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. 2011;7(8):1145-60.
doi: 10.7150/ijbs.7.1145. Epub 2011 Oct 16.

Stability of a Long Noncoding Viral RNA Depends on a 9-nt Core Element at the RNA 5' End to Interact With Viral ORF57 and Cellular PABPC1

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Free PMC article

Stability of a Long Noncoding Viral RNA Depends on a 9-nt Core Element at the RNA 5' End to Interact With Viral ORF57 and Cellular PABPC1

Maria J Massimelli et al. Int J Biol Sci. .
Free PMC article

Abstract

Kaposi sarcoma-associated herpesvirus (KSHV) ORF57, also known as Mta (mRNA transcript accumulation), enhances viral intron-less transcript accumulation and promotes splicing of intron-containing viral RNA transcripts. In this study, we identified KSHV PAN, a long non-coding polyadenylated nuclear RNA as a main target of ORF57 by a genome-wide CLIP (cross-linking and immunoprecipitation) approach. KSHV genome lacking ORF57 expresses only a minimal amount of PAN. In cotransfection experiments, ORF57 alone increased PAN expression by 20-30-fold when compared to vector control. This accumulation function of ORF57 was dependent on a structured RNA element in the 5' PAN, named MRE (Mta responsive element), but not much so on an ENE (expression and nuclear retention element) in the 3' PAN previously reported by other studies. We showed that the major function of the 5' PAN MRE is increasing the RNA half-life of PAN in the presence of ORF57. Further mutational analyses revealed a core motif consisting of 9 nucleotides in the MRE-II , which is responsible for ORF57 interaction and function. The 9-nt core in the MRE-II also binds cellular PABPC1, but not the E1B-AP5 which binds another region of the MRE-II. In addition, we found that PAN RNA is partially exportable in the presence of ORF57. Together, our data provide compelling evidence as to how ORF57 functions to accumulate a non-coding viral RNA in the course of virus lytic infection.

Keywords: E1B-AP5; KSHV; ORF57; PABPC1; PAN; RNA accumulation; RNA stability; long non-coding RNA.

Conflict of interest statement

Conflict of Interests: The authors have declared that no conflict of interest exists.

Figures

Fig 1
Fig 1
PAN expression depends on viral ORF57. (A) ORF57 binds PAN RNA during lytic KSHV infection. RT-PCR was performed by using a PAN-specific primer pair on total RNA isolated from the CLIP complexes obtained with an anti-ORF57 antibody from butyrate-treated JSC-1 cells. (B) PAN expression depends on ORF57 during lytic KSHV infection. Total RNA obtained from BCBL-1 and JSC-1 cells induced with butyrate (Bu, 3 mM) or from Bac36 cells with a wild-type KSHV genome (Bac36 wt) and Bac36-Δ57 cells with an ORF57-null KSHV genome induced with VA (1 mM) was examined by Northern blot for PAN RNA with a PAN-specific probe oJM7. GAPDH RNA served as a loading control. (C) ORF57 enhances PAN accumulation at the posttranscriptional level. Total RNA from HEK293 cells transfected with an expression vector of PAN, K5 or vGPCR (see diagrams) or an empty pcDNA3 vector, together with a FLAG-tagged ORF57 (+) or an empty FLAG vector (-) was examined by Northern blot with a T7 probe for expression of PAN, K5 or vGPCR. (D) ORF57 inactive mutant (mtNLS2+3) is unable to enhance PAN expression. Total RNA from HEK293 cells transfected with a PAN expression vector together with a GFP-tagged ORF57 wt (ORF57 wild type), GFP-tagged ORF57 inactive mutant (mtNLS2+3) or GFP empty vector pEGFP-N1 was examined by Northern blot for PAN RNA.
Fig 2
Fig 2
ORF57-mediated enhancement of PAN expression depends on a 5' PAN element. Shown in (A) are schematic diagrams of wt PAN and its deletion mutants. Shaded and numbered boxes represent the positions of ORF57 interacting regions identified by CLIP in PAN: box 1 (black), a PAN 5' region from nt 28667-28750; box 2 (dark grey), a PAN internal region from nt 28971-29114; box 3 (hatched and solid grey), a PAN 3' region from nt 29544-29658 including the entire ENE (hatched grey, nt 29554-29632). Total RNA from HEK293 cells transfected with each of the PAN constructs in the presence (+) or absence (-) of ORF57 was examined by Northern blot with a T7 probe for PAN expression (B). The same membrane in each panel was stripped and reprobed separately with a 32P-labeled ORF57 or GAPDH probe for ORF57 expression and sample loading. Relative levels of PAN RNA in each sample were shown at the bottom of each Northern blot panel after normalization to the level of the corresponding GAPDH for sample loading.
Fig 3
Fig 3
Stability of PAN depends on MRE and ORF57. (A) Calculation of half life of wt PAN and mt PANΔ1 lacking the 5' MRE in the presence or absence of ORF57. HEK293 cells were transfected with a vector expressing wt or mt PAN together with ORF57 or a vector control. 24 h after transfection, transcription was stopped by addition of 10 μg/ml of actinomycin D and total RNA was extracted from each sample collected over the time as indicated. PAN and GAPDH (for normalization) were quantified by qRT-PCR. The remaining level of PAN RNA in each time point relative to time zero was determined as described in Methods, after normalization to GAPDH RNA for sample loading. Results are presented as mean, minimum and maximum. A non-linear regression analysis on the raw data was performed, choosing an exponential decay model [Fold Percent = α*exp(β*time) ] where α (alpha) is the intercept when time = 0, and β (beta) is the decay rate. (B) Summary of the estimated betas (Est. β) and its standard errors (SE) for each study group. Also included are the adjusted R-squares and calculated half-life for each study group as well as two-tailed p-values for the indicated pair-wise comparisons between the estimated parameters. The p-values for the estimated betas were adjusted using a step-down Bonferroni method.
Fig 4
Fig 4
Motif II (MRE-II) of the PAN 5' MRE is necessary for ORF57 enhancement of PAN expression. (A) Schematic diagrams of the 5' MRE and its mutants. Shown on the top indicates an 84-bp MRE in the 5' PAN which contains three potential motifs (stem-loops) marked as I, II and III, with the sequences underlined. A series of successive 5' to 3' deletions in the 5' MRE, an internal deletion or point mutations in the MRE-II are shown as ΔMRE-a, -c, -d and MRE-PM. Nucleotide substitutions in the MRE-II sequence in an MRE-PM mutant are boxed. (B) Predicted secondary structures of the MRE. On the left is the secondary structure of wt MRE with a folding energy of -19.5 Kcal/mol, consisting of 3 stem-loops I, II, III as described in (A). On the right show the point mutations introduced into the MRE-II loop to create an MRE-PM construct. (C, D) Mapping of a 9-nt core in the MRE-II responsible for ORF57 enhancement of PAN expression. Total RNA of HEK293 cells transfected separately with an indicated PAN construct in (A), together with an ORF57 expression vector (+) or an empty vector (-) was examined for PAN expression by Northen blot with a 32P-labeled T7 probe. (E) The MRE element in the presence of ORF57 promotes PAN accumulation in the context of its native promoter. The upper panel show schematic diagrams of PAN expression driven by a CMV promoter (pJM1) or by a native PAN promoter (PANPr) (pJM53 for wt PAN and pJM54 for mt PAN [MRE-PM]). HEK293 cells transfected with each of these constructs in the presence (+) or absence (-) of ORF57 were analyzed for PAN expression by Northern blot with a 32P-labeled PAN-specific probe oJM7 (Supplementary Material: Table S2). Relative PAN expression levels (fold) were quantified based on the density of each band after normalization to GAPDH for sample loading and were shown at the bottom of each blot, with the PAN level in the absence of ORF57 in (C, D) as 1 for each PAN construct and wt PAN level in the absence of both ORF57 and ORF50 in (E) as 1 for comparison.
Fig 5
Fig 5
The identified PAN MRE does not function efficiently in the expression of heterologous genes vGPCR and Luciferase. (A) Schematic diagrams of the vGPCR constructs. The MRE-II motif or the entire MRE was inserted upstream of vGPCR (pJM6, Fig. 1C) either in the sense or antisense (As) orientation as indicated by the arrow. (B) Effect of the MRE on vGPCR expression in the presence or absence of ORF57. Total RNA of HEK293 cells transfected with each of the constructs in (A), together with an ORF57 expressing vector (ORF57 +) or an empty vector (-), was examined for vGPCR expression by Northern blot with a 32P-labeled T7 probe. Relative vGPCR expression level (fold) is shown at the bottom of Northern blots after normalization to GAPDH. (C) Schematic Diagrams of the firefly luciferase constructs. The entire MRE and/or ENE elements were inserted upstream (MRE) or downstream (ENE) of the firefly luciferase ORF, respectively, to mimic their native locations in PAN. (D) Effect of the MRE on luciferase expression in the presence or absence of ORF57. HEK293 cells were cotransfected with each firefly luciferase reporter construct, together with a Renilla luciferase pRL-TS construct and a FLAG-tagged ORF57 expressing vector or an empty FLAG vector (FLAG). 24 h after transfection cells were lysated and luciferase activity was measured by a dual luciferase assay. Relative luciferase activity in each sample was calculated by dividing the light unit readings obtained from a tested firefly luciferase reporter construct by the light unit readings obtained from the Renilla luciferase reporter (F/R ratio). Luciferase activities (mean ± 95% confidence limits) from a mixed model analysis of variance are presented along with an adjusted two-tailed p-value.
Fig 6
Fig 6
MRE-II RNA interacts in vitro with ORF57 and other cellular proteins. (A) Mapping of an MRE binding site for ORF57. Shown on the top is a PAN MRE sequence lacking the MRE-I region in Fig. 4A, with the 9-nt core of MRE-II loop sequence bolded and underlined and the nt positions of biotinylated RNA oligomers employed in RNA pull-down assays. A cell lysate prepared from TREx BCBL-1-Rta cells induced with Dox for 24 h was used for the RNA pulldown assays with an indicated RNA oligomer. The RNA oligo oNP42 derived from vIL-6 RNA which binds ORF57 was used as a positive control. ORF57 associated with RNA oligos in the pulldowns was immunoblotted using an anti-ORF57 antibody. The cell lysate (10%) before the pulldown was loaded as a Western blot control. (B) Biotinylated RNA affinity pulldown analysis by Western blot using anti-ORF57, PABPC1 and E1B-AP5 antibodies. Total cell extract from TREx BCBL-1-Rta (R) or -vector (V) cells induced by Dox for 24 h was used for the RNA pulldown assays with each biotinylated RNA oligomer. RNA oligos oNP41 and oNP42 derived from vIL-6 RNA were used, respectively, as a negative and positive oligomer control for ORF57 interaction and 10% of the cell lysate from each cell line before the pulldown was loaded for Western blotting control. RNA oligo oJM68 is a copy of oJM35 with the point mutations in the MRE-II loop described in Fig. 4B. Control beads indicate no RNA oligomer on the beads.
Fig 7
Fig 7
A small proportion of PAN are exportable in ORF57-coexpressing cells. Total (T) and fractionated cytoplasmic (C) or nuclear (N) RNA from BCBL1 cells treated with or without valproate (VA, 1 mM) for viral lytic induction (A) or from HeLa cells (B) cotransfected with pJM1 (wt PAN), pJM2 (mt PANΔ1), or pJM4 (mt PANΔ3) in the presence (ORF57+) or absence (an empty vector control, ORF57-) of ORF57, were examined by Northern blot for PAN RNA expression. HEK293 cells cotransfected with pJM1 (wt PAN), pJM2 (mt PANΔ1), or pJM4 (mt PANΔ3) in the presence of ORF57 were also fractionated and examined by Northern blot for PAN RNA expression (C). GAPDH RNA served as a control for sample loading and U6 RNA served as a fractionation efficiency control. The relative levels of PAN in each sample were calculated after normalization to GAPDH.

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