Author Contributions
J.S., N.A.F., D.F., J.Y. and W.Z. conceived the study and planned the analysis. J.S. and N.A.F. performed data analysis. J.S., N.A.F., H.N., S.C., I.M., J.Y. and W.Z. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.
Figure 1.
Alternative splicing (AS) and alternative polyadenylation (APA). (A) Schematic representation of the five major types of alternative splicing in eukaryotes: Skipped Exon (SE), Retained Intron (RI), Alternative 3 Splice Site (A3SS), Alternative 5 Splice Site (A5SS), and Mutually Exclusive Exon (MXE). Light blue boxes represent the constitutive exons, whereas light yellow and pink exons represent the spliced ones. (B) Two types of alternative polyadenylation, i.e., coding region alternative polyadenylation (CR-APA) and 3-UTR alternative polyadenylation (UTR-APA), and their impact on the functional proteome. PAS: polyadenylation signal; TSS: transcription start site; RBP: RNA-binding protein.
Figure 1.
Alternative splicing (AS) and alternative polyadenylation (APA). (A) Schematic representation of the five major types of alternative splicing in eukaryotes: Skipped Exon (SE), Retained Intron (RI), Alternative 3 Splice Site (A3SS), Alternative 5 Splice Site (A5SS), and Mutually Exclusive Exon (MXE). Light blue boxes represent the constitutive exons, whereas light yellow and pink exons represent the spliced ones. (B) Two types of alternative polyadenylation, i.e., coding region alternative polyadenylation (CR-APA) and 3-UTR alternative polyadenylation (UTR-APA), and their impact on the functional proteome. PAS: polyadenylation signal; TSS: transcription start site; RBP: RNA-binding protein.
Figure 2.
(A) KEGG pathways enriched by alternative splicing events. The blue and red bar charts show the pathways enriched by the splicing events in mock-treated cells and SARS-CoV-2 infected cells, respectively. (B) A plot for the SDCCAG3 AS event. The spliced exon is highlighted in orange. The first two subplots show the read coverage of the gene in both groups, whereas the bottom subplot denotes the gene annotation with exon information. The x-axis and y-axis of the plot represent the position of the specific gene and read coverage of that sample, respectively. To show the differential splicing event, the altered exon is highlighted in orange to provide a clear insight into the phenomenon.
Figure 2.
(A) KEGG pathways enriched by alternative splicing events. The blue and red bar charts show the pathways enriched by the splicing events in mock-treated cells and SARS-CoV-2 infected cells, respectively. (B) A plot for the SDCCAG3 AS event. The spliced exon is highlighted in orange. The first two subplots show the read coverage of the gene in both groups, whereas the bottom subplot denotes the gene annotation with exon information. The x-axis and y-axis of the plot represent the position of the specific gene and read coverage of that sample, respectively. To show the differential splicing event, the altered exon is highlighted in orange to provide a clear insight into the phenomenon.
Figure 3.
One example of a gene (HNRNPH3) shows an alternative polyadenylation event in the 3-UTR region. The black vertical line indicates the potential cleavage site. The x-axis and y-axis represent the position of the gene in its chromosome and the read coverage, respectively. The top two subplots show the changes in read coverage surrounding the event, and the cleavage site is indicated by a black vertical line. The bottom part of the plot shows both the full length and truncated 3-UTRs.
Figure 3.
One example of a gene (HNRNPH3) shows an alternative polyadenylation event in the 3-UTR region. The black vertical line indicates the potential cleavage site. The x-axis and y-axis represent the position of the gene in its chromosome and the read coverage, respectively. The top two subplots show the changes in read coverage surrounding the event, and the cleavage site is indicated by a black vertical line. The bottom part of the plot shows both the full length and truncated 3-UTRs.
Figure 4.
RNA-seq data of mock-treated and SARS-CoV-2 infected A549 cells were analyzed for the CR-APA. The x-axis and y-axis represent the CR-truncation ratios ((short mRNA)/(total mRNA)) of a gene. Each dot represents a gene, where the blue dots and red dots show the significant ones in mock and SARS-CoV-2 infected cells, respectively. Upon SARS-CoV-2 infection, 656 genes showed up-regulated CR-APA while 1206 genes showed down-regulated CR-APA. A total of 8832 genes remained unchanged in their CR-APA.
Figure 4.
RNA-seq data of mock-treated and SARS-CoV-2 infected A549 cells were analyzed for the CR-APA. The x-axis and y-axis represent the CR-truncation ratios ((short mRNA)/(total mRNA)) of a gene. Each dot represents a gene, where the blue dots and red dots show the significant ones in mock and SARS-CoV-2 infected cells, respectively. Upon SARS-CoV-2 infection, 656 genes showed up-regulated CR-APA while 1206 genes showed down-regulated CR-APA. A total of 8832 genes remained unchanged in their CR-APA.
Figure 5.
(A) KEGG pathways enriched by CR-APA events. The blue and red bar charts show the pathways enriched by the APA events in mock-treated cells and SARS-CoV-2 infected cells, respectively. (B) One example of a gene (LARP6) shows an alternative polyadenylation event in the coding region. The truncated coding exon is highlighted in orange.
Figure 5.
(A) KEGG pathways enriched by CR-APA events. The blue and red bar charts show the pathways enriched by the APA events in mock-treated cells and SARS-CoV-2 infected cells, respectively. (B) One example of a gene (LARP6) shows an alternative polyadenylation event in the coding region. The truncated coding exon is highlighted in orange.
Figure 6.
COVID-19 A549 cell lines are clustered by the top 100 transcript markers detected by differential transcript expression analysis.
Figure 6.
COVID-19 A549 cell lines are clustered by the top 100 transcript markers detected by differential transcript expression analysis.
Figure 7.
Differentially expressed transcripts enriched KEGG pathways. The blue and red bar charts show the pathways enriched by the up-regulated and down-regulated transcripts in mock-treated samples over SARS-CoV-2 infected samples, respectively.
Figure 7.
Differentially expressed transcripts enriched KEGG pathways. The blue and red bar charts show the pathways enriched by the up-regulated and down-regulated transcripts in mock-treated samples over SARS-CoV-2 infected samples, respectively.
Figure 8.
Scatter plot of APA and differential gene expression. Red dots represent the individual gene in the analysis. Horizontal blue-dashed lines represent the cutoff values for two-fold changes in differential gene expression. Vertical green-dashed lines represent the cutoff values for the (p-value) of UTR-APA (A) determined by the Chi-squared test, and the (p-value) of CR-APA (B) determined by the Student’s t-test.
Figure 8.
Scatter plot of APA and differential gene expression. Red dots represent the individual gene in the analysis. Horizontal blue-dashed lines represent the cutoff values for two-fold changes in differential gene expression. Vertical green-dashed lines represent the cutoff values for the (p-value) of UTR-APA (A) determined by the Chi-squared test, and the (p-value) of CR-APA (B) determined by the Student’s t-test.
Figure 9.
Four-set Venn diagram shows the overlapped genes in three different types of post-transcriptional regulations and differentially expressed genes (DEG).
Figure 9.
Four-set Venn diagram shows the overlapped genes in three different types of post-transcriptional regulations and differentially expressed genes (DEG).
Table 1.
Number of detected significant splicing events in each category between SARS-CoV-2 infected and mock-treated cells.
Table 1.
Number of detected significant splicing events in each category between SARS-CoV-2 infected and mock-treated cells.
Sample | SE | RI | MXE | A3SS | A5SS |
---|
Mock | 51 | 16 | 7 | 13 | 6 |
SARS-CoV-2 | 126 | 30 | 19 | 59 | 100 |
Table 2.
Top 10 significant splicing events between A549 mock-treated and SARS-CoV-2 infected cells.
Table 2.
Top 10 significant splicing events between A549 mock-treated and SARS-CoV-2 infected cells.
Gene Name | Chr | Start | End | p-Value | FDR | Ratio Difference |
---|
TPT1 | chr13 | 45,914,846 | 45,914,920 | 5.81 × 10 | 7.5 × 10 | −0.074 |
C6ORF48 | chr6 | 3,118,955 | 3,119,049 | 9.21 × 10 | 5.94 × 10 | −0.113 |
FKBP1A | chr20 | 1,373,477 | 1,373,525 | 2.25 × 10 | 9.67 × 10 | −0.168 |
PPIA | chr7 | 44,838,346 | 44,838,413 | 1.63 × 10 | 5.25 × 10 | 0.149 |
HNRNPA1 | chr12 | 54,676,862 | 54,677,018 | 1.26 × 10 | 3.26 × 10 | −0.248 |
RPS24 | chr10 | 79,799,961 | 79,799,983 | 5.25 × 10 | 1.13 × 10 | 0.122 |
RPS9 | chr19 | 54,710,420 | 54,710,592 | 4.59 × 10 | 8.46 × 10 | 0.117 |
SRSF2 | chr17 | 74,731,853 | 74,731,957 | 1.24 × 10 | 1.78 × 10 | 0.172 |
CA12 | chr15 | 63,638,728 | 63,638,908 | 1.66 × 10 | 1.99 × 10 | −0.103 |
RPLP1 | chr15 | 69,745,985 | 69,746,060 | 1.69 × 10 | 1.99 × 10 | 0.040 |
Table 3.
Literature review of identified genes with transcript variants and their implication on SARS-CoV-2 pathogenesis and prognosis.
Table 3.
Literature review of identified genes with transcript variants and their implication on SARS-CoV-2 pathogenesis and prognosis.
Category | Gene | Ref. | Description |
---|
Alternative Splicing | BTF3 | [15] | Interacts with the NSP10 CoV protein, which is involved in the pathological function of SARS-CoV in cells. |
| FKBP1A | [16] | FKBP1A causes immunosuppression and is required by CoV for viral growth. |
| G3BP1 | [17] | SARS-CoV-2 N protein undergoes liquid–liquid phase separation, which serves as a scaffold for virus replication and assembly, through its N-terminal intrinsically disordered region (IDR) with G3BP1. |
UTR-APA | ANXA2 | [18] | The upregulation of expression of annexin A2 (ANXA2) by SARS-associated cytokines and the cross-reactivity of anti-SARS-CoV S2 antibodies to annexin A2 may have implications in SARS disease pathogenesis. |
| CAV1 | [19] | Coronaviruses enter cells via the CAV1 dependent pathway. |
| TMEM97 | [20] | TMEM97 forms a complex with ACE2 and modulates its ability to internalize the SARS-CoV-2. |
CR-APA | CTSC | [21] | CTSC activates the elastase-related neutrophil proteases mediated tissue degradation in which it diffuses the alveolar inflammation in acute respiratory distress syndrome. |
| RHOA | [22] | Activation of RhoA GTPase and its downstream effector, Rho kinase (ROCK), contributes to a burst in inflammatory features, immune cell migration, apoptosis, coagulation, contraction, and cell adhesion in pulmonary endothelial cells, leading to endothelium barrier dysfunction and edema as hallmarks of lung injury. |
| CANX | [23] | Calnexin (CANX) strictly monitors the maturation of the CoV S protein by its direct binding. |
Differential Expression | BCL2A1 | [24] | Pro-survival gene mostly present in adult genes, if downregulated, promotes apoptosis in lung tissue. |
| SKP2 | [25] | SKP2 attenuates autophagy through Beclin1-ubiquitination and allowing for the replication of coronaviruses. |
Table 4.
Top 10 significant alternative polyadenylation events in the 3-UTR region (UTR-APA) between A549 mock-treated and SARS-CoV-2 infected cells. The ‘Position’ column refers to the potential cleavage site for the UTR-APA.
Table 4.
Top 10 significant alternative polyadenylation events in the 3-UTR region (UTR-APA) between A549 mock-treated and SARS-CoV-2 infected cells. The ‘Position’ column refers to the potential cleavage site for the UTR-APA.
Gene Name | Chr | Position | p-Value | FDR | Ratio Difference |
---|
ACTN4 | chr19 | 38,730,184 | 6.07 × 10 | 5.71 × 10 | 0.144 |
ALDH1A1 | chr9 | 72,900,986 | 1.34 × 10 | 6.30 × 10 | 0.054 |
S100A6 | chr1 | 153,534,690 | 2.73 × 10 | 8.56 × 10 | 0.045 |
HNRNPA2B1 | chr7 | 26,191,861 | 1.26 × 10 | 2.96 × 10 | 0.088 |
PMEPA1 | chr20 | 57,651,579 | 2.10 × 10 | 3.95 × 10 | 0.153 |
ACAT2 | chr6 | 159,779,033 | 3.11 × 10 | 4.88 × 10 | 0.273 |
ARL4C | chr2 | 234,495,151 | 6.05 × 10 | 8.14 × 10 | 0.231 |
H3F3A | chr1 | 226,071,775 | 8.70 × 10 | 1.02 × 10 | 0.189 |
RPL15 | chr3 | 23,919,537 | 1.32 × 10 | 1.38 × 10 | 0.041 |
MYH9 | chr22 | 36,281,660 | 3.29 × 10 | 2.91 × 10 | 0.177 |
Table 5.
Top 10 significant alternative polyadenylation events in the coding region (CR-APA) between A549 mock-treated and SARS-CoV-2 infected cells.
Table 5.
Top 10 significant alternative polyadenylation events in the coding region (CR-APA) between A549 mock-treated and SARS-CoV-2 infected cells.
Gene | Chr | Truncated Position | p-Value | FDR | Ratio Difference |
---|
GLRX5 | chr14 | 95,544,557 | 2.76 × 10 | 6.12 × 10 | 0.361 |
UBXN6 | chr19 | 4,445,009 | 5.98 × 10 | 1.14 × 10 | 0.020 |
SLC3A2 | chr11 | 62,881,290 | 1.31 × 10 | 2.16 × 10 | 0.256 |
ARIH2 | chr3 | 48,928,674 | 1.37 × 10 | 1.18 × 10 | 0.071 |
DVL3 | chr3 | 184,164,517 | 1.50 × 10 | 2.16 × 10 | −0.269 |
DEF8 | chr16 | 89,959,366 | 1.62 × 10 | 1.18 × 10 | −0.232 |
SIAH2 | chr3 | 150,742,487 | 1.62 × 10 | 2.16 × 10 | −0.362 |
SNHG7 | chr9 | 136,724,659 | 1.84 × 10 | 2.16 × 10 | 0.020 |
LTBP2 | chr14 | 74,506,705 | 1.94 × 10 | 2.16 × 10 | −0.248 |
S100A16 | chr1 | 153,606,974 | 2.29 × 10 | 2.35 × 10 | 0.200 |