Fig 2: p100/TSN enables E2F1 to interact with alternatively spliced transcripts.(A) Schematic representation of exon structure for the SENP7 gene. Each alternatively spliced transcript expressed from this gene is displayed, with primer binding sites used to detect specific transcript variants in subsequent experiments indicated with black arrows. Note that forward primers were designed to span exon junctions. Mining of the RIP-seq dataset for exon spanning peaks identified reads around exons 4 and 7 (indicated by the red numbering), which occurs in SENP7 transcript V5 (highlighted in red text). (B) Anti-E2F1 RIP with U2OS cells treated with siRNA against E2F1, TSN, or nontargeting (NT) control, as indicated, for 72 hours. Cells were then immunoprecipitated with E2F1 antibody, and coimmunoprecipitated RNA was reverse-transcribed before qPCR analysis with primers against specific SENP7 transcript variants as indicated. n = 3. (C) HCT116 cells were treated with 5 μM PRMT5 inhibitor, where indicated, before performing an anti-E2F1 RIP. Coimmunoprecipitated SENP7 V5 transcripts were analyzed by qRT-PCR. Input protein levels are the same as those displayed in Fig. 3E. n = 2. DMSO, dimethyl sulfoxide. (D) U2OS cells were treated for 72 hours with 5 μM PRMT5 inhibitor. RNA was then isolated from cells and analyzed by qRT-PCR using primers targeting specific SENP7 transcript variants or total SENP7 RNA. Average (mean) fold change of each RNA species as compared to untreated U2OS cells was calculated and displayed with SE. Statistical analysis for each condition compared to untreated U2OS cells is also displayed over each bar (i). An immunoblot to demonstrate input protein levels is also included (ii). n = 3. (E) As described above, although the experiment was performed in HCT116 cells. n = 4. ns, not significant. (F) Examination of the promoter region of the SENP7 gene (–2 to +1 kb) identified an E2F1 DNA binding motif within +450 bp of the transcription start site, lying within the first intron (E2F1 motif marked in red) (i). An E2F1 ChIP was performed in the HCT116 E2F1 CRISPR and MCF7 TSN CRISPR cell lines. Immunoprecipitated chromatin was analyzed using primers spanning the identified E2F DNA binding motif in SENP7 or against the known E2F motif in the promoter sequence of CDC6 (ii). An immunoblot is included to demonstrate input protein levels (iii). n = 3

Fig 3: Analysis of N and NSP9 RNA binding on viral RNA, related to Figure 6(A) Comparison of the viral RNA binding pattern observed for NSP9 (cRIP, top) and N (eCLIP, bottom) at 12 hpi in A549ACE2 cells. Alignment of strand-separated cRIP and eCLIP data to the SARS-CoV-2 RNA genome is shown. Relative information in IP vs. SMI is calculated at each position (STAR Methods) and displayed for positive-sense RNA (blue) and negative-sense RNA (magenta). Zoom-in views of the 5′ end (left) and the 3′ end (right) of the viral RNA genome are shown and regions of interest are highlighted by dashed box.(B) Enrichment of reads mapping to the SARS-CoV-2 genome relative to the human genome in N eCLIP experiments with (+XL) and without (−XL) UV-crosslinking, as well as corresponding SMI libraries.(C) Cumulative density function displaying enrichment of crosslinked nucleotides in N IP relative to SMI experiments with and without UV-crosslinking. Inset shows Venn diagram displaying the overlap of true crosslinking sites reproducibly found in N eCLIP experiments with sites found in N eCLIP experiments without UV-crosslinking.(D) Volcano plot displaying SND1-dependent changes in NSP9 binding on peak regions relative to non-peak regions in negative-sense RNA at 8 hpi (SND1 KO vs. CTRL). Color scale reflects localization of peak region relative to the 5′ and 3′ end of the viral RNA genome. Peaks with the statistically most pronounced NSP9 binding change are annotated. Initiation site-proximal peaks are encircled.(E) As in (D), but for positive-sense RNA.

Fig 4: Measuring changes in viral RNA synthesis with SLAM-seq, related to Figure 4(A) Mismatch frequencies observed in SLAM-seq libraries.(B) Principal component analysis of SLAM-seq experiments.(C) Analysis of SLAM-seq experiments at 12 hpi using GRAND-SLAM. Scatter plot of average log2 fold changes in total RNA (x axis) and newly synthesized RNA (y axis) is shown for SND1 knockout (KO) and control (CTRL) cells, relative to wild-type (WT) cells (n = 2 independent experiments). Insets show a zoom-in view on viral genes for improved visibility.(D) Bar graph visualization of log2 fold changes for new RNA (left) and total RNA (right) observed in SLAM-seq experiments shown in (C) and Figure 4C for viral genes at 6 (top) and 12 hpi (bottom).

Fig 5: Expression, localization, and interaction analyses for virus and host proteins, related to Figure 5(A) Scatter plot displaying average log2 fold changes in SND1 coIP experiments (x axis) and average log2 fold changes in total proteome measurements of input lysates used for coIP experiments (y axis). SARS-CoV-2 infected cells are compared relative to uninfected cells in coIPs and total proteome measurements (n = 2 independent infections). Proteins that display a significant interaction and expression change (absolute log2 fold change > 1, FDR < 0.05) upon SARS-CoV-2 infection cells are highlighted.(B) IF staining of SND1 and NSP3 proteins in SARS-CoV-2 infected A549ACE2 cells at 8 hpi (MOI = 10 PFU/cell). Mock-infected cells were stained as controls. Representative images are shown. Overlap between NSP3 and SND1 is quantified by Manders’ co-localization coefficient (right, n = 12 images). Scale bars, 30 μm.(C) Log2 fold changes for viral proteins observed in total proteome measurements of SARS-CoV-2 infected A549ACE2 cells at different infection time points, as previously reported.18(D) As in (B), but for SND1 and NSP9. Overlap between NSP9 and SND1 is quantified by Manders’ co-localization coefficient (right, n = 10 images).(E) Microscale thermophoresis (MST) assay to monitor binding of recombinant NSP9 protein to recombinant SND1 protein in vitro. Unlabeled NSP9 protein (254 pM to 8.33 μM) was titrated against site-specific cysteine-labeled SND1 (10 nM) and thermophoresis was recorded. Change in fluorescence (ΔFnorm) was measured at MST on-time of 10 s. Values are mean ± SD (n = 3 independent measurements).(F) CoIP western blot analysis for epitope-tagged SND1 and NSP9 proteins exogenously expressed in uninfected HEK293T cells. SND1-FLAG constructs are deleted for indicated protein domains. NSP9-V5 served as bait. Input lysates are shown on the left. Anti-FLAG and anti-V5 antibodies are used to detect tagged SND1 and NSP9 proteins, respectively. SN, SNase.