Murine Gammaherpesvirus 68 ORF45 Stimulates B2 Retrotransposon and Pre-tRNA Activation in a Manner Dependent on Mitogen-Activated Protein Kinase (MAPK) Signaling

Gammaherpesviral infection alters the gene expression landscape of a host cell, including through the induction of noncoding RNAs transcribed by RNA polymerase III (RNAPIII). Among these are a class of repetitive genes known as retrotransposons, which are normally silenced elements and can copy and spread throughout the genome, and transfer RNAs (tRNAs), which are fundamental components of protein translation machinery. ABSTRACT RNA polymerase III (RNAPIII) transcribes a variety of noncoding RNAs, including tRNA (tRNA) and the B2 family of short interspersed nuclear elements (SINEs). B2 SINEs are noncoding retrotransposons that possess tRNA-like promoters and are normally silenced in healthy somatic tissue. Infection with the murine gammaherpesvirus MHV68 induces transcription of both SINEs and tRNAs, in part through the activity of the viral protein kinase ORF36. Here, we identify the conserved MHV68 tegument protein ORF45 as an additional activator of these RNAPIII loci. MHV68 ORF45 and ORF36 form a complex, resulting in an additive induction RNAPIII and increased ORF45 expression. ORF45-induced RNAPIII transcription is dependent on its activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) signaling pathway, which in turn increases the abundance of the RNAPIII transcription factor Brf1. Other viral and nonviral activators of MAPK/ERK signaling also increase the levels of Brf1 protein, B2 SINE RNA, and tRNA, suggesting that this is a common strategy to increase RNAPIII activity. IMPORTANCE Gammaherpesviral infection alters the gene expression landscape of a host cell, including through the induction of noncoding RNAs transcribed by RNA polymerase III (RNAPIII). Among these are a class of repetitive genes known as retrotransposons, which are normally silenced elements and can copy and spread throughout the genome, and transfer RNAs (tRNAs), which are fundamental components of protein translation machinery. How these loci are activated during infection is not well understood. Here, we identify ORF45 from the model murine gammaherpesvirus MHV68 as a novel activator of RNAPIII transcription. To do so, it engages the MAPK/ERK signaling pathway, which is a central regulator of cellular response to environmental stimuli. Activation of this pathway leads to the upregulation of a key factor required for RNAPIII activity, Brf1. These findings expand our understanding of the regulation and dysregulation of RNAPIII transcription and highlight how viral cooption of key signaling pathways can impact host gene expression.

Genome-wide mapping studies in murine fibroblasts identified widespread induction of B2 SINEs and upregulation of ;14% of tRNA loci upon MHV68 infection (44,52). This induction initiates prior to viral genome replication and is not a consequence of antiviral signaling, nor does it involve Maf1, a central negative regulator of RNAPIII activity (39,52,53). A partial screen of MHV68 open reading frames (ORFs) identified a role for the conserved herpesvirus kinase ORF36 in B2 SINE activation, potentially through alterations to the chromatin landscape (53). However, MHV68 mutants lacking functional ORF36 still induce modest levels of SINE RNA, suggesting that additional viral activities contribute to RNAPIII activation.
Here, we identify a second MHV68 protein, ORF45, as an activator of B2 SINE and pre-tRNA transcription during infection. We show that MHV68 ORF45 interacts with ORF36 and together these proteins additively increase RNAPIII transcription. Increased RNAPIII activity requires the ability of ORF45 to stimulate the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway, which increases the levels of Brf1, an essential component of the RNAPIII transcription factor complex TFIIIB. We show that other activators of MAPK/ERK signaling also enhance Brf1 expression, suggesting that this is a common mechanism to increase RNAPIII activity under conditions where components of the RNAPIII transcriptional machinery are limiting.

RESULTS
MHV68 ORF45 is sufficient to induce B2 SINE transcription and is required for robust B2 SINE and pre-tRNA transcription during MHV68 infection. Previously, we screened a partial library of MHV68 open reading frames (ORFs) and identified ORF36 as an activator of B2 SINE transcription (53). To determine if any additional MHV68 ORFs induce B2 SINE transcription, we cloned and rescreened a portion of the library that included an additional 19 FLAG-tagged ORFs not represented in our prior screen. These were transfected into NIH 3T3 fibroblasts and B2 SINE ncRNA levels were measured by B2 SINE-specific primer extension (Table S1). Among these, the expression of only one additional MHV68 gene was sufficient to upregulate B2 SINEs. Cells expressing FLAG-ORF45, a virion tegument protein conserved among the Gammaherpesvirinae, showed a ;2.5-fold increase in B2 SINE ncRNA levels compared to a green fluorescent protein (GFP)-expressing plasmid control (Fig. 1A). In line with prior observations that MHV68 specifically activates B2 SINE and pre-tRNA loci (39,44), ORF45 did not alter the levels of another RNAPIII-transcribed small nuclear RNA, 7SK. ORF45-mediated induction of B2 SINEs was less pronounced than the ;8-fold induction of B2 SINEs by ORF36, perhaps due to the lower expression levels of FLAG-ORF45 compared to FLAG-ORF36 (Fig. 1B). Nonetheless, B2 SINE transcriptional activation was consistently specific to only ORF45 or ORF36 and not observed with other MHV68 ORFs such as ORF65 (Fig. 1A, Table S1).
We next sought to assess the contribution of ORF45 toward B2 SINE and pre-tRNA transcriptional activation during infection. ORF45 is an essential MHV68 gene, and we were unable to delete it in the virus, so to deplete it from infected cells we instead generated 3T3 fibroblast cell lines constitutively expressing control or ORF45-targeting shRNAs (54). The ORF45-targeting shRNAs partially reduced ORF45 protein expression 24 h postinfection (hpi) with MHV68 compared to the control shRNA cell line, whereas expression of the ORF65 capsid protein was largely unaffected (Fig. 1C). Reduced ORF45 levels scaled with a reduction in MHV68-induced B2 RNA as measured by primer extension, and in pre-tRNA induction as measured by quantitative reverse transcriptase PCR (RT-qPCR) using primer sets that were specific for the pre-tRNAs produced from the intron-containing tRNA-TYR gene ( Fig. 1D and E). From these data, we conclude that ORF45 contributes to RNAPIII transcriptional activation during MHV68 infection.
MHV68 ORF45 and ORF36 interact and additively upregulate B2 SINE and pre-tRNA transcription. In KSHV, the ORF45 and ORF36 proteins interact (55), which is notable given that these are the two MHV68 ORFs involved in RNAPIII activation. In streptavidinbased affinity purification experiments, we similarly found that N-terminally Strep-tagged MHV68 ORF45 interacted with N-terminally FLAG-tagged MHV68 ORF36 ( Fig. 2A). Also, in line with studies of the KSHV protein homologs (55), expression of MHV68 ORF36 resulted in increased levels of the ORF45 protein (Fig. 2B). This did not appear to be a mutually stabilizing interaction, as ORF36 protein levels were unaffected by cotransfection of ORF45 (Fig. 2B).
The ORF45 NLS and conserved C-terminal domain are dispensable for B2 SINE induction. Both KSHV ORF45 and MHV68 ORF45 contain nuclear localization signals and can traffic between the nucleus and the cytoplasm; however, KSHV ORF45 is predominantly localized to the cytoplasm while MHV68 ORF45 is predominantly localized to the nucleus (54,56). Deletion of the MHV68 ORF45 nuclear localization signal (Strep-ORF45-DNLS) shifted its localization largely to the cytoplasm as measured by immunofluorescence assay (Fig. 3A). Despite this shift in localization, Strep-ORF45-DNLS activated B2 SINE transcription to comparable levels as WT ORF45, suggesting that a predominantly nuclear localization may be dispensable for B2 SINE activation (Fig. 3B).
Gammaherpesvirus ORF45 homologs share very low amino acid sequence identity, although the C-terminal region is the most conserved and is required for transcomplementation of an MHV68 ORF45-null mutant virus (54). The last 23 amino acids of the MHV68 ORF45 C terminus share 58% similarity to the C terminus of KSHV ORF45, 50% to the EBV homolog BKRF4, and 39% to the Rhesus rhadinovirus (RRV) ORF45 homolog FIG 1 MHV68 ORF45 contributes to B2 SINE and pre-tRNA transcription during MHV68 lytic infection. (A) NIH 3T3 cells were transfected with plasmids containing the indicated N-terminally FLAG-tagged MHV68 ORFs or GFP-expressing control for 24 h, whereupon total RNA was subjected to primer extension using primers specific to B2 SINE ncRNAs or 7SK RNA (control). In this and all subsequent primer extensions, the relative induction of B2 SINE ncRNAs for each sample was measured as the ratio of the mean integrated intensity between 7SK RNA and B2 SINE ncRNA levels and normalized to the control (GFP-expressing plasmid) ratio. (B) Protein extracted from cells harvested from panel A was analyzed by Western blotting with antibodies against FLAG and GAPDH (loading control). The asterisk (*) represents a nonspecific band. (C) Stable NIH 3T3 cell lines constitutively expressing a control shRNA or ORF45-targeting shRNAs (shRNA-1 or shRNA-2) were mock-infected or infected with MHV68 at an MOI of 5. At 24 hpi, cells were harvested and lysed to extract total protein and were analyzed by Western blotting with antibodies against MHV68 ORF45, ORF65, and Vinculin (loading control). (D) Cells from panel C were also harvested for total RNA extraction and subjected to primer extension using primers specific to B2 SINE ncRNAs or 7SK RNA (control). (E) Total RNA extracted from panel D was also used to detect pre-tRNA TYR levels with forward and reverse primers with 39 ends complementary to the tRNA intron using AMV RT-qPCR. Expression was normalized to 18S rRNA and compared to values from mock-infected cells. RT-qPCR experiments were done in triplicate. Error bars show the standard deviation (SD), and statistics were calculated using an unpaired t test on raw DC T values. ns, not significant; *, P , 0.05; **, P , 0.01.  (54). However, deletion of the C-terminal 23 amino acids (ORF45-DC23) resulted in only a modest reduction of B2 SINE transcriptional activation (Fig. 3B). WT ORF45 exhibited the characteristic protein doublet indicative of phosphorylation, but the banding pattern of the mutants was different, suggesting that these residues may directly or indirectly influence the modification state of ORF45 (Fig. 3D). Although it remains unclear which phosphorylated form of ORF45 contributes to B2 SINE transcriptional upregulation, these data demonstrate that neither strong nuclear localization nor the conserved C-terminal region of MHV68 ORF45 is essential for RNAPIII activation.
In agreement with data for KSHV ORF45, transfection of MHV68 Strep-ORF45 greatly increased the abundance of phosphorylated ERK1/2, indicative of activated MAPK/ERK signaling (Fig. 4D). Amino acids 29 to 146 of MHV68 ORF45 are most similar to the previously identified RSK/ERK activation domain of KSHV ORF45 (65). Expression of an ORF45 deletion mutant lacking a portion of this region (Strep-ORF45-D46-106) resulted in reduced levels of phosphorylated ERK1/2; however, this large deletion also impaired ORF45 protein expression, confounding the interpretation of its activity (Fig. 4D). We tested additional mutants and ultimately determined that a smaller deletion of amino acids 61 to 75 (ORF45-D61-75) resulted in protein expression levels comparable to the full-length protein yet did not activate ERK1/2 (Fig. 4E, Fig. S1). Unlike full-length ORF45 or the ORF36 control, ORF45-D61-75 failed to induce B2 SINE RNA as measured by primer extension (Fig. 4F).
To determine if the ORF45-mediated activation of ERK1/2 contributed to the transcriptional upregulation of B2 SINEs and pre-tRNAs observed during MHV68 lytic infection, we used a highly selective inhibitor (U0126) of the ERK kinases Mek1/2 (66). Intriguingly, while U0126 treatment reduced the levels of phosphorylated ERK1/2 during infection, it also significantly reduced ORF45 protein levels (Fig. 4G). This suggests that activation of ERK by ORF45 creates a positive feedback loop that boosts ORF45 expression. Notably, the reduction in phosphorylated ERK and ORF45 led to a decrease in B2 SINE and pre-tRNA transcriptional activation during infection relative to the dimethyl sulfoxide (DMSO)-treated controls ( Fig. 4H and I). Overall, these data validate a role for MHV68 ORF45 in the activation of cellular MAPK/ERK signaling, identify an ERK activation domain within MHV68 ORF45, and demonstrate a connection between ORF45-mediated MAPK/ERK signaling and RNAPIII transcriptional activation during infection.
Constitutive activation of MAPK/ERK signaling leads to B2 SINE and pre-tRNA upregulation through increased levels of Brf1. Several connections exist between RNAPIII activity and mitogenic factors such as ERK. Most notably, ERK directly binds and activates components of the essential RNAPIII transcription factor complex, TFIIIB, and the levels of the essential TFIIIB component Brf1 increase in response to cellular growth stimuli in specific cell types (67)(68)(69)(70). We previously showed that depletion of Brf1 completely abrogates the expression of B2 SINEs during MHV68 infection (53). Therefore, one possible mechanism whereby ORF45 might increase RNAPIII activity is by boosting the levels of Brf1. Indeed, while endogenous Brf1 protein was barely detectable in NIH 3T3 fibroblasts transfected with a control GFP plasmid, its levels markedly increased upon transfection of ORF45 (Fig. 5A). This was dependent on the activation of ERK signaling, as Brf1 levels did not increase in cells containing the ORF45-D61-75 mutant (Fig. 5A). It was also notable that MHV68 ORF36 did not alter Brf1 levels, indicating that its RNAPIII induction occurs via a mechanism distinct from that of ORF45 (Fig. 5A).
Finally, to determine whether ERK activation was sufficient to upregulate Brf1 and induce transcription of B2 SINEs and pre-tRNA, we activated ERK signaling through two ORF45-independent mechanisms. We expressed either a constitutively active form of the small GTPase H-Ras (Ras-V12) (71), or the HSV-1 ICP0 protein, which is a predicted stimulator of MAPK/ERK signaling during infection (72). Indeed, compared to the GFP control plasmid, both Ras-V12 and HSV-1 ICP0 transfection resulted in an increase in Brf1 protein levels (Fig. 5B), as well as enhanced expression of B2 SINE ncRNAs and pre-tRNA TYR levels ( Fig. 5C and D). These levels were comparable to the levels of transcriptional enhancement induced by ORF45 (Fig. 2C and D).
Collectively, these data support a model whereby the ERK activation function of ORF45 increases RNAPIII transcription by elevating the levels of Brf1 in cells where its expression is limiting. This mechanism likely extends to other viral activators of MAPK/ ERK signaling that regulate RNAPIII activity during viral infection.

DISCUSSION
RNAPIII transcription is modulated during infection with several DNA viruses, leading to the upregulation of specific cellular RNAPIII transcripts (34, 37-40, 44, 45, 73-75). Together with our prior work (53), we have now screened 90% of the known MHV68 ORFs (76) and identified two, ORF36 and ORF45, as having independent RNAPIII activation functions. Both MHV68 ORF36 and ORF45 are required for efficient viral replication and viral gene expression, and ORF45 is also involved in virion morphogenesis (54,77,78). ORF36 and ORF45 are early and early-late genes, respectively (79,80), consistent with the viral DNA replication-independent early and late kinetics of B2 SINE and pre-tRNA transcriptional activation (39,44). Although the mechanistic details of how ORF36 activates RNAPIII transcription remain unknown, it is proposed to function by inhibiting proteins involved in the maintenance of a repressive chromatin landscape (53). Our mutational analysis of MHV68 ORF45 revealed that its stimulation of RNAPIII transcription is coordinated in the cytoplasm through its ability to activate MAPK/ERK signaling. Cytoplasmic ORF45 may propagate a signaling cascade that enhances Brf1 transcription in the nucleus, or perhaps ORF45-phosphorylated ERK increases the protein stability of Brf1 in the cytoplasm. Notably, while the impact of ORF36 and ORF45 on RNAPIII activation is additive and thus likely to occur via distinct mechanisms, these proteins physically interact, underscoring their potential coordination during infection. Altogether, these data provide novel insight into the function of ORF45 and link cellular MAPK signaling to RNAPIII transcriptional activity in the context of viral infection.

FIG 4 Legend (Continued)
analyzed by Western blotting with antibodies against MHV68 ORF45, ORF65, phospho-ERK1/2, ERK1/2, and Vinculin (loading control). (B) Cells from panel A were also harvested for total RNA extraction and subjected to primer extension using primers specific to B2 SINE ncRNAs or 7SK RNA (control). (C) Total RNA extracted from panel A was also used to detect pre-tRNA TYR levels with forward and reverse primers with 39 ends complementary to the tRNA intron using AMV RT-qPCR. Expression was normalized to 18S rRNA and compared to values from mock-infected cells. RT-qPCR experiments were done in triplicate. Error bars show the standard deviation (SD), and statistics were calculated using an unpaired t test on raw DC T values. ns, not significant; *, P , 0.05; **, P , 0.01; ***, P , 0.001. (D) NIH 3T3 fibroblasts were transfected with indicated Strep-tagged genes or a GFP control for 24 h, whereupon lysates were analyzed by Western blotting with antibodies against Strep, phospho-ERK1/2, ERK, and Vinculin (loading control). (E) NIH 3T3 fibroblasts were transfected with Strep-tagged ORF45, ORF45D61-75, FLAG-tagged ORF36, or a GFP control for 24 h, whereupon lysates were analyzed by Western blotting with antibodies against Strep, FLAG, phospho-ERK1/2, ERK, and Vinculin (loading control). (F) Cells from panel E were also harvested for total RNA extraction and subjected to primer extension using primers specific to B2 SINE ncRNAs or 7SK RNA (control). (G) NIH 3T3 fibroblasts were mock-infected or infected with MHV68 at an MOI of 5 and treated with DMSO (control) or U0126 (25 mM) for 12 h. Cells were harvested and lysed to extract total protein and were analyzed by Western blotting with antibodies against MHV68 ORF45, ORF65, phospho-ERK1/2, ERK1/2, and Vinculin (loading control). (H) Cells from panel G were also harvested for total RNA extraction and subjected to primer extension using primers specific to B2 SINE ncRNAs or 7SK RNA (control). (I) Total RNA extracted from panel G was also used to detect pre-tRNA TYR levels with forward and reverse primers with 39 ends complementary to the tRNA intron using AMV RT-qPCR. Expression was normalized to 18S rRNA and compared to values from mock-infected cells. RT-qPCR experiments were done in triplicate. Error bars show the standard deviation (SD), and statistics were calculated using an unpaired t test on raw DC T values. ns, not significant; *, P , 0.05.

MHV68 ORF45 Induces B2 SINE Expression
Microbiology Spectrum Studies in proliferating mammalian cells have been critical in understanding the connection between cellular regulators of proliferation (e.g., ERK1/2) and RNAPIII transcriptional regulation. Upon mitogenic stimulation, cell growth is accompanied by ERK activation and a rapid increase in RNAPIII transcription (84)(85)(86). Activated ERK2 induces RNAPIII transcription through phosphorylation of Brf1, an essential component of the RNAPIII transcription factor TFIIIB (69). Phosphorylation of Brf1 enhances promoter recruitment of TFIIIB and RNAPIII, increasing the transcriptional output of RNAPIII. Brf1 can be limiting for the type 2 RNAPIII promoters found in tRNAs and B2 SINEs, with several cell types exhibiting low basal levels of Brf1 (67,69,87,88). Indeed, we observed very low basal Brf1 levels in murine fibroblasts and found that ORF45-induced ERK activation led to an increase in Brf1 protein expression. We hypothesize that this may be a viral strategy to overcome limiting levels of Brf1, thereby facilitating RNAPIII activation in MHV68-infected cells.
Many DNA and RNA viruses hijack the MAPK/ERK signaling cascade to mediate viral internalization, dysregulate the cell cycle, regulate viral replication, and prevent cell death (89)(90)(91)(92). MAPK/ERK signaling promotes viral reactivation from latency during KSHV infection, viral replication during lytic MHV68 infection, and the production of Vinculin served as a loading control. (C and D) Total RNA was also extracted from the samples in panel B and subjected to primer extension using primers specific to B2 SINE ncRNAs or 7SK RNA (as a control) (C) or RT-qPCR to detect pre-tRNA TYR levels, whose expression was normalized to 18S rRNA and compared to values from the GFP-expressing control (D). RT-qPCR experiments were done in triplicate. Error bars show the standard deviation (SD), and statistics were calculated using an unpaired t test on raw DC T values. ns, not significant; *, P , 0.05; **, P , 0.01. infectious progeny during both MHV68 and KSHV infection (64,(93)(94)(95)(96)(97)(98)(99)(100)(101). The pro-viral nature of MAPK/ERK activation is consistent with findings that B2 SINE ncRNAs enhance viral gene replication and expression (39). Although ORF45 is only conserved among gammaherpesviruses, the alphaherpesvirus HSV-1 strongly induces RNAPIII transcription of endogenous human SINE elements (Alus) (37). Here, we show one example of an HSV-1 viral protein (ICP0) that has been previously linked to ERK activity during infection (72), activates ERK signaling, increases Brf1 protein levels, and enhances B2 SINE and pre-tRNA transcription upon transfection in murine fibroblasts. Interestingly ICP4, which has been identified to play a role in RNAPIII activation during HSV-1 infection, functionally interacts with ICP0 (37,102). It is possible that there are additional viral activators of MAPK/ERK signaling, whose characterization should yield new insights into the regulation of noncoding RNA production during infection.
Cell lines and transductions. NIH 3T3 (ATCC CRL-1658) and NIH 3T12 (ATCC CCL-164) mouse fibroblast cell lines and HEK293T human cell lines (ATCC CRL-3216) were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco) with 10% fetal calf serum (FBS; VWR) and screened regularly for Twenty-four hours after transfection, the medium was replaced with fresh DMEM supplemented with 10% FBS and 10 mg/mL bovine serum albumin (BSA; Invitrogen). After 48 h, the supernatant was harvested, and syringe filtered through a 0.45-mm pore size filter (Millipore). Polybrene was added to a final concentration of 8 mg/mL to freshly trypsinized NIH 3T3 cells (3 Â 106) and cells were spinoculated with 2 mL of the lentivirus containing supernatant in a 12-well plate for 2 h at 1,000 Â g. After 24 h, the cells were expanded to a 10-cm tissue culture plate and selected for 1 week in media supplemented with 2 mg/mL puromycin (MilliporeSigma).
Transfections. Transfections of MHV68 ORF containing plasmids into NIH 3T3 fibroblasts were completed as follows: 3T3 cells were maintained in DMEM with 10% FBS and grown to 90% confluence. Cells were removed and washed once with Dulbecco's phosphate-buffered saline (DPBS) (Gibco). Transfections were done using the Neon Transfection System (Thermo Fisher). Then, 2 Â 10 6 cells were resuspended in 100 mL of buffer R, to which GFP (30 mg), ORF45 (30 mg), ORF65 (30 mg), ORF36 (5 or 10 mg), ICP0 (30 mg), or Ras-V12 (20 mg) plasmid DNA was added (in the cases where less than 30 mg of DNA was used, empty pcDNA4/TO-vector was added to equal 30 mg of total plasmid DNA). This was loaded into a Neon 100-mL pipette tip and Neon tube with 3 mL of buffer E2 with electroporation parameters set to 1,300 V, 20 ms, 2 pulses. Following electroporation, cells were plated in 2 mL of DMEM with 10% FBS in 6-well TC-treated plates and were incubated at 37°C for 24 h.
Virus preparations and infections. MHV68 was amplified in NIH 3T12 fibroblast cells, and the viral 50% tissue culture infective dose (TCID50) was measured on NIH 3T3 fibroblasts by limiting dilution. NIH 3T3 fibroblasts were infected at the indicated multiplicity of infection (MOI) by adding the required volume of the virus to cells in 5 mL of serum-free DMEM in 10-cm TC-treated plates. Infection was allowed to proceed for 1 h to allow for viral entry followed by removal of the virus-containing media and replacement with fresh DMEM with 10% FBS. Cells were harvested at the indicated time points postinfection and were treated with 25 mM of U0126 (Cell Signaling) or DMSO, as indicated.
Primer extension and RT-qPCR. Total RNA was extracted from cells using TRIzol reagent (Invitrogen). Primer extension was performed on 15 mg of total RNA using a 59-fluorescein-labeled oligonucleotides specific to B2 SINE RNA or 7SK RNA. RNA was ethanol precipitated in 1 mL 100% EtOH, washed in 70% ethanol, and pelleted at 21,130 Â g and 4°C for 10 min. Pellets were resuspended in 18 mL of 1X SuperScript III reverse transcriptase reaction buffer (SSIII-RT; Thermo Fisher) containing 1 mL of each 59-fluorescein-labeled primer (10 pmol/mL) listed in Table 1. Samples were heated to 80°C for 10 min, followed by annealing for 1 h at 56°C. Then, 30 mL of extension buffer (1X SSIII-RT buffer, 40U RNasin RNase Inhibitor [Promega] 2 mM DTT, 1 mM dNTP, 1,000 U of SSIII-RT) was added, and extension was carried out for 1 h at 42°C. Samples were precipitated in 100% ethanol for 20 min at 280°C, and then pellets were briefly air dried and resuspended in 20 mL 1Â RNA loading dye (47.5% formamide, 0.01% SDS, 0.01% bromophenol blue, 0.005% xylene cyanol, and 0.5 mM EDTA). Then, each sample was run on an 8% urea-PAGE gel for 1 h at 250 V. Gels were imaged on a Chemidoc imager (Bio-Rad) with fluorescein imaging capability. Relative induction of B2 SINE ncRNAs for each sample was measured as the ratio of the mean integrated intensity between 7SK RNA and B2 SINE ncRNA level using FIJI (103) and was normalized to the GFP-expressing plasmid control. For RT-qPCR, total RNA was isolated from cells using TRIzol (Invitrogen), treated with Turbo DNase (Ambion), and reverse transcribed with AMV RT (Promega) primed with random 9-mers. Quantitative PCR (qPCR) analysis was performed with iTaq Universal SYBR green supermix (Bio-Rad) using the primers listed in Table 1. qPCR was performed on at least three biological replicates and threshold cycle (C T ) values were measured from three technical replicates per biological sample. Fold change was calculated by the DDC T method.

SUPPLEMENTAL MATERIAL
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