The C-terminal region but not the Arg-X-Pro repeat of Epstein-Barr virus protein EB2 is required for its effect on RNA splicing and transport

Abstract

The Epstein-Barr virus BMLF1 gene product EB2 has been shown to efficiently transform immortalized Rat1 and NIH 3T3 cells, to bind RNA, and to shuttle from the nucleus to the cytoplasm. In transient-expression assays EB2 seems to affect mRNA nuclear export of intronless RNAs and pre-mRNA 3' processing, but no direct proof of EB2 being involved in RNA processing and transport has been provided, and no specific functional domain of EB2 has been mapped. Here we significantly extend these findings and directly demonstrate that (i) EB2 inhibits the cytoplasmic accumulation of mRNAs, but only if they are generated from precursors containing weak (cryptic) 5' splice sites, (ii) EB2 has no effect on the cytoplasmic accumulation of mRNA generated from precursors containing constitutive splice sites, and (iii) EB2 has no effect on the 3' processing of precursor RNAs containing canonical and noncanonical cleavage-polyadenylation signals. We also show that in the presence of EB2, intron-containing and intronless RNAs accumulate in the cytoplasm. EB2 contains an Arg-X-Pro tripeptide repeated eight times, similar to that described as an RNA-binding domain in the herpes simplex virus type 1 protein US11. As glutathione S-transferase fusion proteins, both EB2 and the Arg-X-Pro repeat bound RNA in vitro. However, by using EB2 deletion mutants, we demonstrated that the effect of EB2 on splicing and RNA transport requires the C-terminal half of the protein but not the Arg-X-Pro repeat.

FIG. 1

EB2 inhibits the expression of differentially spliced polyadenylated RNAs expressed from pBLCAT2. (A) Diagrams of the expected processed RNAs generated from plasmids pBLCAT2 and pSV2β. The genetic contents of the pre-mRNAs initiated at the tk promoter (pTK) in pBLCAT2 and at the early SV40 promoter in pSV2β (pSV40E) are indicated. The CAT ORF in pBLCAT2 and the rabbit β-globin sequences in pSV2β are shaded. DNA probes 1 and 2 were used for the Northern blot analysis shown in panel B. The deadenylation and cleavage of the expected processed RNAs by RNase H in the presence of oligo(dT) or oligo(dT) plus oligo(H) and the lengths of the expected RNase H-treated RNA products are also indicated. (B) Northern blot analysis of the RNAs transiently expressed in HeLa cells from plasmids pBLCAT2 (lanes 1 to 5) and pSV2β (lanes 6 to 9). The plasmids transfected, the proteins expressed, and the RNase H treatments are indicated above the panels. The ³²P-labeled DNA probes used are indicated below the panels. M(Kb), molecular size markers (number of kilobases).

FIG. 2

EB2 inhibits the expression of polyadenylated RNAs generated by utilization of alternative cryptic 5′ splice sites. (A) Diagrams of the processed RNAs generated from RNA precursors initiated at the tk promoter and at a cryptic promoter in the vector, after transfection of pBLCAT2β into HeLa cells, and primers used for RT-PCR analysis. (B) ³²P-labeled RT-PCR products obtained with primers P1 and P2. (C) ³²P-labeled RT-PCR products obtained with primers P3 and P4. Twenty PCR cycles were done in the RT-PCR.

FIG. 3

The effect of EB2 on alternative splicing is dependent on cis determinants of 5′ splice site selection. (A) Representation of a 5′ splice site sequence hybridizing perfectly to the 5′ end of U1 snRNA. (B) Diagram of the minigenes transfected into HeLa cells and the primers used for RT-PCR analysis of the cytoplasmic RNAs expressed. The alternative 5′ splice sites are indicated by numbers 1 to 4, and their sequences are presented over the diagram of each construction. Twenty PCR cycles were done in the RT-PCR. (C) Splicing patterns of Paac-ALT1, Paac-ALT1.2, and Paac-ALT1.3 minigenes upon cotransfection into HeLa cells with a vector expressing (lanes 2) or not expressing (lanes 1) EB2.

FIG. 4

Effect of EB2 on alternative splicing versus constitutive splicing. (A) Diagrams of the human β-globin gene and the β-thalassemia gene and primers used for RT-PCR analysis. The expected splicing products are indicated below the wt β-globin gene and the β^thal gene. Twenty PCR cycles were done in the RT-PCR. (B) Patterns of splicing of the wt β-globin gene (lanes 1, 2, 5, and 6) and the β^thal gene (lanes 3, 4, 7, and 8), upon cotransfection with a vector not expressing (lanes 1 and 6) or expressing EB2 (lanes 2 and 7), in the nucleus (lanes 1 and 2) and the cytoplasm (lanes 6 and 7).

FIG. 5

EB2 has no effect on constitutive splicing of an EBV immediate-early pre-mRNA. (A) Diagrams of the BZLF1 reporter gene and the primers used for RT-PCR. Twenty PCR cycles were done in the RT-PCR. The BZLF1 gene product EB1 is also schematically represented, with the epitope stained by the monoclonal antibody mabZ125. EB2 has no effect on the expression of the BZLF1 gene transfected into HeLa cells, and this is seen both at the level of the correctly processed polyadenylated RNAs by RT-PCR (B) and at the level of the EB1 protein, expressed by Western blotting and staining with mabZ125 (C).

FIG. 6

EB2 increases the cytoplasmic accumulation of its own RNA. (A) Diagrams of the reporter genes and the single-stranded S1 probe used. (B) Visualization of the Flag EB2 (mAbM2) protein expressed in HeLa cells transfected as indicated in lanes 1 and 2. (C) Quantitative S1 nuclease analysis of the cytoplasmic RNAs expressed in HeLa cells transfected as indicated above the upper panel. The specific protected bands are indicated by Flag (upper panel), for the RNAs initiated at the CMV promoter in PaacFlagM1 and PaacFlag-EB2, and by SV40 for the internal control (lower panel).

FIG. 7

EB2 does not affect cleavage-polyadenylation. (A) Diagram of the reporter gene used in the assay. (B) Sequences of the CPS inserted in I28 to generate I28-PAPUC, I28-PASV40, I28-PAGLO, and I28-PADNApol. The essential sequence elements for cleavage-polyadenylation are underlined or overlined. Cleavage sites are indicated by arrows. (C) Schematic representation of the RT-PCR assay and the expected size of the RT-PCR product generated. Twenty PCR cycles were done in the RT-PCR. sscDNA, single-stranded cDNA. (D) Semiquantitation by RT-PCR of the cytoplasmic RNAs expressed in HeLa cells transfected as indicated above the panel. Double-stranded DNA size markers are indicated on the left.

FIG. 8

EB2 and the EB2 Arg-X-Pro repeat bind to RNA in vitro as GST fusion proteins. (A) Schematic representations of the RNA probes generated from the Paac-ALT1 minigene. (B) Diagram of the EB2 protein and of the Arg-X-Pro (RXP) polypeptide, which were fused to GST. (C) EMSAs were performed with probes 1 to 6. The ³²P-labeled RNA probes used were loaded alone in lanes 1, 5, 9, 13, 17, and 21 or were loaded after incubation with the GST protein (lanes 2, 6, 10, 14, 18, and 22), the GST–Arg-X-Pro protein (lanes 3, 7, 11, 15, 19, and 23), or the GST-EB2 protein (lanes 4, 8, 12, 16, 20, and 24).

FIG. 9

The C-terminal domain of EB2 is essential for its function, but the Arg-X-Pro repeat is not. (A) Diagrams of the EB2 deletion mutants generated. The names of the mutants and the amino acids deleted are indicated on the left side of the panel. The approximate location of the Arg-X-Pro repeat (RXP) is indicated above the diagram. (B and C) Expression of EB2 and EB2 mutants in HeLa and Cos7 cells evaluated by Western blotting and a polyclonal antibody against EB2. M1 is an EB2 mutant in which two stop codons have been inserted downstream of the BMLF1 AUG and which does not express the EB2 protein. (D and E) Effects of EB2 and EB2 mutants on the splicing pattern of the Paac-ALT1 minigene in Cos7 and HeLa cells. Twenty PCR cycles were done in the RT-PCR.