An RNA-immunoprecipitation via CRISPR/dCas13 reveals an interaction between the SARS-CoV-2 5'UTR RNA and the process of human lipid metabolism

We herein elucidate the function of SARS-CoV-2derived 5'UTR in the human cells. 5'UTR bound host cellular RNAs were immunoprecipitated by gRNA-dCas13 (targeting luciferase RNA fused to SARS-CoV-2 5'UTR) in HEK293T and A549 cells. The 5'UTR bound RNA extractions were predominantly enriched for regulating lipid metabolism. Overexpression of SARS-CoV-2 5'UTR RNA altered the expression of factors involved in the process of the human Mevalonate pathway. In addition, we found that HMG-CoA reductase inhibitors were shown to suppress SARS-CoV-2 5'UTR-mediated translation activities. In conclusion, we deduce the array of host RNAs interacting with SARS-CoV-2 5'UTR that drives SARS-CoV-2 translation and influences host metabolic pathways.


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The outbreak of a novel strain of coronavirus -SARS-CoV-2 causing severe respiratory 56 illness designated as COVID-19, surged as a large-scale global pandemic claiming more than 57 1 million lives while requiring intensive care hospitalizations for about 2% to 10% of the 58 infected cases worldwide [1]. Accumulated data corroborated since a year of the pandemic 59 has shown COVID-19 patients to present diverse symptoms with severe immune 60 dysregulation of unknown incentives [2][3][4][5]. 61 It is noteworthy that a recent meta-analysis establishes a positive link between COVID-19 62 10 contained transcribed SARS-CoV-2-derived 5'UTR RNA and/or luciferase RNA. SARS-142 CoV-2 derived 5'UTR RNA and luciferase RNAs were not detected in cells expressing 143 gRNA that do not target luciferase RNA (nontarget gRNA) (Figure 3-b). Hence we achieved 144 the extraction of 5' UTR bound host RNA clusters that are crucial in the translation of SARS-145 CoV-2 genome by gRNA-dCas13 assisted Trans RNA Immunoprecipitation (TRIP) method. Host regulatory RNA bound to 5'UTR extracted from TRIP were then subjected to ultra-low 150 input RNA sequencing. In the cluster analysis of differentially enriched genes, we found that 151 the RNA group that binds to SARS-CoV-2 5'UTR RNA in A549 cells belong to a different 152 cluster for the RNA group that binds to Luciferase RNA. (Figure 4-a). Similar enrichment 153 differences between RNA group that binds to the 5'UTR RNA of SARS-CoV-2 and the 154 control group that binds only to the RNA of luciferase could be confirmed in HEK293T cells 155 as well. (We confirmed the duplication of genes that are differentially enriched in each group 156 compared to the control group or genes that are differentially enriched in multiple groups). 157 We found that more than 1,000 RNA genes that bind to the 5'UTR of SARS-CoV-2 are shared 158 between A549 cells and HEK293T cells. (Figure 4-b). Further, we performed Gene 159 Ontology enrichment analysis of RNA genes that bind predominantly (p-value <0.01) to 160 5'UTR of SARS-CoV-2 extracted from HEK293T cells and A549. The top 5 GO terms 161 identified in the analysis were lipid metabolic process, the cellular lipid metabolic process, 162 the small molecule metabolic process, the organic acid metabolic process and oxoacid 163 metabolic process. (Figure 4-c & Table 1). These results suggest that the 5'UTR of SARS-164 CoV-2 may interfere significantly with lipid metabolism in human cells. 165 Next, we performed peak calling followed by motif analysis for 5'UTR bound host RNA 166 which enabled us to identify protein binding regions ( Figure 5 & Table 2). From the 167 concentrated RNA motif, respectively 12 and 14 types of RNA-binding proteins were 168 extracted from HEK293T and A549 cells. The proteins commonly observed in both cells 169 were RBM38, BRUNOL4, BLUNOL5, and TARDBP. All of them are essentially metabolic 170 regulators. This observation suggests that the SARS-CoV-2 5'UTR may alter the cellular 171 metabolic process, especially the lipid metabolic process in the host. We found that the 172 SARS-CoV-2 virus uses 5'UTR to interact with various protein and regulatory RNAs 173 to sneak into the human intracellular environment for its translation initiation. It may be 174 thought of as "the takeover of RNA metabolism" mechanism in the human cell by the SARS-175 CoV-2 virus. 176 177

5'UTR-mediated translation influences Mevalonate pathway
178 Following our previous investigation which showed that 5'UTR bound RNAs were enriched 179 for lipid metabolism, we further examined whether factors for lipid metabolism altered upon 180 entry of SARS-CoV-2 into the human cells. Therefore, we introduced pGL3-5'UTR or pGL3-181 Promoter vectors into HEK293T cells and A549 cells, and quantified the RNAs with qPCR. 182 Surprisingly, significant changes in the mRNA expression level were observed 183 for ACAA, HMGCS, and FADS2 genes which are enriched in the Mevalonate pathway, and 184 remarkably, HMGCS expression was highly upregulated (Figure 6-a). Since we had a set of 185 enriched genes that bind to 5'UTR of SARS-CoV-2 in human cells, we further proceeded for 186 drug repositioning by target identification. We used TargetMine program 187 (https://targetmine.mizuguchilab.org) which aided the identification of suitable targets. As a 188 13 result, four interacting compounds (Atorvastatin, Simvastatin, Rosuvastatin and Pravastatin) 189 that can be expected to act (p-value<0.05) on the enriched RNA were identified. Amongst 190 all, statins (p-value <0.01) have been identified as optimal, ready-to-use compounds to treat 191 antiviral growth in COVID-19 patients. (Figure 6-b). To investigate, whether suppression of 192 translational activity by 5'UTR of SARS-CoV-2 can be induced, we added over-the-counter 193 drug HMG-CoA reductase inhibitors after overexpressing the pGL3-5'UTR or pGL3-194 Promoter vector into HEK293T cells and A549 cells. It was interesting to observe that 5'UTR 195 SARS-CoV-2-mediated translational activity in both HEK293T cells and A549 cells were  However two studies suggest preferential loss of lipids and amino acids was associated with 221 enhanced infiltration of cytokines leading to disease severity in 26]. It is 222 anticipated that further understanding of altered lipid metabolism in the virus-infected cells 223 may help uncover the severity pattern in patients. In conclusion, we emphasize that elucidating the function of the SARS-CoV-2 5'UTR region 252 in human cells helps the understanding of the host transcriptome changes which may help to 253 predict the severity of the infection. We also anticipate the dysregulated genes as a result of 254 infection may serve as a biomarker to predict the course of the disease. Our CRISPR / 255 dCas13-based RNA genetic engineering approach as employed in the present study can also 256 be extended to understand host cell response in other infectious diseases as well.    the culture solution at a concentration range of 1nM, 10nM, 100nM and 1,000 nM. Following 573 incubation for 48 hours, the luciferase activity was measured. To assess the effect on the 574 5'UTR, the pGL3-promoter and luciferase activity from each adjacent well into which the 575 pGL3-5'UTR vector was introduced were subtracted. The internal control used was the sum 576 of each value. The results were shown as mean ± SD and were tested for significance by 577 Student's t-test. The internal control used was the sum of the respective values. Results were 578 shown as mean ± SD, and Student's t-test examined significance.   Figure 1 Work ow of the study. Upon introduction of pGL3-5'UTR vector, relative luciferase activity increased more than 10 times to the equivalent amount of pGL3-promoter. Note that the SARS-CoV-2 derived 5'UTR synergistically activated translation in an intracellular factor-dependent manner. Relative luciferase activity was calculated against the activity of endogenous pRL-RSV serving as control (100%). The Mann-Whitney U test was used for statistical signi cance (p <0.05).

Figure 3
RNA-immunoprecipitation. (a) Prediction of luciferase mRNA-5'UTR fusion. Two optimal 28 nt regions gRNAs targeting the luciferase RNA were designed -gRNA1 and gRNA2. (b) gRNA-dCas13 mediated RNA immunoprecipitation of host RNA bound to 5'UTR sequences. RT-qPCR was performed to determine the accumulation of immunoprecipitated 5'UTR sequence (Top) and luciferase RNA (bottom) via gRNA-dCas13 mediated targeting of luciferase RNA. The host RNA bound to 5'UTR was immunoprecipitated and the enrichment factor was calculated as 2 (-ΔΔCt [RIP / background]). The mean and standard error of the three independent experiments is shown. Statistical analysis was performed using a two-sided paired t-test. * Indicates a signi cant difference from the control sample at a value of p <0.05. (b) Venn diagram showing genes common to the unique genes in the test sample (293L2CV, A549L2CV, and A549L1Cv) that were signi cantly enriched (p <0.05) compared to the control (293L2GL3 or A549L2GL3). 293T-XIST is an RNA-immunoprecipitation sample serving as a negative control using HEK293T cells expressing lncRNA XIST-targeting guide RNA and dCas13. The genes enriched by Control-293 vs 293T-L2-CoV2, Control-A549 vs A549-L1-CoV2, and Control-A549-L2-CoV2 are human intracellular factors that interact with 5'UTR derived from SARS-CoV-2. (c) Genes enriched by Control-293 vs 293T-L2-CoV2 dataset were ranked according to their differential enrichment and the resulting enriched GO terms are visualized using a DAG graphical representation with color-coding re ecting their enrichment degree.

Figure 5
Motif analysis and host binding factors to 5'UTR . The motif in the 5'UTR in HEK293T cells (top) and A549 cells (bottom) is crucial for binding the host translational factors.

Figure 6
Overexpression of 5'UTR in SARS-CoV-2 alters genes expression involved in the mevalonate pathway in HEK293T and A549 cells. (a) 1 ug of pGL3-promoter and pGL3-5'UTR vectors were introduced into cells cultured in 6 well plates (0.3 x 106). Expression of lipid metabolism factors from total RNA was measured. Overexpression of 5'UTR affected the expression of ACAA and HMGCS. Results are shown as mean ± SD, and Student's t-test was used as a test of signi cance. (b) Outline of metabolic pathway of Acetyl-CoA by overexpression of 5'UTR of SARS-CoV-2 and inhibitory mechanism of HMG-CoA reductase inhibitors like statins. (c) 1 ug of pGL3-promoter and pGL3-5'UTR vectors was introduced in cells cultured in 24 well plates. HMGCoA reductase inhibitors were added to the culture solution at a concentration range of 1nM, 10nM, 100nM and 1,000 nM. Following incubation for 48 hours, the luciferase activity was measured. To assess the effect on the 5'UTR, the pGL3-promoter and luciferase activity from each adjacent well into which the pGL3-5'UTR vector was introduced were subtracted. The internal control used was the sum of each value. The results were shown as mean ± SD and were tested for signi cance by Student's t-test. The internal control used was the sum of the respective values. Results were shown as mean ± SD, and Student's t-test examined signi cance.