Self-quenched tRNA reporters for imaging tRNA-derived RNA biogenesis

Abstract

tRNA-derived small RNAs (tDRs) are an emerging class of small non-coding RNAs that play crucial roles in various cellular processes. However, there is a paucity of data on their sub-cellular localization due to a lack of tools and reagents to image tDRs. Imaging tDRs remains challenging due to the similar sequences between tDR and its parent tRNA. Here, we describe an innovative tool for studying the formation and localization of tDRs in various biological processes using a self-quenched tDR biogenesis reporter. This method utilizes a full-length tRNA molecule conjugated with both fluorescence and quencher groups at 5’- and 3’- ends. In its intact state, the fluorescence is quenched. Upon cleavage by specific ribonucleases and strand separation, the fluorescence becomes detectable, allowing real-time imaging of tDR biogenesis. This protocol details the design, synthesis, and application of this reporter, including transfection procedures and imaging techniques. The method offers a powerful approach for investigating tDR dynamics in living cells, providing insights into their roles in cellular processes and stress responses.

1. Introduction

As one of the most abundant RNA species in cells, the canonical function of transfer RNAs (tRNAs) in decoding the genetic code during protein translation is well established (Crick, 1968). More recently, it has been shown that full-length tRNA molecules are processed into smaller regulatory fragments, variously termed tRNA fragments and tRNA halves, or tRNA-derived small RNAs (tsRNA, or tDRs) (Holmes et al., 2023), by stress-activated ribonucleases, including DICER1, Angiogenin, ELAC2, and RNASE1, in a stereotypical manner (Fu et al., 2009; Lee, Shibata, Malhotra, & Dutta, 2009). Following initial hydrolysis, these nicked full-length tRNAs may be subjected to helicase-dependent unwinding to produce the functional tDRs (Drino et al., 2023). Although first reported to be functional only a decade ago (Lee et al., 2009; Thompson & Parker, 2009), tDRs have been shown to play versatile roles, such as RNA silencing (Deng et al., 2015; Haussecker et al., 2010; Kuscu et al., 2018; Yeung et al., 2009), translational regulation (Ivanov, Emara, Villen, Gygi, & Anderson, 2011; Kim et al., 2017, 2019), epigenetic regulation (Chen et al., 2016; Dhahbi et al., 2013), and stress granule formation (Ivanov et al., 2014; Lyons, Achorn, Kedersha, Anderson, & Ivanov, 2016) in development and diseases, including intergenerational inheritance (Chen et al., 2016; Dhahbi et al., 2013), neurologic disorders (Ivanov et al., 2014), and various cancers (Pekarsky et al., 2016; Yu et al., 2020).

While emerging data has suggested localization of certain tDRs to specific sub-cellular organelles or compartments, such as cytoplasmic granules in Trypanosome cruzi (Garcia-Silva et al., 2010), these limited methodologies only provide a single snap shot, lack sufficient resolution, and are difficult to implement. Despite some advances in tools to profile and study tDRs (Xie et al., 2020), there remains an unmet need to develop reagents that may allow real-time visualization of tDRs. Fluorescent in situ hybridization (FISH) has been used to study the presence of tDRs in tissues (Yang et al., 2022), but the resolution is not sufficient to assess sub-cellular localization; furthermore, the designed probes may not distinguish parent full-length tRNAs from tDRs. The self-quenched tDR biogenesis reporter described in this protocol offers a novel approach to studying the formation and dynamics of these important RNA species in real time within living cells.

The principle of this method relies on the strategic placement of fluorescence and quencher groups on a whole-length tRNA molecule at 5’- and 3’- ends (Fig. 1). In its intact state, the proximity of the quencher to the fluorophore prevents fluorescence emission. However, when the tRNA is cleaved by specific ribonucleases involved in tDR biogenesis in eukaryote (such as Angiogenin, Dicer, or RNase Z), with the separation of the tRNA halves, the physical separation of the fluorophore from the quencher results in detectable fluorescence (Fig. 1). We have recently reported the cleavage of tRNA-Asp-GTC in HEK293 cells upon hypoxia treatment, which generated both 5’ halves and 3’ halves of tRNA-Asp-GTC (Li et al., 2022). As a proof-of-concept, we describe here the design of such a sensor for 3’ halve of tRNA-Asp-GTC (here referred as tRNA-Asp-GTC-3’tDR) and demonstrate its use in investigating the biogenesis and localization of this tDR in response to hypoxia. The model system used are HEK293 cells subjected to hypoxic stress (0.1 % O₂, 5 % CO₂).

Fig. 1. Designs of tDR biogenesis reporters.

(A) The design and principle of the reporter for a 3’-tDR. The fluorescence group is conjugated to the 3’end of full-length tRNA oligo, and the quencher group is conjugated to the 5’end of the tRNA. (B) The design and principle of the reporter for a 5’-tDR. The fluorescence group is conjugated to the 5’end of full-length tRNA oligo, and the quencher group is conjugated to the 3’end of the tRNA.

2. Materials

This protocol describes the design of a self-quenched tDR biogenesis reporter, the transfection of a self-quenched tDR biogenesis reporter to target cells, and the detection of fluorescent signals. Prepare all buffers and medium used for cell culture and transfection in a sterile environment under a laminar flow hood and store buffers at 4 °C unless indicated otherwise.

2.1. Cell culture

1. Cell culture 24 well plates (Corning, 3524).

2. 12 mm round coverslips (Bellco Glass, 1943-10012A). Autoclaved for cell culture use.

3. CO₂ incubator is set to 37 °C and 5 % CO₂ in the humidified atmosphere. The hypoxia chamber in the CO₂ incubator is set to 37 °C, 0.1 % O₂, and 5 % CO₂ in the humidified atmosphere.

4. Targeted cell line. In this study, we used HEK293 cells (ATCC, CRL-1573).

5. Targeted cell line culture medium. Prepare culture medium as preferred by the desired target cell line. In this study, we used the D10 medium for culturing HEK293 cells, which consisted of DMEM with high glucose and pyruvate (ThermoFisher Scientific, 11995073) supplemented with 10 % fetal bovine serum (FBS) (ThermoFisher Scientific, 10437028) and 1 % Penicillin/Streptomycin (ThermoFisher Scientific, 15140122).

6. Matrigel (Corning, 354234).

7. Phosphate-buffered saline (PBS) without calcium and magnesium (ThermoFisher Scientific, 10010049)

8. TrypLE Express Enzyme (ThermoFisher Scientific, 12605028)

9. Freeze medium. 10 % DMSO in FBS.

2.2. Transfection

1. Self-quenched tDR biogenesis reporter. Basically, this is a full-length tRNA molecule (~76 nucleotides in length) conjugated with both fluorescence and quencher groups at opposite ends. It could be any tRNA of interest. In this study, we focus on tRNA-Asp-GTC. This reporter RNA molecule could be synthesized by Dharmacon, Integrated DNA Technologies, or others. Chemical modifications on the full-length tRNA are not necessary but could be added as needed.

2. Opti-MEM, Reduced serum medium (ThermoFisher Scientific, 31985070)

3. Lipofectamine RNAiMAX (ThermoFisher Scientific, 13778150)

4. RNase-free water.

2.3. Imaging and analysis

1. FluoroBrite D10 medium: FluoroBrite DMEM supplemented with 10 % FBS and 1 % Penicillin/Streptomycin.

2. 4 % Formaldehyde.

3. PBS-T. PBS supplemented with 0.1 % Tween-20.

4. Hoechst 33342,10 mg/mL solution in water (ThermoFisher Scientific, H3570).

5. Antifade Mountant with DNA Stain DAPI (ThermoFisher Scientific, P36935).

3. Methods

All steps should be carried out in a sterile and RNase-free biosafety environment. Use RNase-free pipette tips and tubes when handling tDR biogenesis reporters during resuspension and transfection (See Note 1). Use sterile pipettes and pipette tips to transfer liquids during cell culture.

3.1. Design and synthesis of tDR biogenesis reporter

1. Determine the tDR of interest and its parent tRNA molecule (see Note 2). We have recently reported the cleavage of tRNA-Asp-GTC in HEK293 cells upon hypoxia treatment, which generated both 5’ halves and 3’ halves of tRNA-Asp-GTC (Li et al., 2022). This study will focus on the cleavage of tRNA-Asp-GTC molecules and the generation of tRNA-Asp-GTC-3’tDRs in HEK293 cells during the hypoxic response (Fig. 2A).

2. Design the reporter with the fluorescence group (e.g., FITC) conjugated to the end corresponding to the tDR location and the quencher group (e.g., BHQ-1) at the opposite end. For instance, if the tDR-of-interest is generated from the 3’end of tRNA, the fluorescence group could be conjugated to the 3’end of tRNA reporter, and the quencher group will be conjugated to the 5’end (Fig. 1A). If the tDR-of-interest is generated from the 5’end of tRNA, the fluorescence group could be conjugated to the 5’end of tRNA reporter, and the quencher group will be conjugated to the 3’end (Fig. 1B). In this study, we take the tRNA-Asp-GTC-3’tDR as an example. Therefore, we conjugated the FITC group at the 3’end of tRNA-Asp-GTC and the BHQ-1 group at the 5’end (Fig. 2B).

3. Submit the design to a custom RNA synthesis service (e.g., Dharmacon, Inc.) for synthesis (see Note 3).

4. Upon receiving the synthesized reporter, resuspend in RNase-free water to make a 100 μM stock solution. Aliquot the stock solution and store at −80 °C to avoid repeated freeze-thaw cycles.

Fig. 2.

The application of tDR biogenesis reporter to visualize the generation of tRNA-Asp-GTC-3’tDR. (A) ARM-seq indicates that tRNA-Asp-GTC-3’tDR is upregulated in HEK293 cells upon hypoxia treatment. Data was shown in Mean ± SEM. (B) The design and principle of tDR biogenesis reporter for tRNA-Asp-GTC-3’tDR. (C) Confocal fluorescent microscopy demonstrates the cleavage of tRNA-Asp-GTC reporter and the generation of tRNA-Asp-GTC-3’tDR (FITC positive signals) in HEK293 cells after 24 h of hypoxia treatment. Scale bar, 50 μm.

3.2. Transfection of reporter into target cells

1. Maintain the target cells as needed. HEK293 cells are cultured in the D10 medium on a suitable cell culture plate in the CO₂ incubator with 21 % O₂. When reaching ~80 % confluency, cells are split using TrypLE.

2. Day 0, Coating: One day before plating cells, place an autoclaved round 12 mm coverslip into one well of 24-well plate and coat the coverslip with 0.5 mL 1 % Matrigel overnight (see Note 4). Two 24-well plates with 3–4 coverslips each plate are needed.

3. Day 1, Seeding: Trypsinize the cells using TrypLE and seed 25,000–50,000 cells onto each coated coverslip in the 24-well plate.

4. Day 2, Transfection: For three wells, prepare two tubes:

   • Tube 1: Add 1 μL of 100 μM reporter stock to 100 μL Opti-MEM and mix well.
   • Tube 2: Add 6 μL Lipofectamine RNAiMAX to 100 μL Opti-MEM and mix well.

   Combine the contents of tubes 1 and 2, mix gently with a pipette, and incubate for 5 min at room temperature. Next, add ~54 μL of the transfection mixture dropwise to each well and gently rock the plate to distribute evenly. Then incubate the cells overnight in a 37 °C, 5 % CO₂ incubator (See Note 5).

5. Day 3, Stress treatment: Replace the transfection medium with fresh culture medium. After refreshing the medium, expose cells to desired experimental conditions or stressors known to induce tDR formation (See Note 6). In this study, we place one 24-well plate into the hypoxia chamber with 0.1 % O₂ and 5 % CO₂, and another one stays in the normal CO₂ incubator with 21 % O₂.

6. Day 4: 1–24 h after the treatment, the cells are ready for imaging and analysis (See Note 6).

3.3. Imaging and analysis

Fluorescence signals at appropriate time points after stress treatment could be monitored using live-cell imaging, confocal microscopy, or flow cytometry (See Note 7 and 8).

3.3.1. For live-cell imaging

1. Add Hoechst into the cell culture medium at the final concentration of 1 μg/mL for 5–10 min.

2. Replace the culture medium with FluoroBrite D10 medium.

3. Observe cells using a fluorescence microscope with appropriate filters for the fluorophore used (e.g., FITC) and Hoechst.

4. Capture images at multiple time points to track the dynamics of generated tDR (FITC-positive signals).

3.3.2. For confocal microscopy

1. At desired time points, fix the cells with 4 % formaldehyde for 10 min and wash the cells with PBS-T three times.

2. If desired, stain the cells with any antibody of interest.

3. Mount the coverslips in glass slides with the Antifade Mountant with DNA Stain DAPI and store them in 4 °C overnight.

4. Capture images using a fluorescent confocal microscope with appropriate filters for the fluorophore used (e.g., FITC) and DAPI. As shown in Fig. 2C, we detected significantly increased FITC signals in reporter-transfected HEK293 cells after hypoxia treatment compared to those at the normal culture condition. Moreover, the punctate-like fluorescence in the cytoplasm allowed visualization of the subcellular localization of the tDR.

3.3.3. For flow cytometry

1. At desired time points, stain the cells with Hoechst for 5–10 min.

2. Trypsinize cells and collect them in tubes.

3. Wash cells with PBS and resuspend in an appropriate buffer for flow cytometry.

4. Analyze samples using a flow cytometer with appropriate laser and filter settings for the fluorophore used (e.g. FITC and Hoechst).

3.3.4. Data analysis

1. For microscopy data, quantify fluorescence intensity using appropriate image analysis software (e.g. ImageJ, Cell Profiler).

2. For flow cytometry data, analyze the proportion of fluorescent cells and the distribution of fluorescence intensities using FlowJo.

3. Compare results across different experimental conditions and time points to draw conclusions about tDR biogenesis dynamics (see Note 9 and 10).

4. Notes

1. RNase contamination is a significant concern when working with RNA-based reporters. Always use RNase-free reagents and equipment, and practice good aseptic techniques to minimize the risk of degradation. False-positive fluorescence signals can result from RNase contamination.

2. The design of the tDR biogenesis reporter is crucial for its functionality. If the tDR of interest is derived from the 5' end of tRNA genes, the fluorescence group should be conjugated to the 5′ end of the tRNA molecule. Conversely, for 3′ end-derived tDRs, conjugate the fluorescence group to the 3′ end. For tDRs derived from the middle of tRNA genes, a truncated tRNA exposing the tDR at one end may be more effective, although this approach has not been extensively tested.

3. When ordering the custom synthesis of the reporter, ensure that the synthesis service can accommodate the specific modifications and requirements (e.g. fluorescence group and quencher group conjugations and HPLC/PAGE purification) required for your reporter design. Also, the sensitivity of this method can be affected by the efficiency of the quenching and the brightness of the fluorophore. Consider testing different fluorophore-quencher pairs if you encounter issues with signal-to-noise ratio.

4. Matrigel is temperature sensitive. Always place Matrigel on ice when handling and use cold PBS to dilute Matrigel.

5. The transfection efficiency and reporter uptake can vary between cell types. Optimize the transfection conditions (e.g., reporter concentration, transfection reagent amount, incubation time) for your specific cell line to achieve the best results.

6. Choosing the appropriate stressors is important. Some tDRs may have baseline expression in certain cell types. As a result, you might detect low to medium levels of fluorescence signals even before applying any stressors. It is important to establish a baseline and use appropriate controls to account for this background signal.

7. Live imaging is generally recommended for this protocol, as the fluorescence signal can be sensitive to fixation procedures. If fixation is unavoidable, use 4 % formaldehyde rather than paraformaldehyde (PFA), as it tends to better preserve the fluorescence signal.

8. For quantitative comparisons between different experimental conditions, it is crucial to maintain consistent imaging settings (exposure time, gain, etc.) across all samples.

9. When interpreting results, remember that the fluorescence signal indicates tRNA cleavage, which is a proxy for tDR biogenesis. Additional experiments, such as small RNA sequencing, may be necessary to confirm the identity and abundance of specific tDR species. While we cannot completely exclude some non-specific degradation of the synthetic tRNA to release fluorescence, the lack of fluorescence at baseline (indicating lack of degradation), and our detailed RNA sequencing data in this same model (Li et.al., 2022) that shows predominant biogenesis of the tRNA halves (3’ and 5’) suggests that this would be a minor component of the fluorescence observed.

10. This protocol can be adapted for high-throughput screening applications by scaling down to 96-well or 384-well plate formats. However, optimization of transfection conditions and imaging protocols will be necessary for these smaller-scale formats.

5. Discussion

In this protocol, we present a new reagent and methodology that allows for high-resolution visualization of tDR biogenesis and subcellular localization. Currently available methodologies like FISH or the use of RNA aptamers such as Spinach or Mango have recently enhanced the ability to visualize single mRNA or lncRNA molecules in the cell (Cawte, Unrau, & Rueda, 2020). Fluorescent RNA aptamers that utilize FRET can allow for assessing RNA conformational changes (Jepsen et al., 2018). However, these technologies have not been applied for tDRs.

The unique biogenesis of tDRs from the parent tRNA allowed us to exploit fluorescence-quencher interactions that yield fluorescence only when the tRNA halves separate from each other. The lack of a fluorescence signal in the absence of the stressor (hypoxia) indicates the presence of uncleaved, full-length ‘parent’ tRNA Asp-GTC. The detection of fluorescence in the presence of hypoxia in a time-dependent manner indicates the cleavage of the parent tRNA into 3’ and 5’ halves and physical separation of these halves. While we cannot completely exclude the presence of other smaller tRNA fragments that contain the 3’ end (with the fluorescent probe attached), correlation with other experiments, such as northern blotting or ARM-seq, may answer that question. Finally, the fluorescent synthetic reporter system does not incorporate specific RNA modifications that may be present in endogenous tRNAs, hence would not capture the effects of such modifications on tDR biogenesis. In conclusion, our tDR biogenesis reporter adds to the toolbox necessary for the inter-rogation of tDR biology.