RNA Binding Motif Protein RBM45 Regulates Expression of the 11-Kilodalton Protein of Parvovirus B19 through Binding to Novel Intron Splicing Enhancers

Figures

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  FIG 1. Identification of B19V ISE2-interacting proteins. (A) ISE2 wild-type (WT) and ISE2 mutant (mut3) RNA sequences. The two RNAs used to pull down ISE2-interacting proteins are listed. (B) SDS-PAGE. The proteins pulled down by the biotinylated ISE2-WT and ISE2-mut3 RNAs from nuclear lysates of UT7/Epo-S1 cells were separated by 6%, 10%, or 15% SDS-PAGE as indicated and stained with Coomassie blue to visualize proteins. The bands uniquely stained in the ISE2-WT pulldown sample but not in the ISE2-mut3 sample are indicated.

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  FIG 2. RBM45 knockdown did not alter cell cycle progression in CD36+ EPCs. (A) RBM45 knockdown. Lentiviruses expressing shRNAs against RBM45 were transduced into CD36+ EPCs. At 3 days postransduction, the cells were lysed for Western blot analysis using an anti-RBM45 antibody. The blots were reprobed for β-actin. (B and C) Cell cycle analysis. CD36+ EPCs were transduced with shScramble (shScram) or shRBM45. A BrdU incorporation assay was used to track de novo DNA synthesis. The cells were processed and analyzed for cell cycle progression using flow cytometry. (B) Representative histograms showing cell cycle analysis of the control CD36+ EPCshScram and CD36+ EPCshRBM45 cells. DAPI, 4′,6-diamidino-2-phenylindole. (C) Values representing relative fold changes of the cell population in S phase were calculated for CD36+ EPCs. Each experiment was repeated three times for the calculation of means and standard deviations. n.s, no statistical significance (P > 0.05).

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  FIG 3. RBM45 regulated 11-kDa protein expression in CD36+ EPCs. CD36+ EPCs transduced with shRBM45-expressing and scramble shRNA (shScram)-expressing lentiviruses were infected with B19V at 2 days postransduction. The cells were collected at 2 days postinfection for analyses. (A) RT-qPCR. Total RNA was extracted. cDNAs reverse transcribed from the RNAs were used for qPCR of each viral mRNA, as indicated, using specific primers and probes for each viral mRNA as described previously (37). The quantified viral mRNA level was normalized to the level of β-actin mRNA. The values representing viral mRNA from B19V-infected CD36+ EPCs transduced with shScram were used as controls and arbitrarily set to 1. (B) Western blotting. CD36+ EPCs were collected at 2 days postinfection, lysed, and run for Western blotting. Blots were probed for the VP1, VP2, NS1, and 11-kDa proteins, using their respective antibodies. Blots were reprobed for β-actin as a loading control. (C and D) Southern blotting. At 2 days postinfection, Hirt DNA was extracted from CD36+ EPCs for Southern blot analysis. (C) The blots were probed with the M20 probe (top) and the mitochondrial DNA probe (Mito-DNA) (bottom), respectively.…

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  FIG 4. Identification of a novel intronic splicing enhancer in B19V pre-mRNA that was bound by RBM45 specifically. (A to D) Schematic representations of in vitro transcription and RNA pulldown assay. The B19V DNA region surrounding the A2-2 site is shown. Biotin-labeled in vitro-transcribed RNA (shown in red) was captured by streptavidin-conjugated agarose beads, followed by incubation with UT7/Epo-S1 nuclear lysates. RNA-bound proteins were eluted and analyzed by Western blotting. (A) B19V DNA (nt 4710 to 4905) was used as a template to transcribe RNA (named 11-kDa intron), while its cRNA (named intron com) was transcribed as a negative control. (B) B19V DNA (nt 4890 to 5171) was used as a template to transcribe RNA (named 11-kDa exon), while its cRNA (named exon com) was transcribed as a negative control. (C) B19V DNA (nt 4710 to 4905) was in vitro transcribed to generate 3 overlapped small RNA segments termed RNA P1 (nt 4710 to 4829), P2 (nt 4747 to 4866), and P3 (nt 4786 to 4905). cRNA P1, P2, and P3 (complementary P1 [P1-com], P2-com, and P3-com, respectively) were transcribed as negative controls. (D) B19V DNA (nt 4710 to 4905) was further in vitro transcribed to generate 4 small…

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  FIG 5. RBM45 specifically bound to ISE3 under in vitro conditions. (A) ISE3 RNA sequence. The ISE3 RNA shown was used in the binding assay. (B) RNA pulldown assay. Biotinylated ISE3 and complementary ISE3 (ISE3-com) were incubated with recombinant RBM45-His protein and were then pulled down by using streptavidin-conjugated beads. Eluted RNA-bound proteins were analyzed by Western blotting for RBM45. The recombinant RBM45-His protein (Input) and the no RNA-pulldown sample were loaded in lanes 3 and 4 as input and bead only controls, respectively. (C to E) Biolayer interferometry. (C) Comparison of binding affinities of the RBM45 protein (2 μM) for the ISE3 and ISE3-com RNAs, respectively. Maltose-binding protein (MBP-His) was used as a negative protein control. (D) BLI sensorgrams showing association and dissociation of the RBM45 protein with ISE3 at different concentrations over time as indicated. (E) Binding parameters used to calculate KD values (ratios of dissociation and association rate constants). The binding experiments were repeated at least three times for calculating the means and standard deviations (SD), by using various concentrations of the protein.

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  FIG 6. The RRM2-HOA domain of RBM45 was largely responsible for binding to ISE3 of B19V pre-mRNA. (A) Domain structures of RBM45. The RNA recognition motif (RRM) domains are shown in red. The homo-oligomer assembly (HOA) domain and nuclear localization sequence (NLS; residues 469 to 472) are shown in green. Linker regions between RRMs are diagrammed in yellow. (B) Purification of truncated RBM45 proteins. Purified recombinant proteins (6 μg) were separated on an SDS-10% or 15% PAGE gel and stained with Coomassie brilliant blue dye. Asterisks denote the truncated proteins. (C and D) Biolayer interferometry (BLI) analysis. We used BLI to measure the interactions of ISE3 with different truncated and full-length forms of RBM45. (C) Domain 1–2 (aa 26 to 195), domain 2–3 (aa 121 to 462), domain 1 (aa 26 to 106), domain 2 (aa 121 to 195), and domain 3 (aa 390 to 462) of RBM45 were analyzed for their binding capability with ISE3. Domain 2–3 (aa 121 to 462) was found to be the key domain for RBM45 binding to ISE3. (D) Data representing domain 2-linker (aa 121 to 389) and 2-HOA (aa 121 to 318) were analyzed and compared with data representing binding to the full-length RBM45. KD values are shown…

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  FIG 7. Expression of the ISE3-binding domain of RBM45 decreased expression of 11-kDa at the levels of both mRNA and protein. CD36+ EPCs were transduced with a lentivirus expressing RBM45-RRM2-HOA. At 3 days postransduction, the cells were infected with B19V. After 48 h, the cells were collected for analyses of expression of viral mRNAs and proteins and of viral DNA replication. (A) RT-qPCR. Total RNA was extracted from CD36+ EPCs and reverse transcribed to generate cDNA, which was used for quantification of the viral mRNAs that encode the VP1, VP2, NS1, and 11-kDa proteins by the use of qPCR. The quantified viral mRNA levels were normalized to the level of β-actin mRNA, and the values representing mRNAs quantified from B19V-infected control CD36+ EPCs were arbitrarily set at 1. (B) Western blotting. The cells were lysed and analyzed by Western blotting. The blots were probed for the VP1/2, NS1, and 11-kDa proteins, using their respective antibodies. β-Actin was reprobed as a loading control. (C and D) Southern blotting. At 2 days posttransfection, Hirt DNA samples were prepared from CD36+ EPCs and were subjected to Southern blot analysis. SalI-digested pM20 (10 ng) was loaded as a size…

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  FIG 8. A B19V M20 infectious clone that harbors silent mutations of ISE3 expressed a low level of the mRNA spliced at the D2 and A2-2 sites and of 11-kDa protein in transfected cells. (A) ISE3 and ISE3 mutant 7 (ISE3-mut7) RNA sequences. The two RNAs shown were used in the binding experiment. (B) RNA pulldown assay. Biotinylated ISE3 and ISE3-mut7 were incubated with RBM45-His protein and then pulled down by using streptavidin beads. The RNA-bound proteins were eluted and analyzed by Western blotting for RBM45, as indicated. Approximately 4% of the RBM45-His protein was loaded as input (lane 3). (C) Diagram of pM20ISE3-mut7. Silent mutations in the ISE3 of the M20 clone (pM20ISE3-mut7) are diagrammed and shown with 7 mutations in red, while the coded amino acids of the VP1/2 protein remained unchanged. (D and E) RNA and protein analyses. UT7/Epo-S1 cells were transfected with either M20ISE3-mut7 or the parent M20 clone. (D) RT-qPCR. At 2 days posttransfection, total RNA was extracted from treated UT7/Epo-S1 cells and reverse transcribed. cDNA was used for quantification of the mRNAs that encode the VP1, VP2, NS1, and 11-kDa proteins by the use of qPCR. Quantified viral mRNA levels were…

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  FIG 9. RBM45 was colocalized within the viral DNA replication centers indicated by capsid detection. CD36+ EPCs were mock infected or infected with B19V. At 24 h postinfection, the cells were analyzed by immunofluorescence assay. (A) Confocal colocalization. Mock-infected or B19V-infected CD36+ EPCs were costained and incubated with rabbit anti-RBM45 and mouse anti-B19V capsid antibodies followed by Alexa Fluor 594-conjugated and FITC-conjugated secondary antibody. (B) Proximity ligation assay (PLA). Infected cells were costained with rabbit anti-RBM45 and mouse anti-B19V capsid antibodies, followed by a proximity ligation assay, which detected two proteins detected by two labeled secondary antibodies in close proximity (∼40 nm apart). Images were taken with a Leica TCS SPE confocal microscope at ×63 magnification. Nuclei were stained with DAPI.

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  FIG 10. ISE2 specifically binds the RBM45 protein under in vitro conditions. (A) Alignment of the ISE2 and ISE3 sequences. ISE2 and ISE3 share the 8 nucleotides 5′-GGG ACG GU-3′ indicated in blue. (B and C) RNA pulldown assay. Biotinylated ISE2 and ISE3 and ISE2-mut3 (negative control) (B) and biotinylated ISE38nt, ISE39nt, ISE3, and ISE3-com (C) were incubated with recombinant RBM45-His protein and then pulled down by using streptavidin-conjugated beads. Eluted RNA-bound proteins were analyzed by Western blotting for RBM45. A 3-μg volume of the recombinant RBM45-His protein was loaded as the input. (D to F) Biolayer interferometry (BLI). Data represent results of comparisons of binding affinities of the RBM45 protein at the indicated concentration for ISE2 RNA (D), ISE39nt (E), and ISE38nt (F). BLI sensorgrams show association and dissociation of the RBM45 protein with ISE2 RNA (D), ISE39nt (E), and ISE38nt (F) at different concentrations over time as indicated. (G) KD determination. Binding parameters used to calculate KD values and ratios of dissociation (Kass) and association (Kdiss) rate constants. The binding experiments were repeated at least three times for calculating the means…

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  FIG 11. A proposed model of the role of RBM45 in B19V pre-mRNA processing. RBM45 is a scaffold protein that functions to bring the 5′ and 3′ splice sites into close proximity. The B19V pre-mRNA is depicted, with cis elements of D2 donor, A2-1/2 acceptors, and exon splicing enhancers (ESE) and intron splicing enhancers (ISE) indicated, together with interacted splicing factors, U1, U2, RBM45, and potential mediator proteins (M). Spliced mRNAs that encode VP1, VP2, and 11-kDa protein are diagrammed, with the dashed lines indicating introns. U1 and U2 represent U1-small nuclear ribonucleoprotein (snRNP) and U2-snRNP complexes, and M represents protein mediators associated with U1-snRNP and U2-snRNP complexes. Dotted circles represent ISE-binding proteins (ISE2/3-BPs). Question marks indicate unknown factors. The approximate size of each mRNA is indicated, and the cap probe for Northern blotting is diagrammed.