CPSF6 protein expression is decreased in virus-infected cells

Knockdown of CPSF5 (CFIm25) or CPSF6 (CFIm68), two key subunits of the CFIm tetramer leads to a widespread increase in the use of proximal PASs (pPASs) [40], making this complex a key candidate to reveal the genome-wide landscape of shortened tandem 3’ UTRs upon viral infection. To explore the mechanism of the widespread dynamic changes in APA in host cells after viral infection, we first tested the change in the mRNA expression of CPSF6 and the other two members of CFIm upon viral infection or nucleic acid analogue stimulation. The data indicated that all the members of the CFIm complex exhibited decreased expression upon infection with VSV (vesicular stomatitis virus) or HSV-1 (herpes simplex virus 1) (Fig 1A and 1B) or stimulation with the nucleic acid analogue Poly (I:C) LMW (low molecular weight) or HSV60 (synthetic herpes simplex virus 1 DNA analogue) in BMDMs (mouse bone marrow-derived macrophages) (Fig 1C and 1D). Interestingly, the protein expression of CPSF6 was significantly decreased after viral infection or analogue simulation, accompanied by a decreased RNA abundance, while CPSF5 and CPSF7 (CFIm59) showed no significant changes (S1A and S1B Fig). Similar results were obtained in VSV-eGFP- or SeV (Sendai virus)-infected, or in Poly (I:C)- or Poly (dA:dT)-stimulated A549 and L929 cells (Fig 1E–1H). These data collectively suggested that the protein abundance of CPSF6 protein is reduced in multiple virus-infected cells.

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Fig 1. CPSF6 protein expression is decreased in virus-infected cells.

(A-D) Mouse BMDMs were challenged with VSV (A), HSV-1 (B) poly (I:C) (C) or HSV60 (D) for 12h, and the mRNA levels of core 3’ processing factors were analyzed by qRT–PCR. (n = 3 replicates) (E, F) Immunoblot analyses of CFIm complex expression in A549 (E) and L929 (F) cells infected with VSV or SeV at indicated time points. (G, H) Immunoblot analyses of CFIm complex expression in A549 (G) and L929 (H) cells stimulated with poly (I:C) or Poly (dA:dT) at indicated time points. The expression of RIG-I indicates that viral infection or analogue stimulation has successfully activated the antiviral signaling of cells. (I-L) The relative mRNA expression of CPSF6 in whole blood (I), PBMCs (J), monocytes and DCs (K) from patients with COVID-19 or in peripheral blood cells (L) from patients with influenza are shown. (M-P) The relative mRNA expression of CPSF6 in RV-A16-infected AECs (M), HCV-infected Huh7 cells (N), S. aureus-infected hMDMs (O) and helminth-infected mouse lung tissues (P) are shown. Data are representative of three independent experiments, with one representative shown in (E-H). The values represent mean ± SD with individual measurements overlaid as dots, statistical analysis was performed using a two-tailed Student’s t-test or Mann–Whitney U test in (A-D) and (I-P).

https://doi.org/10.1371/journal.ppat.1012061.g001

To confirm this observation, we conducted statistical analyses of previously published gene expression datasets (GSE213313/149689/150728/236651/27131/226071/20948/13670/3414) and found that the mRNA expression of CPSF6 was significantly lower in SARS-CoV-2- (Fig 1I–1K) or influenza (H1N1)-infected patients (Fig 1L), rhinovirus A16 (RV-A16)-infected primary human airway epithelial cells (AECs) (Fig 1M) and hepatitis C virus (HCV, JFH-1)-infected Huh7 cells (Fig 1N). Not only viral infections, but also bacterial or helminthic infections can lead to downregulation of CPSF6 (Fig 1O and 1P). All these data indicated that downregulated expression of CPSF6 is a common phenomenon in the immune response to infection, implying that CPSF6 may play an important regulatory role in pathogen infection, especially viral infection.

CPSF6 deficiency inhibits virus replication

To explore the role of CPSF6 in antiviral immune response, we next evaluated the effect of CPSF6 on virus replication. First, we silenced the endogenous expression of CPSF6 in A549 and L929 cells (S2A Fig). Then, VSV-eGFP-infected cells were subjected to flow cytometry analyses. The results showed that knockdown of CPSF6 in A549 and L929 cells significantly inhibited the production of VSV virions (S2B Fig), accompanied by a lower VSV RNA abundance (S2C Fig). To further study the function of CPSF6 in regulating the antiviral response, we generated the CPSF6^-/- A549 and L929 cells using the CRISPR/Cas9 system (Fig 2A) and observed lower VSV replication and lower VSV RNA and protein abundances in these cells (Fig 2B–2E). Moreover, lower VSV titers in the cell culture supernatant (Fig 2F) and a significantly decreased VSV-eGFP abundance in CPSF6^-/- A549 and L929 cells were observed (Figs 2G and S2D). Furthermore, the replication of other types of viruses, such as the RNA virus SeV and DNA virus HSV-1, was also reduced in Cpsf6^-/- L929 cells (S2E–S2G Fig). To confirm the role of CPSF6, CPSF6-reconstituted L929 cells (hereinafter referred to as Cpsf6^-/- L929^CPSF6 cells; Fig 2H) were established. Ectopic expression of CPSF6 resulted in increased VSV RNAs and proteins abundances (Fig 2I and 2J) compared with those in Cpsf6^-/- L929 cells. The fluorescence intensity of GFP and the load of VSV titers indicated that more VSV virions were produced in Cpsf6^-/- L929^CPSF6 cells (Fig 2K–2M). In addition, qRT–PCR and immunoblot analyses indicated that the ectopic CPSF6 expression also increased HSV-1 replication (S2H and S2I Fig). All these data indicate that CPSF6 deficiency can inhibit the replication of both DNA and RNA viruses.

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Fig 2. CPSF6 deficiency inhibits virus replication.

(A) Immunoblot analyses knocking-out efficiency of CPSF6 in A549 and L929 cells. (B) qRT–PCR analyses of VSV mRNA abundance in WT and CPSF6^-/- A549 and L929 cells infected with VSV-eGFP at indicated time points. (n = 3 replicates) (C) FACS analyses of VSV replication in WT and CPSF6^-/- A549 and L929 cells infected with VSV-eGFP for 12 h. (n = 3 replicates) (D, E) Immunoblot analyses of VSVG protein expression in WT and CPSF6^-/- L929 (D) or A549 (E) cells infected with VSV-eGFP at indicated time points. (F) Plaque assays of VSV titers in cell supernatants from WT and Cpsf6^-/- L929 cells infected with VSV-eGFP for 12 h. (n = 3 replicates) (G) Fluorescence microscope analyses of GFP intensity in WT and Cpsf6^-/- L929 cells infected with VSV-eGFP for 16 or 24 h. Scale bar, 100 μm. (H) Immunoblot analyses the expression of CPSF6 in WT, Cpsf6^-/-L929^PLVX and Cpsf6^-/- L929^CPSF6 L929 cells. (I) qRT–PCR analyses of VSV mRNA abundance in WT, Cpsf6^-/-L929^PLVX and Cpsf6^-/- L929^CPSF6 L929 cells infected with VSV-eGFP at indicated time points. (n = 3 replicates) (J) Immunoblot analyses of VSVG protein expression in Cpsf6^-/-L929^PLVX and Cpsf6^-/- L929^CPSF6 L929 cells infected with VSV-eGFP at indicated time points. (K-M) Fluorescence microscope (K), FACS (L) and plaque assays (M) analyses of VSV expression in Cpsf6^-/-L929^PLVX and Cpsf6^-/- L929^CPSF6 L929 cells infected with VSV-eGFP at indicated time points. Scale bar, 100 μm. (n = 3 replicates). Data are representative of three independent experiments, with one representative shown in (A), (D), (E), (H) and (J). The values represent mean ± SD with individual measurements overlaid as dots, statistical analysis was performed using a two-tailed Student’s t-test in (B), (C), (F), (I), (L) and (M).

https://doi.org/10.1371/journal.ppat.1012061.g002

Global 3’ UTR shortening caused by CPSF6 deficiency orchestrates the cellular antiviral capacity

CPSF6 is a key component of the CFIm complex, and knocking down of CPSF6 leads to transcriptome-wide shortening of 3’ UTRs due to an increase in the use of pPASs [41]. Given the roles of CPSF6 in APA processing and in regulating viral infection as described above, we hypothesized that CPSF6 acts as an antiviral immune regulator by influencing APA and the 3’ UTR length. To test this hypothesis, we used the IVT-SAPAS method for transcriptome-wide APA profiling in VSV-eGFP-infected L929 cells with knockout or ectopic expression of CPSF6 [38,39]. After high-throughput sequencing, we found that more than 80% of the qualified reads were mapped to the annotated UCSC transcript ends database and Tian’s database (S3A Fig). Moreover, approximately 60% of polyadenylation signals are one of two canonical hexanucleotide polyadenylation signal sequences, the AATAAA or ATTAAA, most of which are located 10–30 bases upstream of the cleavage/polyadenylation site and specifically recognized by the CPSF [42] (S3B Fig). Consistent with previous studies, approximately 50% of genes contained more than one poly(A) site (S3C Fig). Consistent with our previous observation [29], the average 3’ UTR length in cells was decreased after viral infection and decreased even more upon CPSF6 deficiency (Fig 3A), further demonstrating the priority usage of proximal PASs in the absence of CPSF6. After statistical analysis, we found that more than 2000 genes showed shortened 3’ UTRs in CPSF6-deficient cells at rest and after viral infection, while the 3’ UTRs of approximately 1500 genes became longer when CPSF6 was ectopically expressed in CPSF6-deficient cells (Figs 3B, S3D and S3E). Subsequently, we defined the genes with shorter 3’ UTRs upon CPSF6 deficiency and longer 3’ UTRs upon CPSF6 restoration as the target genes of CPSF6, and finally identified 1127 target genes (Fig 3C), most of which are related to virus infection, autophagy, signal transduction, metabolism and cell proliferation, as determined using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (Fig 3D).

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Fig 3. Global 3’ UTR shortening caused by CPSF6 deficiency orchestrates the cellular antiviral capacity.

(A) Notched box plot of the weighted mean of the 3’ UTR length. For each gene with UTR-APA, the length of each 3′ UTR isoform was normalized to the longest 3’ UTR, and the weighted mean of 3’ UTR length was calculated. (B) The number of poly (A) site-switched genes in L929 cells at rest. (C), Venn diagram showing the overlap of genes from (B). (D) KEGG enrichment analysis of the overlapped transcripts presented in (C). (E) The number of differentially expressed genes (DEGs) in L929 cells upon VSV-eGFP infection. (F) Venn diagram showing the overlap of genes from CPSF6 target genes and up-regulated genes after the deletion of CPSF6. (G) GO analysis of the overlapped transcripts presented in (F). (H) The heatmap shows the mRNA abundance of immune-related genes from IVT-SAPAS data.

https://doi.org/10.1371/journal.ppat.1012061.g003

In addition to measuring tandem 3’ UTR lengths, we can also use our IVT-SAPAS data to quantify mRNA expression by totaling the reads mapped to the 3’ UTR of a given gene. We found that more transcripts were profoundly upregulated (>3 fold, FDR-adjusted P<0.01, Fisher’s exact test) in Cpsf6^-/- L929 cells than in WT cells with or without viral infection, while the ectopic expression of CPSF6 inhibited this upregulation (Fig 3E). Next, we identified 228 CPSF6 target genes among the upregulated genes upon CPSF6 deficiency (Fig 3F). Gene Ontology (GO) analysis further indicated that the terms interferon production and regulation, stress response, apoptosis and inflammasome assembly (Fig 3G), which are closely related to host response and resistance to invading pathogens, were significantly enriched in these genes. Along with the global 3’ UTR shortening, significant upregulation of a large number of immune-related genes was observed in CPSF6-deficient L929 cells after viral infection compared with the controls; in contrast, ectopic expression of CPSF6 in Cpsf6^-/- L929^CPSF6 cells suppressed the upregulation of these genes (Fig 3H). Furthermore, we also enriched the non-upregulated genes in CPSF6 target genes and found that these genes were mainly enriched to phagocytosis, autophagy, metabolism, protein synthesis and modification, angiogenesis, cell proliferation and apoptosis and other biological processes (S3F and S3G Fig). All these data indicated that the global 3’ UTR shortening caused by CPSF6 deficiency orchestrates the cellular antiviral capacity to inhibit viral infection.

CPSF6 deficiency increases the use of proximal PASs in many immune-related genes

More than half of the genes involved in the TLR, RLR or Jak-STAT signalling pathways have multiple PASs in their 3’ UTRs and exhibit noteworthy APA switching to use pPAS after viral infection [29]. Here, our IVT-SAPAS data further confirmed that many immune-related genes involved in virus recognition and signal transduction undergo dynamic APA processing and tend to use pPASs. This preference was even more pronounced in Cpsf6^-/- L929 cells and reversed in Cpsf6^-/- L929^CPSF6 cells (Figs 4A and S4A–S4G). Accordingly, the mRNA abundances of these immune-related genes were significantly increased with the shortening of 3’ UTRs in CPSF6^-/- L929 cells (Fig 4B). To further verify the above data, qRT–PCR analysis was performed to test the APA switching in 5 selected immune-related genes (Ddx58, Ccl2, Ifit3, Ddx21 and Trim21) that are deeply involved in the antiviral immune response. By convention, the segment of the 3’ UTR upstream of the proximal PAS is called the constitutive 3’ UTR (cUTR), while the downstream segment that is present only in longer isoforms is designated the “alternative UTR” (aUTR) or “extended UTR” (eUTR) [2]. Then, we used eUTR/cUTR ratio to indicate the ratio of longer 3’ UTR isoforms to total transcripts. The results were consistent with our IVT-SAPAS data indicating that all tested genes exhibited appreciable 3’ UTR shortening upon VSV-eGFP infection or CPSF6 deficiency, and that ectopic expression of CPSF6 partially prevented this trend (Fig 4C–4G). The same results were obtained in HSV-1-infected L929 cells (Fig 4H–4L) and Poly (I:C)-stimulated L929 cells (S4H–S4K Fig). We next measured the mRNA expression of the tested genes and observed that their expression was markedly increased in Cpsf6^-/- L929 cells and reduced in Cpsf6^-/- L929^CPSF6 cells (Fig 4M). In contrast, the transcripts of these genes with long 3’ UTRs did not show significant upregulation or even downregulation in Cpsf6^-/- L929 cells after VSV infection (Fig 4N). Taken together, these results demonstrated that CPSF6 deficiency leads to the preferential use of pPASs in many immune-related genes.

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Fig 4. CPSF6 deficiency increases the use of proximal PASs in many immune-related genes.

(a) The genes with APA involved in virus recognition and signal transduction were shown. The black arrows indicate signal transduction. The x axis of the line graphs denotes infection time points and the y axis denotes normalized 3’ UTR length. Asterisk (*) indicates the APA genes shown in the line graph. (B) The heatmap shows mRNA abundance of immune-related genes from (A). (C-G) qRT–PCR analyses the ratio of longer 3’ UTR isoforms to total mRNA of tested genes Ddx58 (C), Ccl2 (D), Ifit3 (E), Ddx21 (F) and Trim21 (G) in WT, Cpsf6^-/-L929^PLVX and Cpsf6^-/- L929^CPSF6 L929 cells infected with VSV-eGFP at indicated time points. (n = 3 replicates) (H-L) qRT–PCR analyses the ratio of longer 3’ UTR isoforms to total mRNA of tested genes Ddx58 (H), Ccl2 (I), Ifit3 (J), Ddx21 (K) and Trim21 (L) in WT, Cpsf6^-/-L929^PLVX and Cpsf6^-/- L929^CPSF6 L929 cells infected with HSV-1 at indicated time points. (n = 3 replicates) (M, N) qRT–PCR analyses the abundance of total mRNA (M) or transcripts with long 3’ UTR (N) of these tested immune-related genes in WT, Cpsf6^-/-L929^PLVX and Cpsf6^-/- L929^CPSF6 L929 cells infected with VSV-eGFP at indicated time points. (n = 3 replicates). The values represent mean ± SD with individual measurements overlaid as dots, statistical analysis was performed using a two-tailed Student’s t-test in (C)-(L) and (N).

https://doi.org/10.1371/journal.ppat.1012061.g004

The increase in pPAS usage upon CPSF6 deficiency results in increased mRNA stability and protein synthesis

Numerous advances in the past few years have substantially enriched our knowledge that 3’ UTRs are hotbeds for mRNA destabilization elements, which often function through the binding of RNA-binding proteins (RBPs) and microRNAs (miRNAs) [43]. Transcripts using pPASs and therefore having shorter 3’ UTRs are generally thought to produce more proteins due to the exclusion of these regulatory elements that mediate degradation or translational efficiency of mRNAs [10]. To test whether the transcripts of these immune-related genes with shortened 3’ UTRs become more stable or more efficiently translated, we first used a miRNA target prediction program (TargetScan) to identify the miRNA binding sites in the long 3’UTR isoform of specific mRNAs. Interestingly, we found that most of the miRNA binding sites in the tested genes, such as Ddx58, Ccl2, Ifit2, Ddx21, Trim21, Dhx36 and Fos, were localized between the pPAS and the dPAS, a region that specifically presents in the long 3’ UTR (S5A Fig). In agreement with this notion, the extended 3’ UTR region of one tested gene, Ccl2, contains conserved seed matches to miR-124a and miR-33 (S5B Fig), both of which have been reported to directly target the degradation of Ccl2 mRNAs [44,45]. Another tested gene Fos has also been demonstrated to be targeted for degradation and translation inhibition by miR-7b and miR-155 that binds to its extended 3’ UTR region in previous studies [46,47] (S5C Fig). These findings raise the intriguing possibility that CPSF6 depletion leading to 3’ UTR shortening may be a mitigation mechanism for miRNA-mediated degradation. Then, we assessed whether CPSF6-mediated APA is implicated in the mRNA stability of targeted genes. When halting transcription with actinomycin D (Act D), we observed that the decay rate of the tested immune-related genes (Ddx58, Ccl2, Ifit2, Ddx21 and Trim21) was slower in Cpsf6^-/- L929 cells, but faster in Cpsf6^-/- L929^CPSF6 cells (Fig 5A–5E). The above results indicated that the increased usage of pPASs in CPSF6-deficient L929 cells resulted in an increased mRNA stability of those immune-related genes.

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Fig 5. The increase in pPAS usage upon CPSF6 deficiency results in increased mRNA stability and protein synthesis.

(A-E) qRT–PCR analyses the mRNA abundance of Ddx58 (A), Ccl2 (B), Ifit2 (C), Ddx21 (D) and Trim21 (E) in WT, Cpsf6^-/-L929^PLVX and Cpsf6^-/- L929^CPSF6 L929 cells treated with Act D (5 μg/mL) for the indicated times. (n = 3 replicates) (F) Schematic diagram of psiCHECK-3’ UTR recombinant constructs. S, short 3’ UTR; L, long 3’ UTR with mutated proximal PAS. (G-K) Relative luciferase activities in 293T cells after transfection with reporter vectors bearing S-3’ UTR or L-3’ UTR of Ddx58 (G), Ifit3 (H), Ccl2 (I), Ddx21 (J) and Trim21 (K). Renilla luciferase activity was measured and normalized to firefly luciferase activity. (n = 3 replicates) (L) Mechanism diagram of PAS selection affecting luciferase protein production. (M-Q) Relative luciferase activities in WT, Cpsf6^-/-L929^PLVX and Cpsf6^-/- L929^CPSF6 L929 cells after transfection with reporter vectors bearing WT full length 3’ UTR of Ddx58 (M), Ifit3 (N), Ifi35 (O), Ddx21 (P) and Trim21 (Q). (n = 3 replicates). The values represent mean ± SD with individual measurements overlaid as dots, statistical analysis was performed using a two-tailed Student’s t-test in (A)-(E), (G)-(K) and (M)-(Q).

https://doi.org/10.1371/journal.ppat.1012061.g005

To further elucidate the effect of CPSF6-mediated APA switch on the ultimate protein translation from the immune-related genes, 5 target genes (Ddx58, Ifit3, Ccl2, Ddx21 and Trim21) were selected to perform luciferase reporter gene assays. The short 3’ UTRs formed by using the pPAS of each target genes were inserted downstream of the Renilla luciferase translational stop codon in the psiCHECK-2 plasmid, and the resulting plasmid was named the S-3’ UTR construct. Similarly, L-3’ UTR construct contained the long 3’ UTR of the target gene with both the proximal and distal PASs, but the pPAS was mutated to prevent the formation of the S-3’ UTR isoform (Fig 5F). We observed that the expression of short 3’ UTRs of these tested genes increased Renilla luciferase expression (Fig 5G–5K), implying the increased translation efficiency of these target genes with short 3’ UTRs. Finally, we used reporter assays to measure the luciferase activity of reporter plasmids containing the Ddx58, Ifit3, Ifi35, Ddx21 or Trim21 wild-type 3’ UTR in different lines of L929 cells (Fig 5L). The results showed that the luciferase activity of these reporters was increased in Cpsf6^-/- L929 cells but decreased in Cpsf6^-/- L929^CPSF6 cells (Fig 5M–5Q). Together, our data suggested that the increased abundance of proteins translated from immune-related genes upon CPSF6 depletion is mediated by shortening of their 3’ UTRs, which may promote activation of the antiviral immune response.

CPSF6 deficiency promotes IFN-I signalling activation

To further determine the effects of CPSF6 deficiency on virus replication, we evaluated the virus-induced activation of IFN-I signalling in CPSF6^-/- A549 and L929 cells. As expected, the mRNA expression of IFNB, IL6, and a number of ISGs (CCL5, DDX58, IFIT1 and ISG15) was obviously increased in CPSF6^-/- A549 cells infected with VSV-eGFP or SeV compared with the corresponding wild-type cells (S6A Fig). After infection with VSV-eGFP, or treatment with Poly (I:C) or Poly (dA:dT), Cpsf6^-/- L929 cells also exhibited higher expression of IFNB and ISGs, but this increase was suppressed in Cpsf6^-/- L929^CPSF6 cells (Fig 6A and 6B). Furthermore, higher levels of secreted IFN-β were detected in culture supernatant from CPSF6^-/- A549 and L929 cells (Figs 6C–6E and S6B–S6D), and this increasing trend was abolished by ectopic expression of CPSF6 (Fig 6C–6E), demonstrating that CPSF6 deficiency promoted IFN-I responses against viral infection by increasing IFN-β production and ISG expression. Upon viral infection, germline-encoded PRRs are employed to detect viral nucleic acids and then trigger the TBK1-IRF3 signalling cascade to produce IFN-Is and proinflammatory cytokines [21]. To further unveil the function of CPSF6 in virus-triggered innate immune responses, we measured the levels of phosphorylated IRF3 and TBK1 and observed that CPSF6 deficiency markedly facilitated TBK1 and IRF3 phosphorylation after VSV-eGFP infection (S6E Fig). The same results were also found in VSV-eGFP CPSF6^-/- L929 cells (Fig 6F), while Cpsf6^-/- L929^CPSF6 cells with ectopic expression of CPSF6 showed the opposite pattern (Fig 6G). With the marked increase in IRF3 phosphorylation, the expression of ISG-encoded proteins, such as MDA5, RIG-I and ISG15, also increased significantly in CPSF6^-/- A549 and L929 cells compared with wild-type cells (Figs 6H, S6F and S6G). Accordingly, ectopic expression of CPSF6 in Cpsf6^-/- L929^CPSF6 cells inhibited the expression of ISGs upon VSV-eGFP infection and Poly (I:C) or Poly (dA:dT) stimulation (S6H and S6I Fig).

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Fig 6. CPSF6 deficiency promotes IFN-I signalling activation.

(A, B) The heatmap shows the mRNA abundance of IFNB and ISGs in WT, Cpsf6^-/-L929^PLVX and Cpsf6^-/- L929^CPSF6 L929 cells infected with VSV-eGFP (A) or stimulated with poly (I:C) or Poly (dA:dT) (B) at indicated time points. (n = 3 replicates) (C-E) ELISA analyses of IFN-β secretion in cell supernatant from WT, Cpsf6^-/-L929^PLVX and Cpsf6^-/- L929^CPSF6 L929 cells infected with VSV-eGFP (C) or stimulated with poly (I:C) (D) or Poly (dA:dT) (E) at indicated time points. (n = 3 replicates) (F, G) Immunoblot analyses of phosphorylated (p-) TBK1 and IRF3 in WT, Cpsf6^-/-L929^PLVX and Cpsf6^-/- L929^CPSF6 L929 cells infected with VSV-eGFP at indicated time points. (H) Immunoblot analyses of the protein expression of ISGs in WT and CPSF6^-/- A549 and L929 cells infected with VSV-eGFP at indicated time points. (I) Immunoblot analyses knocking-down efficiency of CPSF6 in BMDMs from Cpsf6^+/+ and Cpsf6^+/- mice. (J) qRT–PCR analyses of VSV mRNA abundance in Cpsf6^+/+ and Cpsf6^+/- BMDMs infected with VSV-eGFP at indicated time points. (n = 3 replicates) (K) Plaque assays of VSV titers in cell supernatants from Cpsf6^+/+ and Cpsf6^+/- BMDMs infected with VSV-eGFP for 12 h. (n = 4 replicates) (L) qRT–PCR analyses of Ifnb1 mRNA abundance in Cpsf6^+/+ and Cpsf6^+/- BMDMs infected with VSV-eGFP at indicated time points. (n = 4 replicates) (M) ELISA analyses of IFN-β secretion in cell supernatant from Cpsf6^+/+ and Cpsf6^+/- BMDMs infected with VSV-eGFP at indicated time points. (n = 4 replicates) (N-Q) qRT–PCR analyses of Ddx58 (N), Ifit2 (O), Ifit3 (P) and Isg15 (Q) mRNA abundance in Cpsf6^+/+ and Cpsf6^+/- BMDMs infected with VSV-eGFP at indicated time points. (n = 4 replicates) R, qRT–PCR analyses the ratio of longer 3’ UTR isoforms to total mRNA of tested genes Ddx58, Ddx3x, Ddx21, Ifit2 and Ifit3 mRNA abundance in Cpsf6^+/+ and Cpsf6^+/- BMDMs infected with VSV-eGFP at indicated time points. (n = 3 replicates). Data are representative of three independent experiments, with one representative shown in (F)-(I). The values represent mean ± SD with individual measurements overlaid as dots, statistical analysis was performed using a two-tailed Student’s t-test in (C)-(E) and (J)-(Q).

https://doi.org/10.1371/journal.ppat.1012061.g006

In addition, we also tried to study the in vivo roles of CPSF6 in mice by using CRISPR/Cas9 genome editing to delete the third exon of Cpsf6. However, after zygote microinjection, we only obtained 12 infertile Cpsf6^+/- mice, of which 2 males were dissected and found to have clear epididymides without sperm. After evaluating the expression of CPSF6, we found that it was significantly lower in BMDMs from Cpsf6^+/- mice than in macrophages from their wild-type littermates (Fig 6I). Then, a lower viral load and greater IFN-β production were observed in BMDMs from Cpsf6^+/- mice (Fig 6J–6M). Accordingly, the mRNA expression of many ISGs, such as Ddx58, Isg15, Ifit2 and Ifit3, was also higher in Cpsf6^+/- BMDMs (Fig 6N–6Q). Likewise, the 3’ UTRs of Ddx58, Ddx3x, Ddx21, Ifit2 and Ifit3, were significantly shorter in primary BMDMs from Cpsf6^+/- mice, and such shortening was more pronounced upon VSV-eGFP infection (Fig 6R). These data convincingly demonstrated that the downregulation of CPSF6 promoted interferon production by promoting the use of proximal PASs in immune-related genes, which may amplify IFN-I-dependent antiviral immune responses against both RNA and DNA viruses.

Ethics statement

All animal experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals, with the approval of the Institutional Animal Care and Use Committee (IACUC) of Sun Yat-sen University (Guangzhou, China).

Cell culture

A549, L929 and 293T cells were purchased from ATCC. All these cells were cultured in endotoxin-free DMEM (Gibco), supplemented with 10% FBS (Gibco) and 1% penicillin–streptomycin (Gibco). All cells were incubated at 37°C with 5% CO₂.

Mouse bone marrow derived macrophages (BMDMs) were isolated from the bone marrow of 6–8 weeks old female C57BL/6J mice (Guangdong Medical Laboratory Animal Center) and cultured for 6–8 days with 50 ng/mL macrophage colony-stimulating factor (PeproTech).

Mice

Cpsf6^+/+ and Cpsf6^+/− mice (6–8 weeks old) were generated by Biocytogen Pharmaceuticals (Beijing) Co., Ltd using the CRISPR/Cas9 approach. Mice were housed and bred under specific pathogen-free (SPF) conditions.

Antibodies and reagents

Anti-CPSF5 (1:5000; 10322-1-AP, Proteintech), anti-CPSF6 (1:5000; ab175237, Abcam), anti-CPSF7 (1:2000; 55195-1-AP, Proteintech), anti-RIG-I (1:1000; D14G6, Cell Signaling), anti-MDA5 (1:1000; D74E4, Cell Signaling), anti-IFIT3 (1:2000; 15201-1-AP, Proteintech), anti-ISG15 (1:2000; 15981-1-AP, Proteintech), anti-DDX21 (1:2000; 10528-1-AP, Proteintech), anti-DHX15 (1:2000; 12265-1-AP, Proteintech), anti-CCL2 (1:2000; 66272-1-Ig, Proteintech), anti-IRF3 (1:1,000; sc-33641, Santa Cruz), anti-phosphorylated IRF3 (1:1000; 4D4G, Cell Signaling), anti-TBK1 (1:1000; D1B4, Cell Signaling), anti-phosphorylated TBK1 (1:1000; D52C2, Cell Signaling), anti-VSVG (1:1000; V5507, Sigma), anti-ICP27 (1:1000; ab31631, Abcam), anti-GAPDH (1:20000; 60004-1-Ig, Proteintech), anti-HA (1:5000; clone HA-7, H6533, Sigma), anti-Flag (1:5000; clone M2, F1804, Sigma), Anti-rabbit IgG (H+L), (Alexa Fluor 555 Conjugate) (1:1000, 4413, Cell Signaling), goat anti-mouse IgG-HRP (1:10000; HA1006, HuaBio), goat anti-rabbit IgG-HRP (1:10000; HA1001, HuaBio). The poly(I:C) (LMW) and poly(dA:dT) were purchased from InvivoGen. Lipofectamine 3000 and Lipofectamine RNAiMAX were purchased from Invitrogen. Act D (actinomycin D) was purchased from Sigma.

Generation of CPSF6^−/− cells

CPSF6^−/− A549 and Cpsf6^−/− L929 cells were constructed using the CRISPR/Cas9 genome editing technology. Small guide RNAs (sgRNAs) targeting the genomic sequences of target genes were cloned and inserted into the PX458 vector. These CRISPR plasmids were transfected into A549 or L929 cells using Lipo3000, and single-cell colonies of GFP⁺ cells were picked and validated at the translational level by western blot analysis. The sgRNA targeting sequences were as follows:

sgRNA-1: 5’-CACCGTCCATGTAATCTCGGTCTTC-3’ for A549 cells,

sgRNA-2: 5’-CACCGCGGGCAAATGGCCAGTCAAA-3’ for A549 cells;

sgRNA-1: 5’-CACCGATGCCAACATCAGATAGTCG-3’ for L929 cells,

sgRNA-2: 5’-CACCGATGACGGTATTCTCGCTCT-3’ for L929 cells.

Generation of CPSF6-reconstituted L929 cells (Cpsf6^-/- L929^CPSF6 cells)

The CDS of CPSF6 was subcloned and inserted into the pCDH-CMV-MCS-EF1-Puro plasmid. This vector was transfected into HEK293T cells along with the following two lentiviral packing plasmids: psPAX2 and pMD2.G. Culture supernatants were then collected 48 hr after transfection. The culture supernatant was concentrated by ultracentrifugation before use for infection. Infection-positive CPSF6^−/− L929 cells were selected and enriched on the basis of resistance to puromycin. Monoclonal cells were screened by a limiting dilution assay and confirmed by sequencing of PCR- amplified fragments. Immunoblot analysis of cell lysates was performed with the corresponding antibody.

Analogue stimulation and virus infection

Cells were stimulated with HSV60 (1 μg ml⁻¹), Poly (I:C) (1 μg ml⁻¹) or Poly (dA:dT) (1 μg ml⁻¹) delivered via polyethylenimine (PEI) for the indicated hours. Cells were infected with viruses at the indicated MOI in serum-free DMEM for 1 hr, washed with 1×PBS, and incubated with fresh complete medium for the times shown in the figures.

Plaque assay

Culture the Vero cells in growth medium (DMEM+10% FBS) in a six-well plate to give 90–100% confluence by the following days. Then, dilute the virus suspension tenfold serially from 1:10⁻¹ to 1:10⁻⁶, and infect the confluent monolayers of Vero cells with 400 μL volume of diluted virus and 600 μL volume of serum-free DMEM (total 1000 μL volume/well). After incubation 1 hr, discard the unadsorbed virus and overlay the monolayer with 3 mL of autoclaved agar overlay (at about 40°C). After the gel solidifies, incubate the plates at 37°C and 5% CO₂ for up to 24–48 hr. After 24–48 hrs, stain the plates with 0.1% neutral red in PBS for 1 hr (1 mL/well) followed by counting the plaques. Calculate the virus titer using the following formulas:

Pfu/mL = Average no. of plaques per dilution/(D*V)

D: Dilution factor (for dilution 10⁻²; D is 0.01 and for dilution 10⁻⁴; D is 0.0001).

V: Volume of virus added per well (in mL; 0.4 mL in the above protocol).

Preparation of the IVT-SAPAS library, APA sequencing and analysis

The IVT-SAPAS libraries were constructed based on previously developed SAPAS methods [38,39]. Briefly, 200 ng of total RNA was randomly fragmented by heating, and the first round of reverse transcription was performed using an anchored oligo d(T) primer containing an Illumina A adaptor and the T7 promoter sequence. The second strand was synthesized with RNase H, Escherichia coli DNA polymerase and DNA ligase. Subsequently, the RiboMAX Large Scale RNA Production System-T7 (Promega) was used to perform in vitro transcription according to the manufacturer’s instructions (Promega). RNA products were purified with the Agencourt RNA Clean XP kit (Beckman Coulter). A second round of reverse transcription was conducted using random primers containing a partial Illumina B adaptor sequence. PCR was then performed to amplify the cDNA, 200–500 bp fragments were purified with AMPure XP Beads (Beckman Coulter), and the quality of the library was evaluated with an Agilent 2100 Bioanalyzer. The final pooled libraries were quantified and sequenced with an Illumina HiSeq 2500 Sequencing System.

Raw reads were first trimmed, filtered and mapped to the mouse genome (mm9 downloaded from the UCSC Genome Bioinformatics website). The unique mapped reads were used to filter for internal priming by examining the genomic sequences 1 to 20 bases downstream of the poly(A) cleavage sites. To eliminate bias introduced by unequal read counts at different time points, the total counts at each time point were normalized to one million. Poly(A) site definition and tandem 3’ UTR annotation were performed as previously described. The isoform-weighted 3’ UTR length was defined as the mean 3’ UTR length weighted by the read counts for each tandem 3’ UTR isoform, and the normalized 3’ UTR length was defined as the percentage of the isoform-weighted 3’ UTR length relative to the longest tandem 3’ UTR length across all time points. We used a linear model to assess APA switching events, and genes with an absolute tandem 3’ UTR switching index (TSI) value of 40.1 and a P value of less than the threshold corresponding to a BH-sense FDR of 0.01 was defined as an APA gene.

Luciferase reporter assay

The 3’ UTRs of mouse target genes were amplified from the L929 cell genome by a proofreading Pfu polymerase (Takara) and sequenced to confirm their authenticity. Mutation of proximal poly(A) sites was performed using a Site-directed Gene Mutagenesis Kit (ExCell Bio), and 3’ UTRs of differing lengths were cloned and inserted into the psiCHECK-2 plasmid. For reporter assays, 293T cells were plated at a density of 10⁵ cells per well in a 48-well plate. The cells in each well were transiently transfected for 24 hr with psiCHECK-2 plasmids with different 3’ UTRs inserted downstream of the Renilla luciferase gene using ViaFect Transfection Reagent (Promega). Subsequently, the cells were lysed and collected for luciferase reporter assays. The luciferase activity in cell lysates was measured with a dual luciferase reporter gene assay system (Promega) according to the manufacturer’s instructions. Firefly luciferase reporters in psiCHECK-2 plasmids were used as internal controls. Each experiment was performed in triplicate and repeated three times. The data are presented as the mean ± s.d. values.

Quantitative real-time PCR (qRT–PCR) analysis

For mRNA analysis, total RNA was extracted using TRIzol reagent (Invitrogen) and reverse transcribed with a PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa) according to the manufacturer’s instructions. A Light Cycler 480 instrument (using the 384-well module) with 2×Polarsignal qPCR mix (MIKX) was used for quantitative real-time PCR analysis. Target mRNA expression levels were normalized to the expression level of β-actin in each individual sample. The 2^−ΔΔCt method was used to calculate relative expression changes. For microRNA analysis, 1 μg of total RNA was reverse transcribed with a miRNA First Strand cDNA Synthesis kit (Tailing Reaction) (Sangon Biotech) according to the manufacturer’s instructions. A Light Cycler 480 instrument (using the 384-well module) with 2×Polarsignal qPCR mix (MIKX) was used for quantitative real-time PCR analysis. U6 was used as the control for miRNA expression, and the relative expression of microRNAs was calculated by the 2^-ΔΔCt method. S1 Table lists the primers detail.

RNA interference

LipoRNAiMAX (Invitrogen) was used for transfection of siRNAs (30 nM) into A549 or L929 cells according to the manufacturer’s instructions. The sequences of the siRNAs are as follows:

Human-CPSF6-siRNA: GGATCAAGACGTGAACGAT

Mouse-CPSF6-siRNA: CGACTACATGGATACTCTT

RNA decay assay

L929 cells were seeded at a density of 4 × 10⁵ cells/mL in 12-well plates. After incubation overnight, the cells were treated with the transcription inhibitor Act D (5 μg/mL, Sigma) to block de novo RNA synthesis, and collected at the indicated times. RNA samples were extracted for qRT–PCR to measure the mRNA levels of the indicated genes.

Immunoblotting

Cells were lysed with cell lysis buffer (Cell Signaling Technology) supplemented with protease inhibitor ‘cocktail’ (Roche). Protein concentrations in the extracts were measured by a BCA assay (Pierce). A total of 40 mg of protein in each sample was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and then electrotransferred to a polyvinylidene difluoride membrane (Hybond-P; GE Healthcare Life Sciences). Membranes were incubated with the indicated primary antibodies followed by HRP-conjugated secondary antibodies. Bands were detected with an Immobilon ECL kit (Millipore) and imaged on X-ray films (Kodak, Xiamen, China).

Flow cytometry

A549 and L929 cells were transiently transfected with siRNAs for 48 hr and then challenged with VSV-eGFP for 6 hr at an MOI of 2. The cells were washed once in cold PBS and resuspended in cold staining buffer (PBS with 1% fetal bovine serum). For each condition, 10,000 cells were counted, and subsequent analyses were performed on a flow cytometry system.

Statistics and reproducibility

Statistical analyses were carried out using Microsoft Excel software and GraphPad Prism to assess the differences between experimental groups. Statistical significance was determined using a two-tailed, unpaired Student’s t test or the Mann-Whitney U test with a confidence interval of 95%. P ≤ 0.05 was considered to indicate a statistically significant difference. All experiments were performed three or more times independently under identical or similar conditions.