Angiogenin-mediated tRNA cleavage as a novel feature of stored red blood cells

Red blood cell (RBC) transfusion is the most common therapeutic procedure in hospitalized patients. RBC units can be refrigerated for up to 42 days and undergo various biochemical and morphological changes during storage (storage lesions) (D’Alessandro, et al 2015). However, the effects of storage on the RBC transcriptome have not been well-studied. While once thought to lack nucleic acids, RBCs actually contain diverse and abundant RNA species (Chen, et al 2017). In addition, proteomic analyses of RBCs have identified the presence of Argonaute 2 (AGO2) (Bryk and Wisniewski 2017), supporting the regulatory function of miRNAs. Genomic analyses of the RBC transcriptome have provided insights into the heterogeneity and malaria resistance of sickle cell anaemia (Sangokoya, et al 2010, Walzer and Chi 2017) and several recent studies have begun to probe the effects of storage on the RBC transcriptome (Kannan and Atreya 2010, Sarachana, et al 2015).

To define the storage transcriptome, three AS-1 packed RBC units were collected and processed using a standard blood banking procedure that removed +99.99% of white blood cells and ~80% of plasma. RNA was isolated after 1, 3, 7, 10, 14, 28, 36 and 42 days of storage (Fig S1A; see Appendix S1 for details). During RNA extraction, cel-miR-254 (C. elegans) and osa-miR-442 (O. sativa) were added for normalization. The miRNA transcriptome was interrogated by the nCounter^® v2 assay (NanoString Technologies, Seattle, WA). Storage-associated microRNA changes were identified by comparing the normalized data against the Day 1 sample for each individual, arranged by hierarchical clustering (shown in Fig 1A). While most miRNAs showed modest changes during storage (Fig 1A), we noted two markedly increased (miR-33a-5p and miR-720) and two reduced (miR-563 and miR-582–5p) miRNAs (Fig 1A). When the longitudinal plots of all miRNAs were compared (Fig S1B), miR-720 induction stood out as the most consistent change during storage (Fig S1C). miR-720 was first annotated as a miRNA with miRNA-like functions (e.g., Li, et al 2014, Wang, et al 2015); however, it was also suggested to be a tRNA fragment (Schopman, et al 2010) and removed from miRBase. No consensus has emerged on the naming and most studies still use the term miR-720. Therefore, we referred to this small-sized RNA as “miR-720”.

Figure 1: The increase in “miR-720” expression during RBC storage.

(A) Heat map of storage-associated changes in miRNAs of red blood cells (RBCs) from three individuals. miRNA ratios were normalized to Day 1 of storage of each individual and arranged by hierarchical clustering. The two upregulated (red) and downregulated (green) miRNAs were further expanded. (B) Validation of the storage-associated “miR-720” increase by quantitative polymerase chain reaction (qPCR) when normalized against spike-ins foreign miRNAs (cel-miR-254 and osa-miR-442) or endogenous miR-222. (C-D) RNA samples from RBC stored for the indicated days were separated by polyacrylamide gel electrophoresis and transferred to small RNA Northern blots and probed with (C) miR-720 and (D) tRNA^Thr(TGT) full-length probes. The bands corresponding to the full-length tRNA and expected “miR-720” band (~18 nt) are indicated by arrows. The remaining tRNA fragment after “miR-720” removal is indicated by arrowhead.

Next, we used quantitative reverse transcription polymerase chain reaction (RT-qPCR) to measure “miR-720” during RBC storage and found an increase of ≥10-fold when normalized against either spiked-in foreign miRNAs (cel-miR-254 and osa-miR-442) or against endogenous miR-222 (Fig 1B). Similar “miR-720” induction was noted in all tested stored RBC units. Interestingly, Sarachana et al (2015), listed “miR-720” in a supplemental table of storage-induced miRNAs, further supporting “miR-720” induction as a reproducible feature of RBC storage.

We also performed small RNA Northern blotting. The hybridization of “miR-720” probe (Fig S1E; Table S1) revealed bands corresponding to both a tRNA and ~18 nt “miR-720” (Fig 1C; Fig S1D), consistent with NanoString and RT-qPCR results. Several bands of larger sizes, which may represent processing intermediates or cross-hybridization, were also noted (Fig 1C). Similar “miR-720” induction was reproduced in another Northern blot using a synthetic 18 nt “miR-720” as size marker (Fig S1F). When the blot was stripped and re-probed with full-length tRNA^Thr(TGT) sequence (Fig S1G; Table S1), we noted a storage-associated increase in the “miR-720”, an unidentified ~23 nt band and a ~55nt band, roughly corresponding to the expected tRNA remnants after “miR-720” removal (Fig 1D). No similar storage-associated increase was found for tRNA-alanine (Fig S1H) or tRNA-tyrosine (Fig S1I). Together, these results indicate that RBC storage is associated with a specific cleavage of tRNA^Thr(TGT) resulting in release of the “miR-720” fragment.

Next, we identified the relevant nucleases involved in the tRNA^Thr(TGT) cleavage into “miR-720”. Based on the hypothesis that RBC lysates may contain relevant cleavage activity, we incubated RBC lysates with synthetic tRNA^Thr(TGT) and found release of an ~18 nt fragment “miR-720” (Fig 2A). The cleavage activity was abolished by heat inactivation (Fig S2A), suggesting heat-sensitive molecules, such as protein(s). Considering angiogenin (ANG) and Dicer as candidate nucleases, we first confirmed their presence in RBC by Western blotting (Fig S2B). Next, we incubated the synthetic tRNA^Thr(TGT) with increasing amounts of either recombinant Dicer or ANG protein. Interestingly, ANG, in a dose-dependent manner, produced a product corresponding to the expected size of “miR-720” (Fig 2B). In contrast, Dicer incubation generated a ~25 nt fragment that did not match the size expected for “miR-720”. Neither recombinant DNase I nor RNase H had detectable cleavage activities (Fig S2C). Furthermore, the cleavage products of tRNA^Thr(TGT) fragments generated by ANG or RBC lysates shared an ~18 nt fragment expected of “miR-720” (Fig 2C).

Figure 2: Angiogenin contributes to the “miR-720” increase in stored RBCs.

(A) Northern blot of synthetic tRNA^Thr(TGT) before or after incubating with red blood cell (RBC) lysate shows the conversion of full-length synthetic tRNA^Thr(TGT) to “miR-720”-sized fragment as indicated. (B) Synthetic tRNA^Thr(TGT) was incubated with increasing levels of Dicer or angiogenin (ANG) proteins and resulting fragments were probed by miR-720 probe on Northern blots. The ~18 nt “miR-720”-sized fragment is indicated by an arrow. (C) Comparison of cleavage products of tRNA^Thr(TGT) incubated with ANG or RBC lysates. The ~18 nt “miR-720”-sized fragment is indicated by an arrow. (D) Further enhancement of “miR-720” increases when ANG was added to stored RBCs. Vehicle control or recombinant ANG (2 pg/ml) was added to RBCs weekly and “miR-720” levels were determined by quantitative reverse transcription polymerase chain reaction. (E) Immunodepletion of ANG from RBC lysate reduces the generation of “miR-720” of RBC lysates from synthetic tRNA^Thr(TGT). Mouse IgG (anti-mIgG) serves as the appropriate isotype control for angiogenin antibody (anti-ANG). (D-E) Data shown as mean ± standard error of the mean of technical duplicates and are representative of three independent experiments; **p<0.01, ***p<0.001.

Interestingly, the addition of recombinant ANG added to stored RBCs further enhanced “miR-720” induction (Fig 2D). Reciprocally, removing ANG from RBC lysate via immunodepletion significantly reduced tRNA cleavage (Fig 2E). Together, these data indicate that ANG in RBCs contributes to the tRNA^Thr(TGT) cleavage to generate “miR-720” during RBC storage.

In summary, these data suggest that RBC storage is associated with significant and reproducible “miR-720” induction. While “miR-720” increase was among the induced miRNAs of stored RBC (Sarachana, et al 2015), this is the first study to highlight the dramatic increase in “miR-720”, and to use biochemical characterization to explain the underlying mechanistic role of ANG. Such a reproducible and novel feature of stored RBC may be of use as a biomarker to monitor stored RBCs. In the future, it will be of significant interest to determine any functional role(s) of the highly expressed miR-720 in the stored RBCs as well as in endothelial cells and macrophages exposed to transfused RBCs. Another possible application is in blood doping investigations. While autologous blood transfusion in athletes is very difficult to identify using conventional tests, it may be detectable based on the presence of RBCs with levels of miR-720 significantly higher than the normal circulating cells. Further investigations will be necessary to identify the signals during RBC storage that stimulate ANG activation, and the roles that activated ANG, continuous RNA processing, and/or miR-720 induction play in RBC aging and the development of the RBC storage lesion.