Fig 2: LC-MS/MS identifies members from the ‘Royal family’ as putative m6A readers.(a) Mass spectrometry results of RNA affinity pull-downs using m6A-modified and unmodified (control) biotinylated viral RNA baits. A single RNA affinity biological replicate for each RNA bait was carried out. Statistical significance of enrichment was not determined, instead, all proteins identified for a given methylated and control bait were sorted by total number of peptide spectrum matches (PSM’s) for the m6A bait. Proteins were then considered as enriched in methylated baits if the number of PSM’s assigned to the protein was at least double in the methylated bait compared with the control bait. In this table, the number of unique peptides assigned to each protein is shown for each bait for clarity. (b–f) EMSAs were carried out using recombinant SND1-C-terminus protein (residues 548–910) and biotinylated viral RNA baits. A cropped version of ORF50-1 bait (cORF50-1) and a stable version of ORF50-1 bait (named sORF50-1) in which the beginning of the stem is made to have strong base pairing was also tested. Representative EMSAs from six and two independent protein purifications for b and c-f, respectively.

Fig 3: SND1 binds the essential ORF50 RNA in KSHV-infected cells and SND1 knockdown significantly impairs KSHV lytic replication.(a) RIP-seq identifies KSHV mRNAs as high-confidence SND1 targets at 0 hr (left panel), 8 hr (middle panel) and 20 hr (right panel) post-reactivation. Red dashed line indicates a cut-off of >2 fold change RIP/input. Red arrow points ORF50. Note that lytic genes are also detected in latent cells (0 hr) due to spontaneous KSHV reactivation and the high sensitivity of deep-sequencing. As expected, significantly lower coverage for these lytic genes was detected during latency compared with lytic replication. (b) RIP for endogenous SND1, FXR1, FXR2, PSIP1, normal rabbit IgG, YTHDF1 and YTHDF3 followed by qRT-PCR detection. ORF50-1, ORF50-2, ORF50-3 and ORF50-4 indicate primers that generate an amplicon spanning the first, second, third and fourth m6A peaks of the second exon of ORF50 RNA, respectively. ORF50-intron generates an amplicon spanning the m6A peak in the ORF50 intron. ORF50 negative primers were designed at the start of the second exon of ORF50 RNA. Fold enrichment is relative to the enrichment of the non-target 18S rRNA. Values are averages, error bars present s.d. For SND1 RIPs, n = 4 independent RIPs, for FXR1, FXR2 and normal rabbit IgG n = 3 independent RIPs. For PSIP1 and YTH proteins, n = 2 independent RIPs. (c–h) Stable cell lines expressing scramble shRNA (scr. KD) or two independent shRNAs targeting SND1 were generated using TREx BCBL1-Rta cells (c–e) or BCBL1 cells (f–h). (c) Cellular and viral RNA levels were measured by qRT-PCR 24 hr post-reactivation. n = 3 independent viral reactivations. Values are averages, error bars present s.d. ***p<0.001 using an unpaired t-test. ns = not significant. (d) Immunoblot analysis of protein lysates from latent or 24 hr reactivated cells. ORF57 antibody detects both full length ORF57 protein (black arrow) and the caspase-7-cleaved form (white arrow). For SND1 detection a rabbit polyclonal antibody was used. Western blots are representative of two independent viral reactivations. (e) Volcano plot displaying viral expression of scramble KD and SND1 KD2 cells 24 hr post-reactivation analysed by RNA-seq. The green arrow highlights the quadrant containing upregulated viral ORFs in depleted cells. The red arrow highlights the quadrant containing downregulated ORFs in depleted cells. The red dashed line denotes the FDR < 1% cut-off. (f) Cellular and viral RNA levels were measured by qRT-PCR 24 hr post-reactivation. n = 3 independent viral reactivations. Values are averages, error bars present s.d. **p<0.01, ***p<0.001 using an unpaired t-test. (g) Immunoblot analysis of protein lysates from latent or 24 hr reactivated cells. Western blots are representative of two independent viral reactivations. (h) Volcano plot displaying viral expression of scramble KD and SND1 KD2 cells 24 hr post-reactivation analysed by RNA-seq. The red and purple dashed lines denote the FDR < 1% and FDR < 5% cut-offs, respectively.10.7554/eLife.47261.028Figure 7—source data 1.Source data for qRT-PCR experiments and uncropped western blots.

Fig 4: SND1 is a bona fide RNA-binding protein that targets transcripts bearing m6A modification.(a) Most significantly enriched GO terms amongst the 5061 target RNAs (Ensembl transcripts) identified by RIP-seq which are consistently bound by SND1 at all time points (0 hr, 8 hr and 20 hr post-reactivation) in TREx BCBL1-Rta cells. FDR, false discovery rate. (b) Consistently bound SND1 RNA targets throughout the course of KSHV infection are enriched in m6A-modified RNAs, while non-targets are depleted. Target transcripts are defined by a fold change RIP/input > 2 while non-targets have a fold change input/RIP > 2. A false discovery rate (FDR) < 1% is applied to RIP-seq data. (c) Hierarchical clustering of fold change RIP/input for SND1 targets. (d) No significant correlation between the number of m6A peaks in a given RNA and the binding of SND1 as determined by log2 fold change RIP/input. Target transcripts with >300 mean reads of coverage per RNA were used for the analysis. Analysis using a lower expression cut-off showed similar results. (e, f) High-confidence SND1-bound genes (summarised at HGNC gene symbol annotation level) were defined using a cut-off of FDR < 1% and a fold change RIP/input > 2, while m6A peaks were detected using a FDR < 5% and > 1.5 fold m6A-IP reads over input reads. RNA targets and m6A peaks for YTH readers were mined from publically available PAR-CLIP and m6A-seq datasets from HeLa cells. (e) Overlap of target genes with m6A modifications between SND1 and heterologously expressed YTH reader proteins. (f) Overlap of target genes lacking m6A modifications between SND1 and heterologously expressed YTH reader proteins. (g) For SND1 localised enrichment analysis, introns are collapsed and exons spliced together into a single continuous RNA molecule. Spliced transcripts and introns are then segmented into transcriptomic regions based on changes in their fold change RIP/input. (h) Venn diagram showing the overlap between SND1 enrichment in coding region (CDS) and untranslated regions (UTRs) of SND1 target transcripts identified by localised enrichment analysis.

Fig 5: SND1 stabilises native ORF50 RNA and inhibition of m6A deposition on this RNA abolishes SND1 binding.(a) RNA-seq analysis reveals significant alterations in splicing events between scramble latent and scramble lytic TREx BCBL1-Rta cells (left panel), whilst no significant changes in splicing are observed between scramble and SND1-depleted cells during latency (middle panel) or 24 hr lytic replication (right panel). (b) TREx BCBL1-Rta cells were reactivated for 24 hr into the lytic cycle and transcription was inhibited with the addition of actinomycin D (2.5 µg/ml). Transcripts of interest were measured by qRT-PCR at 0 hr, 3 hr and 6 hr post-transcription inhibition (TI). Each viral gene was normalised against 18S rRNA. Values are averages, error bars present s.d. n = 4 independent viral reactivations. (c) m6A-enrichment was determined by m6A-IP-qPCR. HEK 293 cells were transfected for 24 hr with either wild type (WT) FLAG-ORF50 or a double mutant plasmid in which the GGACT motifs present in the ORF50-1 and ORF50-4 baits were mutated to GGATT. When using DAA, 4 hr after transfection DAA was added at a concentration of 200 µM, and 24 hr post-treatment cells were harvested. % of input was calculated similarly as to ChIP-qPCR analysis. Values are averages, error bars present s.d. *p<0.05, **p<0.01, ***p<0.001 using an unpaired t-test. ns = not significant. For WT ORF50 n = 9 independent m6A-IPs [3 including 0.25% (v/v) DMSO-treatment), for double mutant ORF50 n = 3, for DAA-treated cells n = 3. (d) HEK 293 cells were transfected for 24 hr with either wild type (WT) FLAG-ORF50 or a single mutant plasmid in which the GGACT motif present in the ORF50-1 bait was mutated to GGATT. For DAA-treated cells, 4 hr after transfection DAA was added at a concentration of 200 µM, and 24 hr post-treatment cells were harvested. SND1 enrichment was determined by SND1-RIP-qPCR and is relative to the enrichment found in the non-target 18S rRNA. GAPDH RNA served as an additional non-target RNA. Values are averages, error bars present s.d. For WT and mutant ORF50 n = 4 independent RIPs. For DAA-treated cells n = 3 independent RIPs. *p<0.05, **p<0.01 using an unpaired t-test.10.7554/eLife.47261.031Figure 8—source data 1.Source data for qRT-PCR experiments.