Role of novel SARS-CoV-2-derived svRNAs and the involvement of tRF in COVID-19

In a recent study posted to the bioRxiv* preprint server, researchers demonstrated the involvement of transfer ribonucleic acid (tRNA)-derived RNA fragment (tRf) in coronavirus disease 2019 (COVID-19) and identified various novel small non-coding RNA (sncRNAs) derived from severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2) termed as svRNAs.

Study: Changes of small non-coding RNAs by severe acute respiratory syndrome coronavirus 2 infection. Image Credit: Kateryna Kon/Shutterstock

The underlying molecular mechanisms of the COVID-19 pandemic caused by SARS-CoV-2 are not well established. The sncRNAs have regulatory functions in various biological processes including the regulation of host response to viral infection. sncRNAs include microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), tRFs, and small nucleolar RNAs (snoRNAs). The tRFs are a recently emerged subfamily of sncRNAs and have a significant role in the regulation of host-virus interaction. Due to the limitations in sequencing technologies, not much data about the SARS-CoV-2 impact on sncRNAs expression is available.

The study

In the present study, the researchers identified remarkable differences in the profiles of sncRNA with heterogeneous ends among the COVID-19 infected and non-infected individuals using a modified next-generation sequencing (NGS) called T4 PNK‐RNA‐sequence. A modified quantification real-time polymerase chain reaction (qRT-PCR) was used to quantify and validate the tRFs derived from the nasopharyngeal swabs (NPS) and airway epithelial cells of SARS-CoV-2 patients.

Thirteen anonymous NPS samples including seven SARS-CoV-2 negative samples and six positive samples were collected from patients who were tested for COVID-19 in out-patient clinics of the University of Texas Medical Branch (UTMB) in April 2020. The RNAs were extracted using the mirVana PARIS Kit and were subjected to deep sequencing and qRT-PCR. The impact of SARS-CoV-2 on sncRNAs was determined using the T4 PNK‐RNA‐sequence.

The African green monkey kidney epithelial cells (Vero E6), small airway epithelial cells (SAECs) isolated from the lungs, and human alveolar type II-like epithelial cells expressing human angiotensin-converting enzyme 2 (A549-ACE2) were used in the study.

Additionally, the SARS-CoV-2 strains were obtained from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) at the UTMB and viral titers were determined using plaque assay. A549-ACE2 and SAECs cells were infected with the virus under laboratory conditions. The sncRNAs expression was evaluated using the qRT-PCR and the tRF5-GluCTC was analyzed using hybridization in Northern blot (NB).

Results

The results indicated that there is a change in sncRNA expression followed by SARS-CoV-2 infection. The sequencing data from T4 PNK-RNA-sequence show that NPS piRNAs and tRFs had higher global expression than miRNAs. The svRNA derived from the 346 to 382 genomic sites of SARS-CoV-2 (sv-CoV2-346) had the highest expression. The sequencing data from NB data of tRF5-GluCTC and the qRT-PCR result were also consistent with the T4 PNK-RNA-sequence data.

The four novel tRF5s identified in the study were tRF5-GlyCCC-6-1, tRF5-GluTTC-1-2, tRF5-SecTCA-2-1, and tRF5-LysCTT6-1. These new tRfs lacked first 10-15 nucleotides of the tRNA 5’end and were sub-grouped as tRF5DC.

The comparison between the tRF induction by the respiratory syncytial virus (RSV) and SARS-CoV-2 to demonstrate the virus-specific tRF induction indicated that tRF5-ValCAC were only induced by SARS-CoV-2 whereas tRF5-GlyGCC were only induced by RSV.

The sequencing data revealed the presence of a significant amount of svRNAs in the SARS-CoV-2 patients’ NPS and the amount varied among participants. The svRNA, sv-CoV2-346 was also identified from the SARS-CoV-2 infected A549-ACE2 cells. The qRT-PCR results indicated that sv-CoV2-346 had a non-specific band and a specific band around 55nucleotide.

New SARS-CoV-2 svRNAs, which were longer than miRNAs, were identified from the study. The most abundant svRNA was sv-CoV2-346 and it lacked the 3’-OH as T4 PNK treatment was required for sv-CoV2-346 detection. A 68 nucleotide sv-CoV2-314 was identified which may be a precursor of 36 nucleotide sv413 CoV2-346 and may have structural similarity with tRNA.

Conclusion

According to the authors, this is the first study demonstrating the alteration in the tRF function by SARS-CoV-2. The study revealed new long sv-COV-2 using T4 PNK pretreatment and qRT-PCR and also identified piRNAs and tRFs as the most abundant sncRNAs in COVID-19 positive samples. The authors intend to categorize the biogenesis and mechanisms of the newly detected COVID-19-associated sncRNAs and the relationship between viral load and SARS-CoV-2 disease severity in future studies.

The tRF5 detection data obtained from T4 PNK-RNA-sequence in this study were reliable as there was consistency among the T4 PNK-RNA-sequence data, NB data of tRF5-GluCTC, and qRT-PCR result. Since the newly detected tRF5s span the complete region of the D loop and the first half of the anticodon loop, they were sub-grouped as tRF5DC. The tRF5DC and ordinary tRFs may have different biogenesis mechanisms since they were from different isoacceptor tRNAs.

Further studies are required for a comprehensive understanding of the mechanism of host protein utilization by the virus to produce small viral RNA fragments.

Written by

Shanet Susan Alex

Shanet Susan Alex, a medical writer, based in Kerala, India, is a Doctor of Pharmacy graduate from Kerala University of Health Sciences. Her academic background is in clinical pharmacy and research, and she is passionate about medical writing. Shanet has published papers in the International Journal of Medical Science and Current Research (IJMSCR), the International Journal of Pharmacy (IJP), and the International Journal of Medical Science and Applied Research (IJMSAR). Apart from work, she enjoys listening to music and watching movies.