig. 1B, Fig. S1B-C). To confirm that these tDRs were generated by tRNA cleavage, we designed a self-quenched tDR biogenesis reporter. We conjugated a fluorescent group (FITC) and a quencher group (BHQ-1) to each end of full-length tRNA-Asp-GTC (Fig. 1C), which only releases fluorescent signals upon tRNA cleavage and separation of 5’tDR and 3’tDR (22). HEK cells transfected with this reporter exhibited significantly higher FITC signals under hypoxic conditions when compared with normoxic conditions (Fig. 1D-E), confirming hypoxia-induced tRNA-Asp-GTC fragmentation and tDR biogenesis. Previous studies identified Angiogenin (ANG) as a stress-activated ribonuclease that generates tDRs by cleaving tRNAs at the anti-codon loop (23, 24). Recent work identified ANG as one of the most upregulated ribonucleases under hypoxia (20). To determine ANG’s role in tRNA-Asp-GTC-3’tDR biogenesis, we subjected an ANG-deleted cell line (20) to hypoxia treatment and evaluated the levels of tRNA-Asp-GTC-3’tDR. ANG deletion substantially impaired hypoxia-induced generation of tRNA-Asp-GTC-3’tDR (Fig. S1D-F), establishing ANG as a critical ribonuclease for tRNA-Asp-GTC-3’tDR biogenesis under hypoxic conditions, as expected (25). While ANG cleavage appears to be the dominant mechanism for tRNA-Asp-GTC-3’tDR generation, the residual levels of this tDR in ANG-deleted cells suggest that other effectors may also be involved in its biogenesis.

Figure 1. Hypoxia-induced tRNA-Asp-GTC-3’tDR enhances autophagic flux.

(A) Volcano plot shows significantly regulated tDRs in HEK cells upon hypoxia treatment, with the top 2 significantly upregulated tDRs labeled. (B) Northern blotting validates the upregulation of 5’tDR and 3’tDR of tRNA-Asp-GTC in HEK cells upon hypoxia. (C) Schematic illustration of tDR biogenesis reporter design. BHQ1, black hole quencher 1; FITC, fluorescein. (D) Live imaging of fluorescent signals from the tRNA-Asp-GTC-3’tDR biogenesis reporter in HEK cells with or without hypoxia treatment. (E) Quantification of FITC-positive signals per cell from D. (F) Immunoblot analysis of different protein markers involved in ER stress, mitophagy, and autophagy pathways in HEK cells transfected with control (Ctrl) and tRNA-Asp-GTC-5’tDR and tRNA-Asp-GTC-3’tDR mimics. (G) Quantification of protein levels in F. (H) Live imaging of fluorescent signals in RFP-GFP-LC3B autophagy reporter HEK cells transfected with Ctrl or tRNA-Asp-GTC-3’tDR mimics. (I) Quantification of the number of autophagosomes (GFP⁺ & RFP⁺) and autolysosomes (GFP⁻ & RFP⁺) per cell in H. (J) Immunoblot analysis of autophagic protein levels in HEK cells transfected with Ctrl or tRNA-Asp-GTC-3’tDR mimics and treated with DMSO or Bafilomycin A1 (BafA1) for 6 hours. (K) Quantification of protein levels in J. Data are shown as means ± SEM of at least three independent experiments. The Mann-Whitney test was used in (E) and (I). The unpaired two-tailed Student’s t-test was used in (G) and (K). *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar 50μm.

Hypoxia triggers multiple cellular stress responses, including endoplasmic reticulum (ER) stress, mitophagy, and autophagy, and tDRs have previously been shown to induce translational repression and stress granule formation during cellular stress (12–15, 26–28). To investigate the role of hypoxia-induced tDRs in hypoxic stress response, we transfected synthetic RNA oligos of tRNA-Asp-GTC-5’tDR or tRNA-Asp-GTC-3’tDR into HEK cells and analyzed the resulting protein lysates. Immunoblotting demonstrated that hypoxia activated ER stress (phosphorylation of IRE1a and downregulation of PERK), mitophagy (induction of BNIP3 and BNIP3L), and autophagy (conversion of LC3B-I to lipidated LC3B-II) (Fig. 1F). Neither tDR affected ER stress or mitophagy pathways. However, introduction of tRNA-Asp-GTC-3’tDR, but not tRNA-Asp-GTC-5’tDR, led to the activation of autophagy pathways under normoxic conditions (Fig. 1F-G). Using a GFP-LC3B autophagy reporter cell line, we confirmed that tRNA-Asp-GTC-3’tDR significantly promoted the formation of GFP-positive punctate-like structures within hours of transfection (Fig. S1G-J).

Autophagic flux encompasses autophagosome formation, autophagosome-lysosome fusion, and substrate degradation in autolysosomes (29). To examine how tRNA-Asp-GTC-3’tDR regulates autophagic flux, we built an autophagic flux reporter cell line expressing RFP-GFP tandem fluorescent-tagged LC3B. RFP-GFP-LC3B-labeled autophagosomes show both GFP and RFP signals before fusion with lysosomes and only RFP signals after fusion (29). tRNA-Asp-GTC-3’tDR promoted the formation of both autophagosomes (GFP⁺/RFP⁺) and autolysosomes (GFP⁻/RFP⁺) (Fig. 1H-I), indicating enhanced autophagic flux. Because autophagosome-lysosome fusion is usually associated with activated lysosomal activity (30), we observed increased lysosomal activity in tRNA-Asp-GTC-3’tDR-transfected cells (Fig. S1K-L). We used Bafilomycin A1 (BafA1), which inhibits autophagosome maturation, to measure autophagic flux (29, 31). After BafA1 treatment, the accumulation of lipidated LC3B and P62 protein levels was significantly higher in tRNA-Asp-GTC-3’tDR-transfected cells when compared to controls (Fig. 1J-K). Finally, additionally boosting tRNA-Asp-GTC-3’tDR levels in hypoxic HEK cells further enhanced autophagic flux (Fig. S1M-N). Thus, tRNA-Asp-GTC-3’tDR enhanced autophagic flux.

Inhibition of tRNA-Asp-GTC-3’tDR blocks autophagic flux in different kidney cell types

Synthetic locked-nucleic-acid-modified antisense oligonucleotides (ASO) have been used to specifically block tDR function (19, 32). We thus adopted the ASO inhibition approach to further elucidate the function of endogenous tRNA-Asp-GTC-3’tDR. After screening several designed ASOs, we identified one (Asp-GTC-3’tDR-ASO, Fig. S2A) that could efficiently knockdown tRNA-Asp-GTC-3’tDR levels in hypoxic HEK cells with no significant change in the parent tRNA-Asp-GTC expression, when compared to negative control ASO (NC-ASO) (Fig. 2A-B). The targeting specificity was further confirmed by a computational tube reaction model, which incorporates four components at equal concentrations (1μM): Asp-GTC-3’tDR-ASO, tRNA-Asp-GTC-3’tDR, tRNA-Asp-GTC-5’tDR, and full-length tRNA-Asp-GTC, and accounts for their respective secondary structures. Based on calculated equilibrium complex concentrations, the 3’tDR+ASO hybrid emerged as the most dominant species, reaching a concentration of 0.91 μM, while the ASO+full-length tRNA hybrid achieved only minimal formation, with a concentration of 0.086 μM (Fig. S2A-B). Thus, over 90% of the Asp-GTC-3’tDR-ASO preferentially binds to the 3’tDR, while less than 1% interacts with full-length tRNAs.

Figure 2. Inhibition of tRNA-Asp-GTC-3’tDR blocks autophagic flux.

(A) Northern blotting shows the levels of full-length and 3’tDR of tRNA-Asp-GTC in hypoxic HEK cells transfected with negative control ASO (NC-ASO) or tRNA-Asp-GTC-3’tDR ASO (Asp-GTC-3’tDR-ASO). (B) Quantification of levels of full-length and 3’tDR of tRNA-Asp-GTC in A. (C) Immunoblot analysis of protein levels of LC3B and β-ACTIN in HEK cells transfected with control or tRNA-Asp-GTC-3’tDR ASOs under normoxic or hypoxic conditions. (D) Quantification of protein levels in C. (E) Live imaging of fluorescent signals in RFP-GFP-LC3B autophagy reporter HEK cells transfected with NC-ASO or Asp-GTC-3’tDR-ASO under normoxic or hypoxic conditions. (F) Quantification of the number of autophagosomes (GFP⁺ & RFP⁺) and autolysosomes (GFP⁻ & RFP⁺) per cell in E. (G) Immunoblot analysis of protein levels of LC3B and β-ACTIN in hypoxic HEK cells transfected with NC-ASO or Asp-GTC-3’tDR-ASO and treated with DMSO or BafA1 for 6 hours. (H) Quantification of protein levels in G. Data are shown as means ± SEM of at least three independent experiments. The unpaired two-tailed Student’s t-test was used in (B), (D), and (H). The Mann-Whitney test was used in (F). n.s. non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar 50μm.

We next assayed the effects of tRNA-Asp-GTC-3’tDR knockdown on autophagy pathways. The inhibition of tRNA-Asp-GTC-3’tDR using Asp-GTC-3’tDR-ASO significantly increased LC3B-II protein levels in HEK cells under both normoxic and hypoxic conditions (Fig. 2C-D), suggesting either increased autophagy initiation or a block in autophagic flux. Using the RFP-GFP-LC3B reporter cell line, we found that Asp-GTC-3’tDR-ASO treatment resulted in autophagosome accumulation without maturation into autolysosomes under both normoxic and hypoxic conditions (Fig. 2E-F), aligning with increased LC3B-II protein levels. Treatment with BafA1 caused further LC3B-II accumulation in both groups, but with significantly lower levels in tRNA-Asp-GTC-3’tDR-inhibited cells under hypoxia compared to controls (Fig. 2G-H). Thus, the increased LC3B-II levels noted above resulted from a block in autophagic flux owing to tRNA-Asp-GTC-3’tDR inhibition.

To characterize the physiological roles of tRNA-Asp-GTC-3’tDR, we analyzed its tissue abundance through ARM-seq of different mouse tissues and observed highest abundance in kidney, heart, and brain tissues, particularly of a 38nt isoform (tDR-38:76-Asp-GTC-2-M2) (Fig. 3A). We tested the functionality of this mouse 38nt isoform and another abundant 34nt isoform (tDR-38:72-Asp-GTC-2-M2) detected by ARM-seq and northern blotting (Fig. S2C). As expected, both isoforms activated autophagy flux like the 33nt isoform (Fig. S2D-E). We also detected abundant levels of tRNA-Asp-GTC-3’tDRs, especially the 38nt isoform, in a variety of human kidney cells, including proximal tubular epithelial cells (PTEpC), kidney fibroblasts, glomerular microvascular endothelial cells (GMEC), glomerular epithelial cells (GEpC), podocytes, and glomerular mesangial cells (Fig. 3B, Fig. S3A). Healthy kidney cells, especially PTEpCs and podocytes, maintain high basal autophagy levels, which are essential for maintaining kidney homeostasis (33, 34). Our analysis of autophagy activity and tRNA-Asp-GTC-3’tDR abundance in these cells also suggested that high tRNA-Asp-GTC-3’tDR levels may sustain high basal autophagic flux in these kidney cells (Fig. S3A-C). As expected, tRNA-Asp-GTC-3’tDR inhibition by Asp-GTC-3’tDR-ASO impeded overall autophagic flux in various human kidney cells, including PTEpCs, GEpCs, and GMECs (Fig. 3C-D, Fig. S3D-G). Notably, in contrast to HEK cells, in these kidney cells, inhibition of basal tRNA-Asp-GTC-3’tDR affected generalized autophagy flux, not just autophagosome maturation. Autophagy is essential for the function and survival of renal cells (33). Failure to sustain desired autophagy flux in these kidney cells, resulting from tRNA-Asp-GTC-3’tDR inhibition, triggered apoptosis and cell death (Fig. 3E-H, Fig. S3H-I). Thus, tRNA-Asp-GTC-3’tDR was necessary for maintaining autophagic flux in kidney cells, which is indispensable for their health.

Figure 3. tRNA-Asp-GTC-3’tDR sustains autophagy flux in different kidney cell types.

(A) ARM-seq analysis shows the abundance of tRNA-Asp-GTC-3’tDR across mouse organs. (B) Northern blotting shows the levels of full-length and 3’tDR of tRNA-Asp-GTC across different human kidney cells under hypoxia conditions. PTEpC, primary proximal tubular epithelial cells; Fib, primary kidney fibroblasts; GMEC, primary glomerular microvascular endothelial cells; GEpC, primary glomerular epithelial cells; Podo, immortalized podocytes; GMC, primary glomerular mesangial cells. (C) Immunoblot analysis of autophagic protein levels in PTEpC transfected with NC-ASO or Asp-GTC-3’tDR-ASO and treated with DMSO or BafA1 for 6 hours. (D) Quantification of protein levels in C. (E) Immunoblot analysis of the protein levels of full-length or cleaved Caspase 3 in human primary proximal tubular epithelial cells transfected with NC-ASO or Asp-GTC-3’tDR-ASO. (F) Quantification of full-length or cleaved Caspase 3 protein levels in E. (G to H) Cell viability assessment by MTS assay (G) and cell death assessment by secreted LDH assay (H) in different kidney cells following transfection with NC-ASO or Asp-GTC-3’tDR-ASO. (I) Northern blotting shows the levels of full-length and 3’tDRs of tRNA-Asp-GTC in the obstructed kidney tissues at different time points post-UUO surgery. (J) Quantification of the levels of tRNA-Asp-GTC-3’tDR isoforms (33 nt and 38 nt) in I. Data are shown as means ± SEM of at least three independent experiments. The unpaired two-tailed Student’s t-test was used in (D), (F), (G), and (H). *p < 0.05, **p < 0.01, ***p < 0.001.

Inhibition of tRNA-Asp-GTC-3’tDR exacerbates kidney injuries in mouse models.

To investigate the pathophysiological role of tRNA-Asp-GTC-3’tDR in vivo, we validated its basal abundance in kidneys and found marked increases following various kidney injuries, including uninephrectomy-induced volume/hemodynamic overload and albumin injection-induced protein overload (Fig. S4A-D). In the mouse unilateral ureteral obstruction (UUO) model, which resembles human chronic obstructive nephropathy (35), tRNA-Asp-GTC-3’tDR levels were promptly upregulated in the obstructed kidney within four hours post-surgery and remained elevated for 10 days (Fig. 3I-J), with a switch from the 33nt to 38nt isoform on day one after surgery. We hypothesized that this augmented biogenesis of tRNA-Asp-GTC-3’tDRs enhances kidney autophagic flux as an adaptive response to ameliorate the progression of kidney injury.

To investigate the role of tRNA-Asp-GTC-3’tDR in the UUO-induced kidney injury model, we used Asp-GTC-3’tDR-ASO with phosphorothioate backbones for enhanced stability and self-delivery (36). ASO was administered intravenously into mice every other day from the day before UUO surgery, with pathological analysis on day six (Fig. 4A). The knockdown of tRNA-Asp-GTC-3’tDRs in the obstructed kidney was confirmed using northern blotting (Fig. 4B-C, Fig. S5A). Ureteral obstruction usually results in marked tubular injury and cell death, with interstitial immune cell infiltration and fibroblast activation (35). Consistent with this, UUO surgery led to acute kidney injury, immune cell infiltration, and fibrosis, in the obstructed kidney from NC-ASO-treated group (Fig. 4D). Asp-GTC-3’tDR-ASO administration further increased markers for kidney injury, immune cell infiltration, and fibrosis in the obstructed kidney compared to NC-ASO group (Fig. 4D). This was corroborated by immunohistology, revealing substantial increases in apoptosis, neutrophil and macrophage infiltration, and collagen deposition (Fig. 4E-G), in the obstructed kidney following tRNA-Asp-GTC-3’tDR inhibition. Immunoblotting further validated a consistent phenotype of exacerbated kidney injury following tRNA-Asp-GTC-3’tDR knockdown (Fig. 4H-I). Notably, tRNA-Asp-GTC-3’tDR knockdown inhibited autophagic flux in the obstructed kidney (Fig. 4H-I). We also noticed mild kidney injury following tRNA-Asp-GTC-3’tDR inhibition in the sham surgery group (Fig. 4D), which may have been because of disordered autophagy at baseline. To validate this further, we employed a four-week chronic knockdown approach by weekly subcutaneous injection of ASOs into healthy mice. Upon confirming the knockdown of tRNA-Asp-GTC-3’tDR (Fig. S5B-C), we examined the kidney tissues using transmission electron microscopy. Indeed, inhibition of tRNA-Asp-GTC-3’tDR impaired autophagosome maturation, as indicated by deficient autophagosome-lysosome fusion in kidney tissues (Fig. S5D). Thus, tRNA-Asp-GTC-3’tDR abundance is increased across kidney injury models and may be part of a compensatory response to maintain autophagic flux, and its inhibition consequently exacerbates kidney injury.

Figure 4. Inhibition of tRNA-Asp-GTC-3’tDR exacerbates kidney injuries in mouse UUO models.

(A) Schematic illustration of experimental design for tRNA-Asp-GTC-3’tDR inhibition in mouse UUO model. (B) Northern blotting shows the levels of full-length and 3’tDR of tRNA-Asp-GTC in kidney tissues from Sham or UUO (obstructed kidney) mice treated with NC-ASO or Asp-GTC-3’tDR-ASO. (C) Quantification of levels of tRNA-Asp-GTC-3’tDR in B. (D) Analysis of gene expression related to acute kidney injury, immune cell infiltration, and fibrosis in kidney tissues from Sham or UUO (obstructed kidney) mice treated with NC-ASO or Asp-GTC-3’tDR-ASO (n=6–10 per group). (E to F) Immunostaining analysis of Cleaved-Caspase 3 (E) and Ly6B.2 (Neutrophil) and F4/80 (Macrophage) (F) in kidney tissues from Sham or UUO (obstructed kidney) mice treated with NC-ASO or Asp-GTC-3’tDR-ASO. (G) Sirius Red staining analysis of collagen levels in kidney tissues from Sham or UUO (obstructed kidney) mice treated with NC-ASO or Asp-GTC-3’tDR-ASO. (H) Immunoblot analysis of protein markers associated with immune cells, fibrosis, apoptosis, and autophagy in kidney tissues from Sham or UUO (obstructed kidney) mice treated with NC-ASO or Asp-GTC-3’tDR-ASO. (I) Quantification of protein levels in H. Data are shown as means ± SEM of at least three independent experiments. The unpaired two-tailed Student’s t-test was used in (C) and (I). The Mann-Whitney test was used in (D). *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar 250μm.

Delivery of tRNA-Asp-GTC-3’tDR alleviates renal ischemia/reperfusion-induced kidney injury in mice

We further conducted GOF/LOF studies in a renal ischemia/reperfusion injury (IRI) model. tRNA-Asp-GTC-3’tDR levels initially increased 1 day post-IRI but returned to baseline by day 7 (Fig. 5A-B). Similar to the UUO model, inhibition of tRNA-Asp-GTC-3’tDR using Asp-GTC-3’tDR-ASO worsened kidney injury, immune cell infiltration, and fibrosis in the kidney post-IRI (Fig. S3A). We next examined whether further elevating tRNA-Asp-GTC-3’tDR levels could boost autophagy and attenuate kidney injury in this IRI model. We evaluated two non-viral vehicles for tDR delivery and identified that polymer-based Polyethylenimine (PEI) efficiently delivered synthetic tDR mimics to kidneys (Fig. S6A-B), aligning with previous studies (37, 38). We administered tDR mimics using PEI in this IRI model (Fig. 5C). Northern blotting confirmed the successful kidney delivery of tRNA-Asp-GTC-3’tDR mimics (Fig. 5D-E). During the typical course of renal IRI, acute tubular injury, cell death, and immune cell infiltration occur within the first two days, followed by gradual repair after day 7, while the resulting fibrosis shows only partial resolution (39). Delivery of tRNA-Asp-GTC-3’tDR mimics significantly alleviated IRI-induced kidney injury compared to the control mimics-treated group, as demonstrated by decreased expression of makers for tubular injury, immune cell infiltration, and fibrosis, along with increased expression of Klotho (Fig. 5F-G). Immunohistology analysis showed reduced apoptosis, and attenuated fibroblast activation and collagen deposition (Fig. 5H-J). Importantly, tRNA-Asp-GTC-3’tDR administration enhanced the autophagy pathway in both sham and IRI kidneys compared to the control group (Fig. 5K-L). Thus, the tRNA-Asp-GTC-3’tDR/autophagy axis serves a crucial protective function in kidneys.

Figure 5. Delivery of tRNA-Asp-GTC-3’tDR mimics protects against renal ischemia/reperfusion injury in mice.

(A) Northern blotting shows the levels of full-length and 3’tDR of tRNA-Asp-GTC in the kidney from the mouse renal IRI model at different time points. (B) Quantification of the levels of full-length and 3’tDR of tRNA-Asp-GTC in A. (C) Schematic illustration of the experimental design for tRNA-Asp-GTC-3’tDR delivery in the mouse renal IRI model. (D) Northern blotting shows the levels of full-length and 3’tDR of tRNA-Asp-GTC in kidney tissues from Sham or IRI mice treated with Ctrl or tRNA-Asp-GTC-3’tDR mimics. (E) Quantification of tRNA-Asp-GTC-3’tDR levels in D. (F) Analysis of Klotho expression in kidney tissues from Sham or IRI mice treated with Ctrl or tRNA-Asp-GTC-3’tDR mimics. (G) Analysis of gene expression related to acute kidney injury, immune cell infiltration, and fibrosis in kidney tissues from Sham or IRI mice treated with Ctrl or tRNA-Asp-GTC-3’tDR mimics (n=4–7 per group). (H to I) Immunostaining analysis of Cleaved-Caspase 3 (H) and Periostin (I) in kidney tissues from Sham or IRI mice treated with Ctrl or tRNA-Asp-GTC-3’tDR mimics. (J) Picrosirius Red staining analysis of collagen levels in kidney tissues from Sham or IRI mice treated with Ctrl or tRNA-Asp-GTC-3’tDR mimics. (K) Immunoblot analysis of the autophagy pathway in kidney tissues from Sham or IRI mice treated with Ctrl or tRNA-Asp-GTC-3’tDR mimics. (L) Quantification of protein levels in K. Data are shown as means ± SEM of at least three independent experiments. The Mann-Whitney test was used in (E), (F), (G), and (L). *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar 250μm.

tRNA-Asp-GTC-3’tDR assembles stable intermolecular G-quadruplex structures

We next explored the molecular mechanism underlying the regulation of autophagic flux by tRNA-Asp-GTC-3’tDR. tRNA-Asp-GTC-3’tDR is a guanine-rich RNA (~50%) containing two 4-guanine motifs (Fig. S2C). Previous studies have reported G-quadruplex (G4) structure formation by two other 5’tDRs containing terminal oligo-guanine motifs (40). To examine potential G4 formation by tRNA-Asp-GTC-3’tDR, we employed complementary analytical approaches. We first assembled tRNA-Asp-GTC-3’tDR mimics under G4-permissive (K⁺) or G4-non-permissive (Li⁺) conditions and analyzed assemblies using native gel electrophoresis. Under G4-permissive conditions, tRNA-Asp-GTC-3’tDR formed multiple intermolecular assemblies, which were absent under G4-non-permissive conditions (Fig. 6A). Circular dichroism (CD) spectroscopy revealed characteristic G4 structural signatures: a monovalent cation-responsive peak at ~263 nm (high intensity in K⁺ environment and low intensity in Li⁺ environment) and a trough at ~240 nm (Fig. 6B). Finally, we stained these tDR assemblies with NMM, a G4-specific fluorescent probe (41) and found that tRNA-Asp-GTC-3’tDR formed various NMM-positive multimolecular G4 structures only under the G4-permissive conditions (Fig. 6C). We also confirmed the G4-forming ability of the 38 nt isoform through electrophoretic gel mobility, CD spectrum, and thermostability analyses (Fig. S7A-C). Thus, the different isoforms of tRNA-Asp-GTC-3’tDR assembled intermolecular G4 structures.

Figure 6. tRNA-Asp-GTC-3’tDR assembles stable intermolecular G-quadruplex structures.

(A) Electrophoretic mobility shift analysis of intermolecular assemblies under G4 permissive (K+) or G4-non-permissive (Li+) conditions for mimics of Ctrl, tRNA-Asp-GTC-3’tDR, and three 4-guanine motif mutants, with tRNA-Ala-AGC-5’tDR serving as positive control. SYBR gold was used to stain the gels. 1GU, mutation of the first 4-guanine motif. 2GU, mutation of the second 4-guanine motif. 12GU, mutation of both 4-guanine motifs. (B) CD spectral analysis of monovalent cation responses for mimics of Ctrl, tRNA-Asp-GTC-3’tDR, and three 4-guanine motif mutants, with tRNA-Ala-AGC-5’tDR serving as positive control. (C) NMM staining of intermolecular assemblies under G4 permissive (K+) or G4-non-permissive (Li+) conditions for mimics of Ctrl, tRNA-Asp-GTC-3’tDR, and three 4-guanine motif mutants, with tRNA-Ala-AGC-5’tDR serving as positive control. (D) SYBR staining shows the stability of assembled tDR mimics following 16 hours of exposure to crude cell lysate. (E) Immunoblot analysis of protein levels of LC3B and β-ACTIN in HEK cells transfected with wild type (WT) or mutants of tRNA-Asp-GTC-3’tDRs. (F) Quantification of autophagic protein levels in E. Data are shown as means ± SEM of at least three independent experiments. The unpaired two-tailed Student’s t-test was used in (F). n.s. non-significant, *p < 0.05, **p < 0.01.

To identify the role of 4-guanine motifs in G4 formation, we generated variants with two 4-guanine motifs mutated individually (1GU and 2GU mutants) or simultaneously (12GU mutant) by replacing the second guanine with uridine in each motif. Comprehensive biochemical analyses demonstrated that 1GU and 2GU mutants exhibited decreased capacities to form G4 structures, while 12GU mutants completely abolished G4 structure assembly (Fig. 6A-C, Fig. S7D). G4 structure assembly significantly enhanced its molecular stability, as evidenced by the detection of intact tRNA-Asp-GTC-3’tDR mimics following a 16-hour incubation with crude cell lysate (Fig. 6D). In contrast, 4-guanine motif mutants assembled under G4-permissive conditions, control mimics, and tDR mimics assembled under the G4-non-permissive condition, all failed to maintain their stability (Fig. 6D). Functionally, 1GU and 2GU mutants showed reduced ability to regulate autophagy compared to wild-type (WT), while 12GU mutation completely eliminated its regulatory capacity (Fig. 6E-F, Fig. S7E). Thus, tRNA-Asp-GTC-3’tDR assembled stable intermolecular G4 structures, which were critical for its stability and functionality.

tRNA-Asp-GTC-3’tDR sequesters PUS7 proteins to activate autophagy

Functions of tDRs depend on their protein interactions (12–15). To identify functional proteins binding to tRNA-Asp-GTC-3’tDR, we transfected biotin-conjugated tRNA-Asp-GTC-3’tDR mimics into HEK cells and performed streptavidin pulldown followed by proteomics analyses. The analysis revealed that pseudouridine synthases (PUS1, PUS4, and PUS7) and DHX36, a G4-specific helicase, are the top 4 binding proteins of tRNA-Asp-GTC-3’tDR (Fig. 7A). This was confirmed by the probability of protein interaction analysis (Table S2) using the SAINTexpress algorithm (42) and validated by immunoblotting (Fig. 7B). Analysis of human kidney single-cell RNA sequencing data (43) showed that these genes are abundantly expressed across various kidney cell types (Fig. S8A). We then generated stable cell lines expressing two distinct shRNAs targeting each candidate. We found that PUS7 knockdown resulting from two separate shRNAs with different knockdown efficiency led to a significant PUS7-dose-dependent elevation in LC3B lipidation, and the additional tRNA-Asp-GTC-3’tDR transfection showed diminished ability to augment LC3B lipidation compared to control conditions (Fig. 7C-D, Fig. S8B). In contrast, knockdown of PUS1, PUS4, and DHX36 did not appear to notably impact tRNA-Asp-GTC-3’tDR-mediated autophagic flux activation, although we could not exclude a modest effect of PUS4 knockdown (Fig. S8C-H). Thus, PUS7 is the key downstream effector of tRNA-Asp-GTC-3’tDR in modulating autophagy flux.

Figure 7. tRNA-Asp-GTC-3’tDR activates autophagic flux through PUS7 protein sequestration.

(A) Proteomics analysis of binding proteins of tRNA-Asp-GTC-3’tDR in HEK cells. Significant binding candidates (p-value <0.05) are highlighted in orange (log₂[FoldChange] < −1) or blue (log₂[FoldChange] >1). (B) Immunoblot validation of the top 4 tRNA-Asp-GTC-3’tDR binding proteins, following pull-down of biotin-conjugated Ctrl or tRNA-Asp-GTC-3’tDR mimics from HEK cells using streptavidin beads. (C) Immunoblot analysis of PUS7 and LC3B protein levels in scramble shRNA (shScr) and PUS7-silenced (shPUS7–1 and shPUS7–2) HEK cells transfected with Ctrl or tRNA-Asp-GTC-3’tDR (3’tDR) mimics. (D) Quantification of protein levels in C, with the right Y axis showing the fold change of LC3B-II/LC3B-I ratio between tRNA-Asp-GTC-3’tDR and Ctrl groups. (E) Immunoblot analysis of PUS7 and LC3B protein levels in Ctrl and PUS7-silenced cells treated with DMSO or BafA1 for 6 hours. (F) Quantification of protein levels in E. (G) Immunoblot analysis of PUS7 and LC3B protein levels in PUS7 overexpressing cells treated with DMSO or BafA1 for 6 hours. (H) Quantification of protein levels in G. (I) Schematic illustration of three T-arm mutants of tRNA-Asp-GTC-3’tDR. (J) Immunoblot analysis comparing PUS7 binding affinity of three T-arm mutations (4CA, 12UA, and Pusbm) versus wild-type tRNA-Asp-GTC-3’tDR. (K) Immunoblot analysis comparing PUS7 binding affinity of three T-arm mutations (4CA, 12UA, and Pusbm) versus Ctrl mimics. (L) Immunoblot analysis of LC3B protein levels in HEK cells transfected with different mutants of tRNA-Asp-GTC-3’tDR and treated with DMSO or BafA1 for 6 hours. (M) Quantification of protein levels in L. Data are shown as means ± SEM of at least three independent experiments. The unpaired two-tailed Student’s t-test was used in (D), (F), (H), and (M). *p < 0.05, **p < 0.01, ***p < 0.001.

We next assayed autophagic flux in PUS7-deficient and overexpressing cells and found that PUS7 deficiency phenocopies the effects of tRNA-Asp-GTC-3’tDR in enhancing autophagic flux (Fig. 7E-F, Fig. S9A-B). Conversely, PUS7 overexpression blocked autophagic flux (Fig. 7G-H), similar to tRNA-Asp-GTC-3’tDR inhibition. Given that tRNA-Asp-GTC-3’tDR did not affect PUS7 protein levels (Fig. 7C), these results suggested that tRNA-Asp-GTC-3’tDR enhances autophagic flux through binding and inhibiting PUS7 function. Analysis of cellular fractions from hypoxic HEK cells showed that both PUS7 proteins and endogenous tRNA-Asp-GTC-3’tDR localized to the cytoplasm (Fig. S9C), further supporting the hypothesis.

To investigate the essential sequences responsible for PUS7 protein interaction, we first examined the role of G4 structures. Cell-free competitive binding assays revealed that G4-structured tRNA-Asp-GTC-3’tDR markedly increased its binding affinity for PUS7 proteins compared to non-G4-structured tDRs (Fig. S10A-B). The G4-deficient 12GU mutant showed reduced binding affinity for PUS7 (Fig. S10C). We further investigated sequence specificity by generating several tRNA-Asp-GTC-3’tDR mutants, including a 4CA mutant (four continuous cytosines in the T-stem substituted with adenines), a 12UA mutant (four potential pseudouridylated uridines in the T-loop replaced with adenines), and a Pusbm mutant (the putative PUS7-RNA binding GUUCGA motif (44) mutated to CAAGCU) (Fig. 7I). Initial characterization demonstrated that all three mutants successfully formed stable G4 structures (Fig. S10D-G). However, all three mutants exhibited significantly reduced, but not abolished, binding affinities for PUS7 proteins compared to WT (Fig. 7J-K), highlighting the crucial role of the T-arm region in PUS7 protein interactions. Functionally, these mutants demonstrated diminished capacities to enhance autophagic flux compared to WT (Fig. 7L-M), correlating with their reduced (but not abolished) PUS7 binding capacity. Thus, the functionality of tRNA-Asp-GTC-3’tDR was critically dependent on both G4 structure assembly and PUS7 protein interaction.

tRNA-Asp-GTC-3’tDR blocks PUS7-mediated pseudouridylation of histone mRNAs and triggers RNA autophagy

Because PUS7 silencing phenocopies tRNA-Asp-GTC-3’tDR’s function, we hypothesized that PUS7’s targeting of other RNA species (including mRNAs, tRNAs, rRNAs, and snRNAs (45)) rather than tDR itself, may play a crucial role in regulating autophagy flux. We therefore conducted transcriptomic analyses of both long and small RNAs from HEK cells transfected with either control or tRNA-Asp-GTC-3’tDR mimics. While OTTR-seq, a low-bias small RNA sequencing method (8), revealed no substantial changes in small RNA species, including tRNAs, rRNAs, and snRNAs (Fig. S11A-C), differential expression analysis of long RNAs identified significant changes, with histone transcripts representing the majority of the most significantly downregulated long RNAs (Fig. 8A). While tRNA-Asp-GTC-3’tDR triggered a reduction in histone mRNA levels (Fig.8B), histone protein levels remained stable (Fig. S11D), indicating robust maintenance of histone protein homeostasis. The downregulation of these histone mRNAs was also observed in HEK cells following hypoxia treatment or PUS7 silencing (Fig. S11E-F). PUS7 preferentially modifies uridine within the UNUAR consensus sequence on target RNAs (46–48). Analysis revealed that 17 out of 26 downregulated histone mRNAs contain one or many UNUAR motifs (Table S3), indicating that these histone mRNAs are preferential direct targets of PUS7. Cell-free competitive binding assays confirmed that tRNA-Asp-GTC-3’tDR exhibits stronger binding affinity for PUS7 proteins compared to histone mRNAs (Fig. S12A-B). RNA pseudouridylation plays crucial roles in regulating RNA function and stability (49), typically enhancing mRNA stability (50). As expected, T-arm mutations with reduced PUS7 binding affinity showed diminished ability to induce histone mRNA degradation compared to WT, and the G4-deficient mutant completely lost its capacity to downregulate histone mRNAs (Fig. S12C). Thus, the sequestration of PUS7 by tRNA-Asp-GTC-3’tDR resulted in pseudouridylation deficiency in histone mRNAs, leading to their degradation.

Figure 8. tRNA-Asp-GTC-3’tDR inhibits PUS7-mediated histone mRNA pseudouridylation and activates RNA autophagy.

(A) Volcano plot shows the differentially expressed genes in HEK cells transfected with tRNA-Asp-GTC-3’tDR mimics compared to Ctrl mimics. (B) Validation of the top significantly regulated genes by tRNA-Asp-GTC-3’tDR treatment. (C) Expression analysis of tRNA-Asp-GTC-3’tDR-downregulated histone mRNAs in HEK cells transfected with tDR mimics and treated with or without chloroquine (CQ) for 2 hours. (D) RNA FISH assay shows the accumulation of histone H1–2 mRNA (DNA probe, in red) in lysosomes (LAMP1 antibody, in green) within HEK cells following tRNA-Asp-GTC-3’tDR transfection and CQ treatment. (E) Pseudouridylated RNA immunoprecipitation followed by qRT-PCR analysis of H4C3 mRNA pseudouridylation levels at UGUAG and non-UGUAG regions in HEK cells transfected with vectors expressing GFP (control), PUS7 (wild-type, WT), or PUS7 D294A (catalytically inactive mutant), and mimics of control or tRNA-Asp-GTC-3’tDR. (F) The expression levels of histone mRNAs in kidney tissues from renal IRI mice treated with Ctrl or tRNA-Asp-GTC-3’tDR mimics. (G) Northern blotting shows the levels of tRNA-Asp-GTC-3’tDR in kidney tissues from human patients with high eGFR (>90) or low eGFR (<60). (H) Quantification of levels of tRNA-Asp-GTC-3’tDR in G. (I) Expression analysis of histone H4C3 mRNA levels in kidney tissues of human patients with high eGFR (>90) or low eGFR (<60). Data are shown as means ± SEM of at least three independent experiments. The unpaired two-tailed Student’s t-test was used in (B), (C), and (E). The Mann-Whitney test was used in (F), (H), and (I). *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar 50μm.

Because tRNA-Asp-GTC-3’tDR activates autophagic flux through sequestering PUS7 from histone mRNAs, we conjectured that pseudouridylation-deficient histone mRNAs are directed towards the autophagy pathway for degradation, thereby triggering RNA autophagy. To test this hypothesis, we treated tRNA-Asp-GTC-3’tDR-transfected cells with chloroquine (CQ), an inhibitor that blocks autophagosome-lysosome fusion (51). Remarkably, 2 hours of CQ treatment substantially slowed tRNA-Asp-GTC-3’tDR-induced histone mRNA degradation (Fig. 8C), indicating an autophagy-associated degradation of these histone mRNAs. We then employed an in-situ RNA hybridization assay following CQ pretreatment to examine if the pseudouridylation-deficient histone mRNAs were transported to the autophagosome/lysosome pathway for degradation. We detected a significant accumulation of H1–2 mRNAs in LAMP1-positive lysosomes in tRNA-Asp-GTC-3’tDR-transfected cells (Fig. 8D). Next, pseudouridylated RNA pulldown using a pseudouridine-specific antibody revealed that the pseudouridylation levels in the UGUAR region of histone mRNAs were significantly diminished in tRNA-Asp-GTC-3’tDR-transfected HEK cells after CQ pretreatment compared to the control (Fig. S12D), and the decreased pseudouridylation levels were restored by the overexpression of wild-type but not dominant negative PUS7 (Fig. 8E). To investigate whether pseudouridylation-deficient histone mRNAs directly trigger RNA autophagy, we generated in vitro transcribed H1–2 mRNAs with either fluorouridine or pseudouridine incorporated in the places of uridine, to model pseudouridine deficiency or constitutive pseudouridylation, respectively. When transfected to HEK cells, fluorouridine-modified H1–2 mRNAs significantly enhanced autophagic flux compared to pseudouridine-modified H1–2 mRNAs (Fig. S12E-F). Thus, tRNA-Asp-GTC-3’tDR/PUS7 interaction resulted in pseudouridylation deficiency of histone mRNAs, triggering RNA autophagy.

We also investigated this RNA autophagy pathway in our UUO and IRI models. The initial day following UUO surgery showed elevated tRNA-Asp-GTC-3’tDR levels accompanied by a significant reduction in histone mRNA expression (Fig. S12G). By day 3 post-surgery, histone mRNA levels began to recover as tRNA-Asp-GTC-3’tDR levels moderately decreased (Fig. S12G). However, these recoveries were subsequently hindered by the renewed increase in tRNA-Asp-GTC-3’tDR levels after day 5 in the UUO model (Fig. S12G). Notably, in our IRI kidney tissues, tRNA-Asp-GTC-3’tDR administration significantly decreased histone mRNA abundance (Fig. 8F). Thus, tRNA-Asp-GTC-3’tDR negatively regulated histone mRNA levels in mouse kidneys.

Lastly, we examined this tDR/histone mRNA axis in human urine and kidney tissues. We analyzed tRNA-Asp-GTC-3’tDR levels in urine samples collected from a cohort of pregnant women with or without preeclampsia. Proteinuria, a hallmark of preeclampsia associated with renal dysfunction (52) was present in preeclamptic patients. We observed elevated levels of tRNA-Asp-GTC-3’tDRs in the urine of pregnant women with preeclampsia compared to those without (Fig. S13A-B).

Additionally, within patients undergoing total kidney nephrectomy enrolled in the PRECISE cohort (53–55), patients with early-stage chronic kidney diseases (CKD stage 3) with an eGFR lower than 60 ml/min per 1.73 m² were compared to patients with an eGFR higher than 90 ml/min per 1.73 m² matched for age, gender, and BMI (Table S4). Kidney tissue RNAs from these groups were then analyzed. Notably, the levels of tRNA-Asp-GTC-3’tDR were markedly elevated in patients with CKD stage 3 compared to patients with normal eGFR (Fig. 8G-H). Moreover, the expression of histone H4C3 mRNA was significantly reduced in patients with CKD stage 3 compared to patients with normal eGFR (Fig. 8I). Together, these data provided validation for the regulation of tRNA-Asp-GTC-3’tDR in human kidney disease and supported for an association between tRNA-Asp-GTC-3’tDR and the mRNA autophagy pathway.

DISCUSSION

Although 5’tDRs, encompassing 5’tRNA halves and 5’tRNA fragments, have been detected across tissues and biofluids and extensively studied (12, 16, 56, 57), investigations into 3’tDR biology remain scarce. This knowledge gap stems partly from base modifications at the 3’end of tRNAs, particularly 1-methyladenosine on position 58, which can impede reverse transcription during small RNA sequencing (5, 20). By employing ARM-seq, we identified abundant tRNA-Asp-GTC-3’tDR in kidney cells and tissues. While tDRs have been functionally linked to translation, ribosome biogenesis, gene silencing, stress granule assembly, and apoptosis (9, 11), we discovered an important role for 3’tDR in autophagy regulation. We showed that tRNA-Asp-GTC-3’tDR enhanced autophagic flux, while its silencing blocked autophagic flux. The abundance of tRNA-Asp-GTC-3’tDR in kidney cells and tissues, even at basal conditions, aligns with kidneys’ high metabolic activity and physiological hypoxia (58). Kidneys maintain the highest levels of baseline autophagy activity among organs and rely on autophagy for maintaining kidney homeostasis, structure, and function (33, 59). Autophagy in kidneys further increases during stress conditions, such as nephrotoxins, sepsis, and ischemia/reperfusion (60). Indeed, we found increased tRNA-Asp-GTC-3’tDR levels across multiple murine kidney disease models and in human samples from patients with kidney diseases. Inhibition of tRNA-Asp-GTC-3’tDR in injured kidney tissues using ASOs blocked the autophagy pathway and exacerbated kidney injury, while increasing its abundance using synthetic tDR mimics enhanced autophagy activity and was reno-protective. These data support a role for tRNA-Asp-GTC-3’tDR in maintaining basal autophagy in kidney cells and enhancing autophagy as a compensatory response to injury.

While miRNAs and long non-coding RNAs have been recently implicated in autophagy regulation, primarily by modulating ATG gene expression (61), our study unveiled a distinct mechanism for tDRs. First, tRNA-Asp-GTC-3’tDR is a 3’tDR that is dependent on two internal 4-guanine motifs to form G4 structures. Previously, only two 5’tDRs were known to form G4 structures through the 5’-terminal oligo-guanine motif (12, 40). The G4 structures were essential for the stability and functionality of this tDR, and mutations that disrupted formation of G4 structures had diminished capacity for regulating autophagy. Second, tRNA-Asp-GTC-3’tDR’s interaction with PUS7 was crucial for enhancing autophagic flux. Mutations in the T-arm of tRNA-Asp-GTC-3’tDR significantly reduced both PUS7 binding affinity and the ability to enhance autophagic flux, despite their capacity to form G4 structures. Third, tRNA-Asp-GTC-3’tDR induced RNA autophagy. tRNA-Asp-GTC-3’tDR sequestered PUS7 from its target histone transcripts, reducing their pseudouridylation. These pseudouridylation-deficient mRNAs were trafficked into the autophagosome-lysosome pathway for degradation, triggering RNA autophagy. The absence of polyadenylation in histone mRNAs (62) may explain their higher sensitivity to pseudouridylation as a mechanism for being targeted by RNA autophagy. The induction of RNA autophagy was associated with enhanced general autophagy flux and appears to be an important adaptive response in kidney cells.

There are several limitations of our study. First, while ANG-mediated tRNA cleavage typically produces a 5’-OH on 3’tDR and a 3’-cP on 5’tDR and retains extensive modifications from parent tRNAs (63), our synthesized tDR mimics conclude with 5’-OH and 3’-OH without the modifications presented on endogenously generated tDRs, owing to technical challenges and poorly characterized modification patterns and terminal structures. Similarly, the intermolecular G4-forming ability was only determined for synthetic tDR mimics. Future strategies to generate endogenous tDRs may definitively validate these findings. Second, while providing robust evidence for tRNA-Asp-GTC-3’tDR’s role in mediating autophagic flux in HEK and other kidney cells, we observed distinct cellular responses between HEK cells and primary kidney cells. Under hypoxia, tDR silencing in HEK cells led to impaired autophagosome maturation, while tDR silencing in human kidney cells led to an additional impairment in autophagy initiation. This may reflect inherent differences between HEK cells and adult kidney cells (64–66). Third, our investigation of the tDR/PUS7 axis focused primarily on histone mRNAs, leaving the effects on other downregulated transcripts unexplored. We also observed upregulation of other RNA species, including mRNAs, long non-coding RNAs, rRNAs, and snRNAs, though the underlying mechanisms, whether through pseudouridylation or other pathways, require further investigation. Finally, the definitive mechanistic connection between RNA autophagy and general autophagy remains to be elucidated.

MATERIALS AND METHODS

Cell Culture

Human embryonic kidney 293 (HEK) 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# 16000044) and 1% Penicillin/Streptomycin (Thermo Fisher, Cat# 15140122). For the hypoxia treatment, HEK cells were fed with complete D10 medium and maintained in hypoxia chamber with 0.1% oxygen for 24 hours. Human primary renal proximal tubule epithelial cells were purchased from Lonza (Lonza, CC-2553) and cultured in the renal epithelial cell growth medium (Lonza, CC-3190). Human primary kidney fibroblasts were purchased from Cell Biologics (Cell Biologics, H-6016) and cultured in the complete fibroblast medium (Cell Biologics, M2267). Human primary glomerular microvascular endothelial cells were purchased from Cell Systems (Cell Systems, ACBRI 128 V) and cultured in the complete classic medium with serum and Culture-Boost (Cell Systems, 4Z0–500). Primary human renal glomerular epithelial cells were purchased from Cell Applications (Cell Applications, 942–05n) and cultured in the renal epithelial cell growth medium (Lonza, CC-3190). Primary human glomerular mesangial cells were purchased from Cell Systems (Cell Systems, ACBRI 127 V) and maintained in the complete classic medium with serum and Culture-Boost (Cell Systems, 4Z0–500).

Human immortalized podocyte cell line (CIHP-1) (67) was a gift kindly provided by Dr. Moin A. Saleem from the University of Bristol and cultured in R10 medium, which consists of RPMI-1640 basal medium with GlutaMAX (Thermo Fisher, Cat# 61870127) supplemented with 10% FBS (Thermo Fisher, Cat# 16000044), 1% Insulin-Transferrin-Selenium (Thermo Fisher, Cat# 41400045), and 1% Penicillin/Streptomycin (Thermo Fisher, Cat# 15140122). Podocytes were first amplified at the 33 °C CO₂ incubator and matured by culturing them at a 37 °C CO₂ incubator for two weeks before experiments.

To block the autophagic flux in these cells, 20 nM Bafilomycin A1 (BafA) or 50 μM Chloroquine (CQ) were added to the cell culture medium for 6 hours or 2 hours, respectively, before collecting the cells for RNA or protein analysis.

In Vitro Nucleic Acid Oligo Transfection

For the transfection of RNA and DNA oligos, 50 nM mimics (synthesized by Integrated DNA Technologies), 50 nM tDR biogenesis reporter (synthesized by Dharmacon), or 100 nM ASOs (synthesized by Qiagen) were transfected into the cells using siPORT NeoFx (Thermo Fisher, Cat# AM4511) or Lipofectamine RNAiMax (Thermo Fisher, Cat# 13778150) by following the manufacturer’s instructions, unless otherwise specified. tDR mimics synthesized at Integrated DNA Technologies at default mode end with 5’-OH and 3’-OH and no additional modification was added.

The concentration of 50 nM for tDR mimics was determined by comparing the levels of transfected tRNA-Asp-GTC-3’tDR mimics at different concentrations with its physiological levels in hypoxic HEK cells. The analysis indicated that the physiological level of tRNA-Asp-GTC-3’tDR in hypoxic HEK cells corresponds to approximately 1 nM (Fig. S14). Considering that lipid nanoparticle-mediated RNA delivery has limited endosomal escape efficiency of only 1~2%, with the majority of RNA/particle complexes remaining trapped in the endosomal vesicles before eventual lysosomal degradation (68, 69), our transfection protocol using 50 nM tDR mimics with Lipofectamine translates to an effective cytoplasmic delivery of 1 nM (2%) functional tDRs,

Generation of Stable Transgenic Cell Lines

For gene knockdown, shRNAs targeting the mRNA of PUS1, PUS4, PUS7, or DHX36 were designed and cloned into the pLKO.1-TRC cloning lentiviral vector, a gift from David Root (Addgene plasmid # 10878). A scramble shRNA was also designed as a control. The shRNA sequences are listed in Table S5. For building autophagy reporter cell lines, GFP-LC3B and RFP-GFP-LC3B DNA fragments were synthesized by GenScript and subcloned into pLV-EF1a-IRES-puro lentiviral vector, a gift from Tobias Meyer (Addgene, Plasmid #85132). For PUS7 overexpression, GFP, RFP-PUS7, and RFP-PUS7 D294A DNA fragment were synthesized by GenScript and subcloned into the pLV-EF1a-IRES-puro lentiviral vector. For lentivirus packaging, lentiviral vector DNA (10 μg) was transfected into HEK 293T cells (one of 100 mm dish) together with the packaging plasmids, ps-PAX2 (7.5 μg) and pMD2.G (5 μg), using the Lipofectamine 3000 (Thermo Fisher, Cat# L3000015) or PEI-max (Polysciences, Cat# 24765) as previously described (70). The supernatant containing the lentiviruses was concentrated using the Lenti-X concentrator (Takara, Cat# 631232) and then added to HEK cells supplied with 8 μg/ml polybrene. Stable transgenic cell lines were selected by 10 μg/ml puromycin for 3–6 days. Angiogenin knockout HEK cells were generated using paired gRNAs, as mentioned previously (20).

Animal Studies

Wild-type C57BL/6 mice were purchased from Jackson Laboratory (strain# 000664) and maintained under pathogen-free conditions. All procedures conformed to the animal welfare regulations of the Massachusetts General Hospital or Brigham and Women’s Hospital Sub-Committees on Animal Research Care and under the guidelines on the use and care of laboratory animals for biomedical research published by the National Institutes of Health (No. 85–23, revised 1996). All mice used for experiments were within 8–14 weeks of age. For examining tDR abundance in different mouse models, 3–4 mice were used per group. For tDR GOF/LOF studies in mouse models, 4–7 mice were used per sham group, and 6–10 mice were used per surgery group. The detailed number of mice used per experiment is indicated in the figures or figure legends.

To perform uninephrectomy, male 10–12 weeks of age C57BL/6 mice were anesthetized with isofluorane by inhalation. A 1 cm lumbar incision was performed, and one of the kidneys was exposed after separating the muscle and fascia. The renal vein and ureter from the kidney were occluded together by two ligature knots of 6.0 Polyglycolic acid absorbable suture. The kidney was then removed by cutting the renal vessels between the two knots. The muscle layer was then closed using absorbable surgical sutures vicryl and the outer skin was closed with nylon sutures. For the sham surgery, perform the same surgery procedures, including exposure of the kidney, dissection of tissue, and wound closure, but without the removal of the kidney.

To induce a protein-overloaded model, male 10–12 weeks of age C57BL/6 mice were intraperitoneally injected with endotoxin-free BSA (Sigma-Aldrich, Cat# A9430) for 7 consecutive days with increasing doses (2, 4, 6, 8, 10, 10, 10 mg/g body weight). PBS was used as the vehicle to dissolve the BSA.

To induce the unilateral ureteral obstruction (UUO) model, male 10–12 weeks of age C57BL/6 mice were anesthetized with pentobarbital sodium (50 mg/kg, i.p.), 1 cm of incision was performed on the left back of the mice and the ureter of the left kidney was ligated with 2 silk threads 3.0. After ureteral obstruction, the muscle layer was closed using absorbable surgical sutures vicryl, and the outer skin was stapled using sterile staples or closed with nylon sutures. Mouse kidneys were collected on day 6 post-UUO surgery. For the sham surgery, perform the same surgery procedures, including exposure of the kidney, dissection of tissue, and wound closure, but without ureteral obstruction.

To induce the renal ischemia/reperfusion injury (IRI) model, male 10–12 weeks of age C57BL/6 mice were anesthetized with pentobarbital sodium (50 mg/kg, i.p.). Uninephrectomy was first performed to remove the right kidney. A week later, mice were anesthetized with isoflurane by inhalation. A 1 cm of incision was performed on the left back of the mice, and the pedicle of the left kidney was clamped with non-traumatic microaneurysm clamps (Roboz) for 18 minutes and then released. After reperfusion, the muscle layer was closed using absorbable surgical sutures vicryl, and the outer skin was stapled using sterile staples or closed with nylon sutures. Mouse kidneys were collected on day 6 post-IRI surgery. For sham surgery, perform the same surgery procedures, including exposure of the kidney, dissection of tissue, and wound closure, but without uninephrectomy or pedicle clamping.

In Vivo ASO Administration

ASOs with fully phosphorothioate-modified backbones were synthesized by Qiagen. The sequences of ASOs are the same as those used in vitro and detailed in Table S5. Modified ASO targeting tRNA-Asp-GTC-3’tDR or negative control was injected (30 mg/kg body weight, i.v.) into mice undergoing sham or UUO surgery beginning one day before surgery and continuing every other day until day 6 post-UUO surgery. For baseline inhibition, modified ASO targeting tRNA-Asp-GTC-3’tDR or negative control was injected subcutaneously (30 mg/kg body weight) into mice without surgery once a week for 4 weeks. Modified ASO targeting tRNA-Asp-GTC-3’tDR or negative control ASO were injected (30 mg/kg body weight, i.v.) into mice beginning five days before IRI surgery and continuing every other day until day 4 post-UUO surgery.

In Vivo Delivery of tDR Mimics

RNA mimics of tRNA-Asp-GTC-3’tDR and control sequences were synthesized by Integrated DNA Technologies using standard parameters, yielding molecules with 5’-OH and 3’-OH termini without additional modifications.

For DharmaFECT-mediated in vivo delivery, tDR mimics (1.5 mg/kg body weight) were combined with DharmaFECT1 transfection reagent (Dharmacon, Cat# T-2001–03) in RNase-free water, incubated for 15 minutes at room temperature, and subsequently resuspended in 100 μl RNase-free water for intravenous injection.

For PEI-mediated in vivo delivery, transfections were performed following the in vivo jetPEI protocol (Polyplus, Cat# 101000030), maintaining a PEI nitrogen to RNA phosphate molar ratio of 8. For a 30g mouse receiving 3 mg/kg body weight: tDR mimics (9 μl of 10 mg/ml stock in TE buffer) were diluted in 91 μl of RNase-free 5% glucose solution in one tube, while 14.4 μl of in vivo jetPEI or 150 mM PEI-MAX (nitrogen molar concentration; Polysciences, Cat# 24765) was diluted in 85.6 μl of RNase-free 5% glucose solution in a separate tube. The contents of both tubes were mixed thoroughly and incubated at room temperature for 15 minutes. The resulting 200 μl mixture was administered via intravenous injection. For the 1.5 mg/kg body weight dose, a 100 μl mixture was used. Injection volumes were proportionally adjusted according to individual mouse weights. In the renal IRI model, tDR/PEI mixtures were administered (3 mg/kg body weight, i.v.) to sham or IRI mice beginning five days before IRI surgery and continuing every other day until day 6 post-IRI surgery.

Patient Sample Collection

Kidney tissue samples were obtained from patients undergoing total nephrectomy enrolled in the PRECISE cohort at the University of Michigan (institutional review board numbers: HUM00165536 and HUM00052918). Patient information is described in Table S4. Kidney tissue was placed in preservatives shortly after extraction of the kidney from patients to minimize ischemic time. Tissue samples were stored in RNAlater (Thermo Fisher, Cat# AM7021) and frozen at −80°C until use. Clinical data were abstracted using the honest broker system as described (53) or from the participant’s electronic medical record.

Urine samples were collected from patients with and without preeclampsia with written informed consent under IRB protocol #181917, approved by the Human Research Protections program at the University of California, San Diego (UC San Diego). Proteinuria was defined as ≥1+ on urine dip or urinalysis on 2 occasions, ≥0.3 on P/C ratio, or ≥300 mg total protein on 24-hour urine collection. Clean-catch urine samples were collected into sterile containers, centrifuged at 2000 X g for 10 minutes at room temperature to pellet cells and cellular debris, and the supernatant was removed, aliquoted, and stored at −80 °C within 2 hours of collection until further processing. The miRCURY Exosome Cell/Urine/CSF Kit (Qiagen, Cat# 76743) was used to precipitate extracellular vesicles from 4 mL of urine using the manufacturer’s protocol, followed by RNA extraction using Qiazol and miRNeasy Micro Kit spin columns using the manufacturer’s protocol (Qiagen, Cat# 217084). Small RNA libraries were prepared for sequencing as previously described (71), using the NEB Next Small RNA Library Prep Set for Illumina (New England Biosciences, Cat# E7330L) according to the manufacturer’s protocol with the following modifications. All reactions were conducted at 1/5th the volume with 1.2 μL of RNA input, adapters were diluted 1:6 of the supplied concentration, and 18 PCR cycles were conducted. Small RNA libraries were cleaned using the Zymo DNA Clean and Concentrate kit (Zymo Research, Cat# D4103), pooled, and size-selected using the Pippin Prep HT with a 125–150bp size limit to remove adapter dimers. Size-selected libraries were sequenced on the Illumina NovaSeq 6000 as 75bp single-end reads by the Institute for Genomic Medicine at UC San Diego (funded by National Institutes of Health SIG grant S10OD026929). Small RNA-Seq data were processed using the Genboree exceRpt Small RNA-Seq Pipeline (72). Briefly, FASTQ files underwent adaptor trimming and mapping to the endogenous human genome (hg38) using STAR v2.4.2a, with a minimum insert length set to 15 nucleotides and no mismatches permitted on the Genboree workbench, and tRNA counts were extracted.

Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)

qRT-PCR was performed as previously described (20). Briefly, cells or tissues were immediately lysed by adding TRIzol^™ (Thermo Fisher, Cat# A33251) after collection, and total RNAs were isolated by following the manual. 1 μg of total RNA was reverse transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher, Cat# 4368813) or The SuperScript III First-Strand Synthesis System (Thermo Fisher, Cat# 18080051). 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 S5.

Northern Blotting

Northern blotting was performed as previously reported (20). Briefly, denatured 1 ~5 μg total RNAs were separated by 15% Criterion TBE-Urea PreCast Gels (Bio-Rad, Cat# 3450092 or 3450093). The gels were stained with SYBR-Gold (Thermo Fisher, Cat# S11494) and transferred onto positively charged Nylon membrane (Sigma-Aldrich, Cat# 11209299001) using Trans-Blot Turbo Transfer System (Bio-Rad). The membranes were then crosslinked with EDC (Sigma-Aldrich, Cat# E7750) at 60°C for 1~2 hours and prehybridized with ULTRAhyb Ultrasensitive Hybridization Buffer (Thermo Fisher, Cat# AM8669). 50 pmol/ml Biotin-labeled LNA-modified DNA probes (synthesized by Qiagen) were used for hybridization at 37°C overnight. After washing sequentially with low-stringent buffer, high-stringent buffer, and 1× SSC, the blots were then processed and developed using Chemiluminescent Nucleic Acid Detection Module Kit (ThermoFisher, Cat# 89880). For all northern blots, we used a single probe capable of detecting both full-length tRNA-Asp-GTC and its corresponding tDRs within one intact plot. The complete northern blot images from all figures are provided in Fig. S15-16. The sequences for probes are provided in Table S5.

Immunoblotting

Immunoblotting was performed as previously described (20). 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. Tissue samples were cut into small pieces and homogenized in RIPA buffer containing Protease and Phosphatase Inhibitor Cocktail and PMSF, followed by sonication. The protein concentration of clarified cell or tissue lysate was then measured using the BCA protein assay kit (Thermo Fisher, Cat# 23227). 5 ~ 20 μg of total protein per lane was separated by SDS-PAGE gel and transferred to PVDF membrane. 5% BSA in TBST was used for blocking and primary antibody incubation. HRP-conjugated secondary antibodies were then applied, and the blots were developed using SuperSignal^® West Femto Maximum Sensitivity Chemiluminescent Substrate (Thermo Fisher, Cat# 34094). The antibodies used are listed in Table S6.

Live imaging

For live imaging of lysosomal activity (using Lysosomal Intracellular Activity Assay Kit, Abcam, ab234622), autophagy (GFP-LC3B reporter cells), and autophagic flux (RFP-GFP-LC3B reporter cells), cells were incubated with Hoechst 33342 (Thermo Fisher, Cat# H3570) for 5–15 min and fed with fresh medium, within which the basal medium was replaced with FluoroBrite^™ DMEM (Thermo Fisher, Cat# A1896701). Images were captured using the Leica SP8 confocal laser scanning microscope system.

Fluorescent In Situ Hybridization (FISH)

For the RNA FISH assay, cells were pretreated with CQ for 2 hours, followed by fixation in 3.7% formaldehyde (in PBS) and permeabilization in ice-cold methanol at 4°C for 20 min. After washing, cells were incubated with LAMP1 antibody (ThermoFisher, Cat# MA5–29385) diluted in 1X PBST at 4°C overnight, followed by the incubation of fluorescent secondary antibody. Immunostained cells were fixed again in 3.7% formaldehyde (in PBS) and washed with 1X PBST twice and 1X Stellaris RNA FISH Wash Buffer A (LGC Biosearch Technologies, Cat# SMF-WA1–60) once. Cells were then incubated with 1X Stellaris RNA FISH Hybridization Buffer (LGC Biosearch Technologies, Cat# SMF-HB1–10) containing 125 nM Cy3-labeled histone H1–2 DNA probe (synthesized by GenScript) at 37°C for 4–16 hours, followed by washing with 1X Stellaris RNA FISH Wash Buffer A once and 1X Stellaris RNA FISH Wash Buffer B (LGC Biosearch Technologies, Cat# SMF-WB1–20) once. The cells were then mounted by ProLong^™ Glass Antifade Mountant with NucBlue^™ Stain solution and subjected to Leica SP8 for imaging.

Mouse Kidney Tissue Histology

Kidney tissues of UUO models with the injection of LNA antisense were perfused with cold PBS before harvesting. Half of the kidneys were fixed in formalin, dehydrated in 70% EtOH, and embedded in paraffin. Paraffin-embedded tissues were cut into 5- to 7-μm sections and stained with the Hematoxylin and Eosin Staining Kit (Abcam, Cat# ab245880) or the Picrosirius Red staining kit (Polysciences, Cat# 09400; or Abcam, Cat# AB150681). Another half of the kidneys were fixed in 4% PFA, dehydrated in 20% Sucrose, and embedded in optimal cutting temperature (OCT). OCT-embedded tissues were cut into 5- to 10-μm sections and subjected to immunofluorescent staining. Briefly, the frozen sections were permeabilized in 1× PBS containing Triton X-100 (0.1%) for 5–15 minutes and labeled with primary antibodies, followed by the incubation of secondary fluorescent antibodies. ProLong^™ Glass Antifade Mountant with NucBlue^™ Stain solution (Thermo Fisher, Cat# P36981) was then used for nuclei staining and mounting.

Transmission Electron Microscopy (TEM)

TEM was performed in the PMB Microscopy Core at Massachusetts General Hospital. Briefly, following perfusion/immersion fixation with periodate-lysine-paraformaldehyde reagent (4% paraformaldehyde, 75 mM lysine, 10 mM periodate fixative in 0.15 M sucrose, 37.5 mM sodium phosphate), small pieces of kidney tissues with the injection of LNA antisense were immersed in 2.0% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4. They were postfixed for 1 h in 2% osmium tetroxide, dehydrated through a graded series of ethanol solutions up to 100%, and embedded in Epon (Electron Microscopy Sciences, Fort Washington, PA). Thin (~70 nm) sections were collected onto copper slot grids and stained with uranyl acetate and Reynold’s lead citrate before examination. Sections were examined in a JEM-1011 transmission electron microscope (JEOL, Tokyo, Japan) at 80 kV.

Electrophoretic Gel Shift Assay

tDR mimics were purchased by Integrated DNA Technologies and dissolved in RNase-free water to prepare 100 μM stock solutions. tDR mimics were further diluted to 10 μM in the indicated salt solution containing either 150 mM KCl or 150 mM LiCl (in 10 mM Tris-HCl pH 7.4, 0.1 mM EDTA buffer). Samples were heated to 95 °C for 5 min and then slowly cooled to room temperature using a Touchdown program in a Bio-Rad PCR cycler to facilitate proper folding. For native gel analysis, 20 pmol of folded RNA samples were electrophoresed on a 20% non-denaturing gel. Gels were post-stained in a solution with 1X SYBR Gold (Thermo Fisher, Cat# S11494) or 50 μM NMM (Santa Cruz Biotechnology, Cat# sc-396879A) in 0.5X TBE for 10 min and visualized using a 305 nm UV transilluminator.

Circular dichroism (CD) spectroscopy

CD spectra were acquired using a JASCO J815 spectropolarimeter. tDR mimics were prepared at 10 μM in the buffer containing either 150 mM KCl or LiCl in TE buffer, as described above. Measurements were performed in quartz cuvettes (1 mm path length) using 200 μL sample volumes. Spectra were recorded from 200 to 320 nm at 20°C, with each spectrum representing an average of three scans. G-quadruplex formation was indicated by enhanced peak intensity at 260–265 nm and a trough at 240 nm in K⁺ conditions, with reduced peak intensity observed in Li⁺ conditions.

Test Tube Equilibrium Complex Concentration Analysis

To evaluate the targeting specificity of ASO designed for targeting tRNA-Asp-GTC-3’tDR, we developed a computational tube reaction model incorporating four components at equal concentrations: ASO targeting tRNA-Asp-GTC-3’tDR, tRNA-Asp-GTC-3’tDR, tRNA-Asp-GTC-5’tDR, and full-length tRNA-Asp-GTC. The secondary structure calculations for this four-component interaction network were carried out using the NUPACK Python package (version 4.0.1.7). The rna06 parameter is utilized. The tube analysis mode was employed, with each component set to a universal initial concentration of 1 μM under physical and chemical conditions of 37°C and 0.15M NaCl to mimic the cellular environment. For the analysis, the equilibrium complex concentrations were calculated using the tube analysis with the maximum complex size set to 2.

Total RNA-seq

Total RNA-seq was performed as previously described (73). Briefly, total RNAs from HEK cells transfected with control mimics or tRNA-Asp-GTC-3’tDR mimics were isolated with TRIzol^™ (Thermo Fisher, Cat# A33251) by following the manual. cDNA libraries were constructed using the SMARTer Stranded Total RNA-Seq Kit v2 Pico Input Mammalian (Takara Bio, San Jose, CA, USA) and sequenced using the Illumina NextSeq 2000 platform using a resolution at 100 base pairs with paired-end reads. Reads were trimmed to remove adapter sequences using Cutadapt (v2.10) and aligned to the Gencode GRCh38.p13 genome using STAR (v2.7.8a). Gencode v38 gene annotations were provided to STAR to improve the accuracy of mapping. Quality control on both raw reads and adaptor-trimmed reads was performed using FastQC (v0.11.9) (www.bioinformatics.babraham.ac.uk/projects/fastqc). featureCounts (v2.0.2) was used to count the number of mapped reads to each gene. Heatmap3 was used for cluster analysis and visualization. Significantly differential expressed genes with absolute fold change >= 1.5 and FDR-adjusted p-value <= 0.05 were detected by DESeq2 (v1.30.1).

Ordered Two-Template Relay Small RNA sequencing (OTTR-seq)

From HEK cells transfected with control or tRNA-Asp-GTC-3’tDR mimics, total RNAs were purified from TRIzol (ThermoFisher, Cat# 15596026) treated cells and then incubated with T4 PNK (NEB, Cat# M0201L) to resolve 2’−3’ cyclic phosphate ends, and then deacylated by bringing the reaction to pH 9.0 for 30 minutes at 45 °C using 100 mM Na2B4O7. Treated RNA was recovered using the RNA clean and concentrate kit (Zymo Research, Cat# R1013). OTTR-seq libraries were prepared according to previously described methods (8) with modifications. Briefly, 40 ng of the processed total RNA was used as input. RNA was labeled for 2 hours at 30 °C using only dideoxyATP (ddATP). Similarly, cDNA synthesis was conducted using only the RNA-DNA primer duplex with +1T overhang. cDNA was recovered using the MinElute reaction cleanup kit (Qiagen, Cat# 28204), then size selected on a 1×Tris-borate-EDTA (TBE, 89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.3) buffer, 8M urea, and 9% acrylamide gel to remove adapter dimers. cDNA was recovered by diffusion at 70 °C for 1 hour with shaking in a solution of 10 mM Tris-HCl pH 8, 300 mM NaCl, 1mM EDTA, and 0.25% SDS, followed by precipitation in 66% ethanol with 0.6 M ammonium acetate and 1ug/mL Glycoblue (ThermoFisher, Cat# AM9515). cDNA was amplified with Q5 polymerase (NEB, Cat# M0493L) for 13 cycles using the Illumina NebNext sRNA index adapters (NEB, Cat# E7600S) then size selected on a 1 × TBE, 6% acrylamide gel to remove remaining adapter dimer. Libraries were recovered from the gel by diffusion at 70 °C for 1 hour with shaking in a solution of 10 mM Tris-HCl pH 8, 300 mM NaCl, and 1 mM EDTA, followed by precipitation in 70% ethanol with 80 mM sodium acetate and 1 μg/mL Glycoblue (ThermoFisher, Cat# AM9515). The recovered libraries were quantified using the qubit dsDNA High Sensitivity kit (Invitrogen, Cat# Q32854), Agilent High Sensitivity DNA bioanalyzer kit (Agilent, Cat# 5067–4626), and finally by qPCR. qPCR was conducted using PrimeTime Gene Expression Master Mix (IDT, Cat# 1055772) according to a previously published protocol40. Libraries were sequenced on the NextSeq 1000/2000 platform to generate 100 bp single-end reads. Raw fastq files were trimmed using Cutadapt with options -j 8 -m 15 -a GATCGGAAGAGCACACGTC. Reads were further processed to remove an 8NT UMI using umi_tools extract with options --extract-method=string --bc-pattern=NNNNNNN. Trimmed reads were processed through the tRNA Analysis of eXpression pipeline (tRAX)41, using a custom reference database generated for the HG38 assembly which includes sequence and annotations for the scrambled Asp-GTC 3’ tDR sequence. Resulting BAM files were split into reads above or below 60bp in length, and those above 60bp were processed through the tRAX pipeline again (using the --lazyremap option) to analyze the expression of mature tRNA transcripts.

In Vitro Transcription

Human histone H1–2 cDNA, together with an exogenous tag sequence, was synthesized by GenScript and cloned into a pcDNA3.1 vector under a T7 promoter. The linear template T7/H1–2 DNA was then amplified using PrimerSTAR GXL DNA polymerase (Takara Bio, Cat# R050B) and cleaned up using Wizard^® SV Gel and PCR Clean-Up System (Promega, Cat# A9282). H1–2 mRNAs with different uridine modifications were in vitro transcribed using the MEGAscript T7 Transcription Kit (ThermoFisher, Cat# AM1334) with some modifications. Uridine-5’-triphosphate was replaced with Pseudouridine-5’-triphosphate (Advnt Biotechnologies, Cat# 13944) or Fluorouridine-5’-triphosphate (Sierra Bioresearch, Cat# FUTP) to generate Pseudouridine-modified or Fluorouridine-modified H1–2 mRNAs, respectively. The reactions were carried out at 37°C for 16 hours, and the template DNA was removed by TURBO DNase treatment. The H1–2 mRNAs were cleaned up using the Qiagen RNeasy Midi Kit (Qiagen, Cat# 75144).

tDR Pull-down and Proteomics

To identify the binding proteins of tRNA-Asp-GTC-3’tDR, 50 nM Biotin-conjugated tRNA-Asp-GTC-3’tDR or control mimics were transfected to HEK cells using Lipofectamine RNAiMAX. 24 hours after transfection, the cells were pelleted after washing and proceeded with RNA pull-down using part of the Pierce^™ Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher, Cat# 20164). Binding proteins were directly eluted using 1x Laemmli sample buffer (BioRad, Cat# 161–0747) and subjected to in-solution protein digestion using the SP3 protocol (74, 75). The resulting tryptic peptides were directly loaded on a 50 cm C18 EasySpray column and separated by acidic reverse phase chromatography over a 75 min gradient. Data acquisition was carried out on a Thermo Orbitrap Eclipse mass spectrometer equipped with a FAIMS Pro device using the following settings: FAIMS CV at −40V, −60V, and −80V, MS1 resolution of 120K, full scan range of 375–1575 m/z, precursor isolation window of 1.6, MS2 scans in the iontrap from HCD fragmentation of precursor ions with 32% NCE and dynamic exclusion setting of 60 seconds. Data were acquired in top-speed mode with a cycle time of 1 sec per FAIMS CV. Data were searched in Proteome Discoverer v2.4 running Mascot v2.6 against a UniProtKB/Swiss-Prot human protein sequence database (downloaded May 2020). Search parameters included: Cys carbamidomethylation as a fixed and Met oxidation as a dynamic modification; precursor mass tolerance of 10 ppm and a fragment mass tolerance of 0.6 Da. PSM and peptide identifications were filtered to 1% FDR using the Percolator node in Proteome Discoverer v2.4. The probability of protein interaction to tRNA-Asp-GTC-3’tDR was calculated using SAINTexpress (48) and compared against the control group. As recommended by SAINTexpress, high-confidence interactors were thresholded at the average probability of interaction (AvgP) >0.7.

Competitive PUS7 Binding Assay

To compare PUS7 binding affinities between histone mRNAs and tDR variants relative to wild-type tRNA-Asp-GTC-3’tDR, biotin-conjugated tRNA-Asp-GTC-3’tDR mimics (50 nM) were transfected into HEK cells. The biotin-tDR/PUS7 complexes were isolated using the Pierce^™ Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher, Cat# 20164). After washing three times with 1X Washing Buffer, the bead-immobilized biotin-tDR/PUS7 complexes were incubated with either G4-structured tRNA-Asp-GTC-3’tDR mimics, non-G4-structured tRNA-Asp-GTC-3’tDR mimics, control mimics, or varying concentrations of in vitro transcribed histone H1–2 mRNAs. The incubation was performed at 37°C for 30 minutes in 1X Protein-RNA binding buffer, followed by three additional washes with 1X Washing Buffer. Both the flow-through fraction (containing displaced PUS7) and the bead-bound fraction (containing remaining PUS7) were analyzed by immunoblotting.

Pseudouridylated RNA Immunoprecipitation (IP)

HEK cells transfected with control or tRNA-Asp-GTC-3’tDR mimics were treated with 50 μM CQ for 2 hours before RNA collection, and the total RNAs were isolated using Qiagen RNeasy Midi Kit (Qiagen, Cat# 75144). Total RNAs were fragmented using RNA fragmentation Reagent (ThermoFisher, Cat# AM8740) at 95°C for 5 minutes, immediately followed by the addition of EDTA and rapid cooling on ice. Fragmented total RNAs were then cleaned up using the RNA Clean and Concentrator kit (Zymo Research, Cat# R1018) and ~35 μg fragmented total RNAs were then incubated with 15 μg anti-Pseudouridine antibody (MBL Life Science, Cat# D347–3) or control mouse IgG (R&D systems, Cat# MAB002) in 1x RNA IP buffer (10 mM Tris-HCl pH 7.5, 250 mM NaCl, and 0.5% (vol/vol) Igepal CA-630) supplemented with 1 U/μl RNase Inhibitor. The RNA/antibody mixtures were head-over-tail rotated for 2 hours at 4°C. While the RNA/antibody mixtures were incubating, 100 μl Protein A+G magnetic beads (ThermoFisher, Cat# 88803) were washed three times with 1 x RNA IP buffer and blocked in 0.5 mg/ml BSA in 1 x RNA IP buffer supplemented with 1 U/μl RNase Inhibitor with head-over-tail rotation for 2 hours at 4°C. Next, the RNA/antibody mixtures were transferred into the bead-containing tubes prepared, and the reaction mixtures were incubated with head-over-tail rotation for 2 hours at 4°C. After incubation, the immunoprecipitated bead/antibody/RNA mixtures were washed three times with 1 x RNA IP buffer supplemented with 1 U/μl RNase Inhibitor. The immunoprecipitated RNAs were then extracted using the Qiagen RNeasy micro kit (Qiagen, Cat# 74004) and subjected to qRT-PCR.

Statistical Analysis

Quantitative densitometric analysis of immunoblotting and northern blotting images was performed using BioRad ImageLab (Version 6.1.0). Statistical analyses were performed using GraphPad Prism software (Version 9). The data of immunoblotting, northern blotting, and qRT-PCR is expressed as mean ± SEM, and their statistical significance was assessed by the two-tailed unpaired Student’s t-test. CellProfiler (Version 4.2.5) was used to count LC3B puncta or fluorescent signals from immunofluorescent images, and the statistical significance was calculated using the Mann-Whitney test. For the transcriptomics data, differential expression analysis was performed using DESeq2 to generate Benjamini–Hochberg-corrected P values (padj) to assess the statistical significance. For proteomics data, protein abundances were normalized using variance stabilization normalization (vsn package) in R v3.5.2. Statistical significance in protein abundances between the control and tRNA-Asp-GTC-3’tDR pulldown experiment was assessed using a student’s t-test. Proteins with p-value <0.05 and log₂ fold-change (tRNA-Asp-GTC-3’tDR/control) >1 were selected as significantly interacting proteins in the tRNA-Asp-GTC-3’tDR pulldown experiment. For patient characteristics data, the Mann-Whitney test was used to assess the statistical significance of means between the two groups. Fisher’s exact test was used to compare the proportions between the two groups. The criterion for statistical significance was P < 0.05 (* P < 0.05, ** P < 0.01, *** P < 0.001).

Data and materials availability:

The human kidney tissue scRNA-seq data were obtained from GSE183279 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE183279). The ARM-seq data of HEK cells treated with different stressors were obtained from GSE173806 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE173806). The long RNA-seq data and OTTR-seq data of HEK cells transfected with the control or tRNA-Asp-GTC-3’tDR mimics are deposited in NCBI GEO (accession numbers: GSE295814 and GSE289124). Proteomics data were deposited in PRIDE (accession number: PXD052235). Other materials are available from Dr. Saumya Das under a material transfer agreement with Massachusetts General Brigham.