Supplementary figures 1-9 Supplementary Table 1 from Long Noncoding RNA MALAT1 Regulates Cancer Glucose Metabolism by Enhancing mTOR-Mediated Translation of TCF7L2

Supplementary Information: Figure S1: MALAT1 affects cancer glucose metabolism Figure S2: Glucose metabolism in HCC cell lines with MALAT1 knockdown. Figure S3: Regulation of TCF7L2 protein expression by MALAT1 in HCC cell lines. Figure S4: A non-phosphorylatable mutant of 4EBP1 inhibits TCF7L2 protein expression and expression of glycolytic genes. Figure S5: SRSF1 regulates TCF7L2 levels post-transcriptionally. Figure S6: TCF7L2 modulates glucose metabolism in a HCC cell line. Figure S7: MALAT1 and TCF7L2 regulate gluconeogenesis through the same pathway. Figure S8: Oncogenic properties of HCC cell lines with TCF7L2 knockdown. Figure S9: TCF7L2 protein, Gluconeogenesis and Glycolytic enzyme expression in livers from mouse HCC model Mdr2-/-. Table S1: List and sequences of shRNAs, siRNAs and PCR primers used in the paper</p

Transcription and translation coordinates, timing of expression, and function of latent, early lytic and delayed early lytic ORFs.

<p>Transcription and translation coordinates for the primary viral ORFs were annotated from in silico predictions and/or by manually curating mRNA-seq and Ribo-seq data. Timing was determined according to the time of transcription. Functions were gathered from the literature. U: uORF in leader region; Str: Strand; TSS: transcription start site; N/A, not determined in silico or by manual annotation; Ann: Annotation; P: predicted; M: manual annotation; Sp. Var: ORF with splice variant, Alt. Start: ORF with alternative starts; Internal: ORF with internal ORFs; AS-ORF: Antisense ORFs; MHV68: Function annotated in MHV68.</p

Dynamic changes in translation efficiency of cellular genes during HCMV infection.

<p><b>A.</b> TE was calculated by dividing RPKM of ribosome footprints with RPKM of mRNA measurements and the correlation in TE of host genes between biological replicates is represented. <b>B.</b> TE of 731 human transcripts after interferon (IFN) treatment, infection with inactivated virus (5hr UV) and during HCMV infection were calculated relative to TE in uninfected cells. The TE ratios were subjected to partitioning clustering and shown is the heat map of log₂ TE ratios and the corresponding footprints and mRNAs ratios. The five clusters are marked and for each of these clusters the pathway enrichment (P-val < 1E-4) is labeled on the left. <b>C.</b> HSP90AB1 mRNA and footprints levels as measured in our RNA-seq and ribosome profiling experiment (left panel) and verification of these measurements by real-time PCR and western blot analysis (right panel). Real-Time PCR data was normalized by the amount of <i>polr2l</i> mRNA. <b>D.</b> Schematic representation of the live cell translation reporter. An inducible degron (DHFR-Y100I) fused to sfGFP-NLS is separated from an NLS-mCherry protein by a P2A ribosome skipping sequence, which allows these two proteins to be synthesized separately from a single transcript. In the absence of the stabilizer, trimethoprim (TMP), sfGFP-NLS is degraded, while NLS-mCherry is stable. Upon addition of TMP, DHFR-sfGFP-NLS is stabilized and GFP fluorescence increases over time due to GFP protein synthesis, thus providing readout for TE. <b>E.</b> HFF cells stably expressing the reporter were treated with 3 μM TMP and then imaged every 60 min. As can be seen in representative images the GFP intensity increases over time, while the mCherry signal remains constant. <b>F.</b> 5’ UTRs of indicated genes were cloned upstream of the GFP, and HFF cells stably expressing these constructs were created. To measure translation cells were either infected with HCMV for 24hr or left uninfected, then TMP was added to the infected and uninfected cells and the cells were imaged for 3hr. The increase in GFP fluorescence was measured and normalized (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005288#sec010" target="_blank">Methods</a>). Shown boxplot represent the GFP accumulation, cells expressing the 5’UTRs of RPS19, SMC2 and RAD50 showed significant higher accumulation of GFP in infected cells compared to uninfected (* P-val < 0.05).</p

Transcription and translation coordinates, timing of expression, and function of late lytic ORFs.

<p>Transcription and translation coordinates for the primary viral ORFs were annotated from in silico predictions and/or by manually curating mRNA-seq and Ribo-seq data. Timing was determined according to the time of transcription. Functions were gathered from the literature. U: uORF in leader region; Str: Strand; TSS: transcription start site; N/A, not determined in silico or by manual annotation; Ann: Annotation; P: predicted; M: manual annotation; Sp. Var: ORF with splice variant, Alt. Start: ORF with alternative starts; Internal: ORF with internal ORFs; AS-ORF: Antisense ORFs; MHV68: Function annotated in MHV68.</p

KSHV employs multiple strategies to expand and regulate its coding repertoire.

<p>(A–C) Novel splice variant of ORF57 detected by mRNA-Seq. Tracks for mRNA-Seq and Ribo-Seq in CHX treated cells (48 hpi) show the new, shorter-splice variant of ORF57, ORF57A (red box). Splicing of the second intron in ORF57 removes the UAA stop codon, resulting in the generation of a 33 aa C-terminal exon. (B) Zoom in of the region highlighted in (A). Note that ribosomes accumulate downstream of the ORF57 stop codon, indicating that the new exon may be translated. (C) Verification of ORF57A splicing by end-point PCR in lytic iSLK-219 (72 hpi) and LEC-219 cells. The primers used for amplification flank the intron boundaries and are shown in panel (A) as solid black lines. The high molecular weight product (∼800 bp) corresponds to the unspliced form of ORF57. The low molecular weight product (165 bp), corresponds to ORF57A, from which the second intron has been removed. (D–E) mRNA editing in the RTA and Kaposin transcripts. Sanger sequencing of RTA (D) and Kaposin (E) genomic DNA (top panel) and cDNAs (48 hpi) (bottom panel) from iSLK-219 cells. The edited nucleotide is bold, italic font. (F–G) ORF54 translation starts from two alternative initiation codons. (F) Ribo-Seq tracks for cycloheximide (CHX blue) and harringtonine (Harr teal) treated cells at 72 hpi. The dotted lines indicate the two translation initiation sites used for ORF54 translation. (G) Immunoblot for ORF54 in latent and lytic (48 hpi) iSLK cells, infected with Wt KSHV or an ORF54 knock-out virus (ΔORF54). *Non-specific product. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003847#ppat.1003847.s006" target="_blank">Figures S6</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003847#ppat.1003847.s007" target="_blank">S7</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003847#ppat.1003847.s008" target="_blank">S8</a>.</p

Host cis-regulatory elements are conserved in viral transcripts.

<p>(A) mRNA-Seq of the locus encoding the ORF8/9/10 and 11 genes at 72 hr post reactivation. Three TSS sharing a common PAS were mapped within this polycistronic locus. (B) Sequence analysis of 50 nucleotides flanking the predicted TSS site for 49 viral genes. Note the presence of the TATA box 30 bp upstream of the TSS. (C) Sequence analysis of 30 nt flanking the 42 polyA sites mapped in KSHV. Note the conserved AA/UUAAA motif 5–20 nt upstream (top panel), and the GU rich region (middle panel) 5–20 nt downstream of the cleavage site. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003847#ppat.1003847.s010" target="_blank">Tables S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003847#ppat.1003847.s011" target="_blank">S2</a>.</p

Differences in translation efficiency between viral and human genes along HCMV infection.

<p><b>A.</b> Cumulative TE distribution among well-expressed human and viral genes shows that viral genes are translated less efficiently than cellular genes at 5hpi but more efficiently at 72hpi. P-values were calculated by Kolmogorov Smirnov test. <b>B.</b> The percentage of mRNA and footprints reads that maps to the virus are plotted along infection.</p

Changes in cellular gene expression during HCMV infection.

<p><b>A.</b> Ribosome footprints and mRNA read densities (reads per kilobase million, RPKM) of well-expressed human transcripts after treatment with interferon (IFN), infection with inactivated virus (5hr UV) and across four time points during HCMV infection were calculated relative to expression in uninfected cells (mock). Shown is heat map of log₂ expression ratios after partitioning clustering. The ten main clusters are marked and for each of these clusters the pathway enrichment (Benjamini < 1E-5) is labeled on the left. <b>B.</b> Cells transfected with a control or siRNAs targeting different host genes were infected with the Merlin strain (MOI = 3). After 5 days supernatants were collected and viral titers were calculated by TCID50. Each experiment was performed in triplicates and results shown are representative. <b>C.</b> Samples from experiments detailed in (B) were analyzed by western blot for viral proteins expression from immediate early, early and late stages of infection (IE1/IE2, UL44 and pp28, respectively).</p

Identification of viral proteins responsible for BTN2A1 and IGSF8 degradation.

<p><b>A.</b> HFF cells stably expressing BTN2A1-HA or IGSF8-HA were transduced with lentiviral vector expressing either US2, US9 or empty vector. BTN2A1 and IGSF8 protein levels were measured by immunoblotting. Right panel represents the quantification of IGSF8 expression relative to GAPDH amount from the same cells. <b>B.</b> HFF cells stably expressing BTN2A1-HA or IGSF8-HA were mock-infected or infected with HCMV AD169VarL, AD169VarL-BAC, AD169VarL-BACdeltaUS9 or AD169VarL-BAC deltaUL40 in MOI = 5 for 72 hr. BTN2A1 or IGSF8 protein levels were analyzed along infection by immunoblotting detection of the HA signal.</p

Integration between ribosome footprints and protein abundance measurements allows detection of cellular proteins that are degraded during HCMV infection.

<p><b>A.</b> Ribosome profiling measurements of ROCK1 and ERC1 compared with protein abundance [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005288#ppat.1005288.ref012" target="_blank">12</a>].<b>B.</b> Real-time PCR analysis of <i>rock1</i> and <i>erc1</i> with parallel measurements of protein levels by western blot analysis. Real-time PCR data was normalized by the amount of <i>mfge8</i> mRNA. Each experiment was performed in triplicates. Western blot analysis was performed on cell lysates and GAPDH was used as loading control. <b>C.</b> Cells were mock-infected or infected with HCMV for 48hr and cycloheximide (25μg/ml) was added to the medium to stop protein translation. Samples were taken at the indicated time points and the abundance of ROCK1 and ERC1 was determined by western blotting. <b>D.</b> Expression of ROCK1 and ERC1 along infection with AD169 HCMV strain. Protein levels were analyzed by western blotting.</p