Human cytomegalovirus UL37 immediate early target minigene RNAs are accurately spliced and polyadenylated

Yan Su1, James R. Testaverde1,{dagger}, Candice N. Davis1, Wail A. Hayajneh1,2,{ddagger}, Richard Adair1 and Anamaris M. Colberg-Poley1,3

1 Center for Cancer and Immunology Research, Room 5720, Children's Research Institute, George Washington University School of Medicine and Health Sciences, 111 Michigan Avenue, NW, Washington, DC 20010, USA
2 Department of Infectious Diseases, Children's National Medical Center, George Washington University School of Medicine and Health Sciences, 111 Michigan Avenue, NW, Washington, DC 20010, USA
3 Department of Pediatrics, George Washington University School of Medicine and Health Sciences, 111 Michigan Avenue, NW, Washington, DC 20010, USA

Correspondence
Anamaris Colberg-Poley (at Children's Research Institute)
acolberg-poley{at}cnmcresearch.org


   ABSTRACT
Top
ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
The human cytomegalovirus (HCMV) UL36–38 immediate early (IE) locus encodes proteins required for virus growth. The UL37 IE promoter drives production of differentially spliced and unspliced RNAs. To study their post-transcriptional processing, we generated target minigenes encoding each UL37 RNA splicing substrate. Target 1 RNA, spanning UL37 exon 1 (x1) donor and 2 (x2) acceptor as well as adjacent intronic sequences, but not the UL38 gene, accurately reproduced UL37 x1/x2 RNA splicing in transfected permissive cells. Surprisingly, deletion of distal intronic sequences nt -82 to -143 from the UL37x2 acceptor resulted in aberrant splicing to an upstream non-consensus exonic donor. Target 1 RNAs carry the UL37x1 polyadenylation (PA) signal and site as well as a downstream SV40 early PA signal. Both the UL37x1 and SV40 PA signals are used in wild-type target 1 RNAs but inhibited in UL37x1 PA signal mutants. Alternative RNA splicing of UL37 exons 2 to 3 or 3A as well as exons 3 to 4, observed in HCMV mature UL37 and UL36 spliced RNAs, is accurately reproduced with target minigene RNAs carrying the corresponding UL37 exonic and intronic sequences. Moreover, alternative splicing using two novel UL37 exon 3 consensus splice donors (di and dii) was found in target and in HCMV-infected cell RNA. These results demonstrate that: (i) target minigene RNAs accurately recapitulate the processing of UL37 IE RNAs in the HCMV-infected cell; (ii) precise UL37x1 donor selection is modulated by 3'-distal UL37 intronic sequences; and (iii) UL37 exon 3 contains multiple alternative consensus splice donors.

{dagger}Present address: Center for Community-Based Health Strategies, Academy for Educational Development, 1825 Connecticut Ave, NW, Washington, DC 20009, USA.

{ddagger}Present address: Jordan University of Science and Technology, Irbid, Jordan.


   Introduction
Top
ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
Human cytomegalovirus (HCMV) UL36–38 immediate early (IE) locus encodes biologically significant proteins for its DNA replication and for anti-apoptosis and growth (Colberg-Poley, 1996; Colberg-Poley et al., 1998; Goldmacher et al., 1999; Hayajneh et al., 2001a, b; Skaletskaya et al., 2001; Smith & Pari, 1995). The UL37 exon 1 (UL37x1) open reading frame (ORF) encodes the amino termini of three UL37 IE proteins (Chee et al., 1990; Goldmacher et al., 1999; Kouzarides et al., 1988) and appears to be essential for HCMV growth in humans as its sequences diverge minimally in HCMV primary strains (Hayajneh et al., 2001a). Conversely, the UL37 exon 3 (UL37x3) ORF appears to be non-essential for HCMV growth in culture (Borst et al., 1999; Goldmacher et al., 1999). Nonetheless, similar to the mouse CMV UL37 ORF (Lee et al., 2000), the HCMV UL37x3 C-terminal ORF appears to be important for HCMV growth in vivo as it diverges minimally (Hayajneh et al., 2001b).

The UL36–38 region encodes at least five transcripts from three different transcriptional promoters (Fig. 1a). Despite being initiated at the same IE promoter, three UL37 RNAs, UL37x1, UL37 and UL37M, are generated by differential RNA splicing and polyadenylation (PA) and show dramatically different temporal expression during HCMV infection (Goldmacher et al., 1999; Kouzarides et al., 1988; Tenney & Colberg-Poley, 1991a, b). The UL37x1 unspliced RNA is expressed abundantly at IE times and remains abundant until late times of infection. In contrast, the UL37 spliced RNAs are expressed at low abundance during IE times.



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Fig. 1. (a) HCMV UL36–38 transcripts. The RNA map indicates the direction of transcription, exons (solid boxes), introns (cross-hatched boxes), the 3' untranslated region (thin lines) and poly(A) tails (arrowheads). The bent and straight arrows on the scale bar represent promoters and PA signals, respectively. The table on the right shows the gene, RNA sizes and kinetic class. A novel splice variant, UL37M, differing in a 3' splice site (exon 3A, x3A) has been identified in transfected cells (Goldmacher et al., 1999). The HCMV sequences contained in the UL37x1 (x1) and UL37x3 (x3) antisense riboprobes are shown below the UL37 RNAs. (b) Similar regulation of UL37x1 and UL37 RNA expression in transfected HFF cells and in HCMV-infected cells. HFF cells were lipofected with a plasmid (p240) carrying the HCMV genomic UL36–38 IE locus (lanes 1, 2, 7 and 8). Control cells were uninfected (lanes 4 and 10) or HCMV-infected (lanes 3 and 9). All cells were anisomycin-treated for 16 h prior to harvesting. Total RNAs were examined by RPA with UL37x1 or UL37x3 biotinylated antisense riboprobes (a). RNA from p240-transfected (5 µg, lanes 1 and 7; 10 µg, lanes 2 and 8), HCMV-infected (5 µg, lanes 3 and 9), or uninfected cells (5 µg, lanes 4 and 10) was hybridized with UL37x1 (lanes 1–4) or UL37x3 riboprobes (lanes 7–10). Control RNase-digested (lanes 5 and 11) or undigested (lanes 6 and 12) riboprobes are shown. Migration of protected UL37x1 (filled arrowhead), UL37x3 (filled circle), undigested UL37x1 (open arrowhead) and UL37x3 (open circle) fragments are indicated. Positions of the RNA markers are indicated.

 
All UL37 RNAs share exon 1 sequences but differ downstream of the first 5' splice site (Fig. 1a). The unspliced UL37x1 RNA, which encodes the UL37x1 and UL38 ORFs, is polyadenylated within intron 1 upstream of UL37 exon 2 (UL37x2). Mature UL37 spliced RNAs contain the UL37x1 donor spliced to the UL37x2 acceptor, thereby removing the UL38 ORF and the UL37x1 PA signal. The UL37x2 donor is alternatively spliced to an upstream (UL37) or a downstream (UL37M) acceptor in exon 3 (Goldmacher et al., 1999; Kouzarides et al., 1988; Tenney & Colberg-Poley, 1991a). Finally, the UL37x3 donor is spliced to the exon 4 (UL37x4) acceptor in UL37, UL37M and UL36 spliced IE RNAs.

The mechanisms underlying the differential regulation of the HCMV UL37 spliced and unspliced IE RNAs are currently not known. Unique to UL37 spliced RNAs are retention of exons 2, 3 and 3A as well as the removal of introns 1, 2 and 2A. Thus, the potential sites of regulation of post-transcriptional processing include inefficient or regulated splicing or efficient polyadenylation of UL37x1 unspliced RNA.

Splicing of pre-mRNA involves the assembly of the spliceosome, which contains small nuclear ribonucleoprotein (snRNP) particles and many non-snRNP proteins (Burge et al., 1998; Krämer, 1996). The spliceosome brings together the reactive groups resulting in intron excision and exon ligation (Wu & Green, 1997). A major challenge for the splicing machinery is to target splicing accurately on nascent transcripts. In higher eukaryotes, three major signals direct RNA splicing: the 5' splice site, branch point (BP) and 3' splice site (Wu & Green, 1997). U1 snRNA base-pairs with the 5' splice site and is displaced by U6 snRNP binding (Wassarman & Steitz, 1992). Cooperative interaction between U2AF65 and mBBP/SF1 facilitates BP recognition (Berglund et al., 1998). The 3' end of the intron comprises a polypyrimidine tract (PPT) followed by an AG dinucleotide at which cleavage occurs (Umen & Guthrie, 1995). Arg/Ser (SR) proteins are essential splicing factors; they recruit U1 snRNP to the 5' splice site and mediate interactions between the 5' and 3' splice sites in early pre-splicing complexes (Fu, 1995). Several splicing factors are increased in abundance at early times of HCMV infection (Zhu et al., 1998).


   Methods
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ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
Cell culture and lipofection.
Human diploid fibroblast (HFF) cells were transfected with LipofectAmine or LipofectAmine 2000 (Invitrogen) as previously described (Colberg-Poley et al., 2000). Briefly, 7–9x106 HFF cells were lipofected with 10–20 µg of a plasmid (p240) carrying the HCMV EcoRI Q fragment (Tenney & Colberg-Poley, 1990) or the indicated target vector. The lipofected cells were incubated at 37 °C for 32 h. Control cultures were uninfected or infected with HCMV (strain AD169) at an m.o.i. of 2–3. All cultures were treated with 100 µM anisomycin for 16 h prior to harvesting.

Target plasmids.
Target minigenes containing the known UL37 splice sites and introns were generated by PCR amplification of HCMV (AD169) DNA using primers containing EcoRI and BamHI sites for cloning. Target 1 (p935) spanned part of UL37x1 (nt 52271–52219) and adjoining intron 1 sequences (nt 52218–52165). The splice donor was joined by an XbaI linker to downstream intronic sequences (nt 51132–50990) and UL37x2 (nt 50989–50949), thereby deleting the UL38 ORF (nt 52123–51130) but retaining the UL37x1 PA signal (nt 51020–51015) and site. Multiple independent target 1 clones carried 9, 10, or 11 Ts within the intronic region nt 51083–51092 (R. Adair & A. M. Colberg-Poley, unpublished results). Target 1 p935 and its derivatives carried only nine Ts in this region, although similar results were obtained with target 1 vectors (p1021 and p1023) carrying 10 Ts, as in the published AD169 sequence (Y. Su & A. M. Colberg-Poley, unpublished results). Target 1S removed an additional 62 nucleotides (nt 51132–51071) of UL37 intron 1 sequences upstream of the UL37x2 acceptor. Target 2 (nt 50987–50788) spanned exon 2, the complete intron 2 and the upstream exon 3 acceptor and adjacent exonic sequences. Target 2A (nt 50987–50521) spanned exon 2, the complete introns 2 and 2A and both the upstream (nt 50842) and downstream (nt 50587) exon 3 acceptors and adjacent exon 3 sequences. Target 3 (nt 49610–49421) spanned the exon 3 donor, the complete intron 3 and the exon 4 acceptor and downstream exonic sequences. For optimal expression, target DNAs were cloned into the mammalian expression vector p394 (Colberg-Poley et al., 1992). Transcription of the small target RNAs in p394 is under the control of the HCMV major IE (MIE) promoter and polyadenylation is directed by the SV40 early PA signal.

Site-specific mutagenesis.
The QuikChange site-directed mutagenesis kit (Stratagene) was used to insert a point mutation (AAUAAA->AAGAAA) into the UL37x1 PA signal in target 1 and 1S vectors, thereby generating p974 and p883. The identity of the PA mutants was confirmed by DNA sequencing.

RNA isolation.
Total cellular RNA was isolated using either the filter-based RNAqueous method (Ambion) or by guanidinium thiocyanate extraction and pelleting through 5·7 M caesium chloride cushions (Colberg-Poley et al., 1985).

RT-PCR.
Total RNA (5–200 ng) was treated with DNA-free (Ambion) and reverse-transcribed as previously described (Tenney et al., 1993) using oligo(dT) primers (Invitrogen) and SuperScript II reverse transcriptase (RT; Invitrogen). Control reactions containing no RT were assayed in parallel. PCR fragments were not observed in the absence of RT addition confirming the use of RNA as templates (Y. Su & A. M. Colberg-Poley, unpublished results). Target cDNAs were amplified by PCR using HotStarTaq DNA polymerase (Qiagen) with the primers shown in each figure. The products were detected by ethidium bromide staining following electrophoresis on non-denaturing 5 or 7 % polyacrylamide gels. DNA markers (123 bp ladder; Invitrogen) served as molecular size standards. Gel photographs were digitized using ScanWizard Pro version 1.21 and importation into Adobe Photoshop 5.0 LE and Microsoft PowerPoint 2000. The predicted sizes of the PCR products, based on the AD169 sequence, are listed in Table 1.


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Table 1. Predicted processed target RT-PCR fragment sizes

 
Nested PCR was performed as previously described (Hayajneh et al., 2001a). The sequencing strategy involved RT, nested PCR, purification of the nested PCR products using QIAquick gel extraction kit (Qiagen) and PCR sequencing using the Beckman CEQ DTCS Quick Start kit and primers 222 (MIE nt 173701–173723) and 223 (SV40 nt 2701–2725).

Biotinylated riboprobes.
UL37x1 (p274)- and UL37x3 (p280)-specific antisense riboprobes (Fig. 1a) were generated by in vitro transcription as previously described (Tenney & Colberg-Poley, 1991a, b) using the MAXIscript kit (Ambion). Full-length riboprobes were purified using 5 % polyacrylamide/8 M urea gels and biotinylated using the BrightStar Psoralen-Biotin kit (Ambion). All the riboprobes were detected by chemiluminescence (BioDetect; Ambion) in the range of 1–2 pg (J. R. Testaverde & A. M. Colberg-Poley, unpublished results) indicating their comparable specific activities.

RNase protection assays (RPAs).
RPAs were performed using the RPAIII kit (Ambion). Briefly, 500 pg biotinylated riboprobes were hybridized with total RNA (5–10 µg) overnight at 45 °C, RNase-digested and ethanol-precipitated. Protected fragments were resolved by electrophoresis on 5 % polyacrylamide/8 M urea gels, electroblotted onto nylon membranes and detected with a chemiluminescent substrate as above. Biotinylated Century RNA markers (Ambion) were included on gels for size comparison. The relative abundance of the UL37x1 and UL37x3 RNAs was quantified by comparison of the corresponding digitized protected fragments using the NIH Image program.


   Results
Top
ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
Regulated production of UL37 spliced and unspliced RNAs from an HCMV genomic IE plasmid
To test whether the production and processing of UL37 spliced and unspliced RNAs from the HCMV genomic UL36–38 locus is regulated comparably in transfected and in HCMV-infected cells, HFF cells were lipofected with an EcoRI Q plasmid (p240) that carries the HCMV genomic UL36–38 locus and treated with anisomycin (Fig. 1b). Control cultures were anisomycin-treated and either uninfected or HCMV-infected. Anisomycin, a protein synthesis inhibitor, was used to facilitate the detection of UL37 IE transcripts during HCMV infection and block switching to subsequent temporal gene classes, preventing confounding results.

The sizes of the UL37x1 (383 nt) and UL37x3 (344 nt) fragments protected by RNA from p240-transfected HFF cells (Fig. 1b, lanes 1, 2, 7 and 8) were similar to those from HCMV-infected cells (Fig. 1b, lanes 3 and 9). The UL37x1 and UL37x3 protected fragments were not detected in uninfected cells (Fig. 1b, lanes 4 and 10), verifying the specificity of the UL37x1 and UL37x3 riboprobes. Moreover, UL37x1 RNA was more abundant than UL37x3 spliced RNAs in p240-transfected HFF cells (~2·5–3·0-fold, lanes 1/7, 2/8) and in HCMV-infected cells (~3·4-fold, lanes 3/9). From these results, we concluded that UL37 and UL37x1 RNA processing, including splicing and polyadenylation and its regulation, are similar in anisomycin-treated, transfected cells and in anisomycin-treated, HCMV-infected HFF cells.

Accurate UL37x1/x2 splicing and polyadenylation of target 1 RNA
To examine UL37 RNA post-transcriptional processing, we generated minigene expression vectors spanning each known UL37 RNA splicing substrate. Target 1 spans the UL37x1 donor and UL37x2 acceptor and adjacent intron 1 sequences. As the UL38 ORF ends at nt -141 and most BP sites are generally located 11–40 nucleotides from the 3' splice site (Burge et al., 1998), most of UL37 intron 1 sequences (nt 52164–51133), including the complete UL38 ORF (nt 52123–51130), were deleted during the generation of target 1. We tested the processing of target 1 RNA in transfected HFF cells by RT-PCR and compared its products with UL37 RNA in HCMV-infected HFF cells (Fig. 2a). Using primers 176 and 179, RT-PCR products of 305 bp (unspliced) and 106 bp (spliced) were detected in target 1-transfected HFF cells (Fig. 2a, lane 5). The UL37x1/x2 spliced product was indistinguishable from that observed with RNA from HCMV-infected cells (Fig. 2a, lane 3), suggesting that UL37x1 and x2 within target 1 RNA are accurately spliced. Because of its large size (1573 bp), the RT-PCR fragment corresponding to full-length UL37x1 unspliced RNA was not observed in HCMV-infected cell RNA (Fig. 2a, lane 3). The RT-PCR products were not observed in uninfected cells (Fig. 2a, lane 1) or cells transfected with an irrelevant target control (Fig. 2a, lane 7), demonstrating the specificity of the PCR primers. PCR fragments were not observed in the control reactions lacking RT (Fig. 2a, lanes 2, 4, 6 and 8), verifying their identities as cDNAs.



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Fig. 2. (a) Accurate UL37 exon 1/exon 2 splicing of target 1 RNA. (b) Accurate polyadenylation of target 1 RNA at the UL37x1 PA site. HFF cells were transfected with vectors expressing target 1 (1) (lanes 5 and 6) or target 2A (2A) (lanes 7 and 8). Control cells were uninfected (Un) (lanes 1 and 2) or HCMV-infected (HCMV) (lanes 3 and 4). All cultures were treated with anisomycin 16 h prior to harvesting RNA. Total RNA (5 ng) was analysed by RT-PCR (+) using primers 176 and 179 (a) or primers 176 and 213 (b). Control reactions (-) did not contain RT (lanes 2, 4, 6 and 8). The positions of DNA markers, unspliced and spliced (a) and polyadenylated (b) RT-PCR products are indicated. Target 1 and target 1 PA Mut RNAs as well as primers 176, 179, 213, 239 and 240 are represented below (b). The cross represents the PA signal mutation. (c) Abrogation of target 1 RNA polyadenylation at nt 50998 by mutation of the UL37x1 PA signal. (d, e) Accurate splicing of target 1 PA Mut RNA. HFF cells were lipofected with vectors expressing target 1 (1) (lane 3), target 1 PA Mut (1PA Mut) (lane 4) or control target 2A (2A) (lane 5). Control cells were uninfected (Un) (lane 1) or HCMV-infected (HCMV) (lane 2). Total RNA (5 ng) was analysed by RT-PCR using primers 176 and 213 for polyadenylated RNA (c), primers 176 and 179 for spliced and unspliced RNA (d) or primers 239 and 240 for unspliced RNA (e). The positions of DNA size markers, polyadenylated (c), unspliced and spliced (d) and unspliced (e) RT-PCR products are indicated.

 
We verified the identities of the unspliced and spliced target 1 cDNAs by direct sequencing. The spliced junction of the target 1 RNA was between nt 52219 and 50989 (Table 2). Thus, the 5' and 3' splice sites used in target 1 RNA corresponded to the authentic splice sites used during UL37x1/x2 splicing in HCMV-infected cells.


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Table 2. Accurate splicing and polyadenylation of UL37 target minigene RNA substrates

 
In addition to the cis elements required for RNA splicing, target 1 RNA carries the UL37x1 PA signal within its intronic sequences at nt -31 to -26 upstream of the UL37x2 acceptor. To determine whether the UL37x1 PA signal and cis elements present in target 1 RNA are sufficient for accurate polyadenylation at the UL37x1 site, we further examined its processing (Fig. 2b). For these studies, RT-PCR was performed using primers 176 and 213, which spans the junction created by UL37x1 RNA cleavage and polyadenylation (Tenney & Colberg-Poley, 1991a). Primer 213 anneals to UL37 RNAs polyadenylated at nt 50998 but not to those lacking a poly(A) tail at that site. A 261 bp fragment corresponding to polyadenylated target 1 RNA in transfected cells was observed (Fig. 2b, lane 5). As expected, the 261 bp fragment was not detectable in HCMV-infected cell RNA (Fig. 2b, lane 3) because of the UL38 ORF and the correspondingly larger size of its RT-PCR product. These results demonstrated that target 1 RNA is polyadenylated at nt 50998, the site used for UL37x1 IE RNA polyadenylation during HCMV infection.

To determine whether the UL37x1 PA signal is required for target 1 RNA polyadenylation at nt 50998, target 1 PA mutant (PA Mut) RNA carrying a point mutation (AAGAAA) in the highly conserved PA signal was studied (Fig. 2c). This mutation is known to eliminate binding of cleavage/polyadenylation specificity factor (CPSF) and block polyadenylation downstream of the mutant PA signal (Bienroth et al., 1991). Polyadenylation at nt 50998 was detected in target 1 RNA (Fig. 2c, lane 3) but not in target 1 PA Mut RNA (Fig. 2c, lane 4). The use of the UL37x1 polyadenylation site by target 1 was verified by cloning and sequencing of the target 1 cDNA (Table 2). The inhibition of target 1 RNA polyadenylation at nt 50998 by the AAGAAA mutation verified the UL37x1 PA signal requirement for its processing at that site.

To determine whether accurate splicing of target 1 RNA is maintained in target 1 PA Mut RNA, its spliced products were examined using primers 176 and 179 (Fig. 2d). RT-PCR products of 305 bp and 106 bp, corresponding to unspliced and spliced target 1 RNA, respectively, were detected in target 1-transfected (Fig. 2d, lane 3) and in target 1 PA Mut-transfected (Fig. 2d, lane 4) HFF cell RNA. As expected, only the spliced (UL37x1/x2) RT-PCR fragment was detected in HCMV-infected cell RNA using primers 176 and 179 (Fig. 2d, lane 2). To increase the detection of target 1 unspliced RNA, we used primers 239 and 240, which are more closely located than primers 176 and 179 (Fig. 2e). The unspliced target 1 RNA was readily detected in both target 1- and target 1 PA Mut-transfected cells as a 101 bp product (Fig. 2e, lanes 3 and 4). As it spans the junction in target 1 created by deletion of UL38 ORF, primer 240 did not hybridize to or detect unspliced UL37 RNA in HCMV-infected cells (Fig. 2e, lane 2). Taken together, these results indicated that the UL37x1 PA signal mutation does not alter the accuracy of target 1 x1/x2 RNA splicing even though it blocks target 1 RNA polyadenylation at nt 50998.

3' Splice site intronic sequences from nt -82 to nt -143 are required for accurate UL37x1 5' splice site selection but not for target 1 RNA polyadenylation at nt 50998
Based on the BP consensus YURAY sequence (Burge et al., 1998), we predicted that the target 1 intron 1 had two potential BPs (at nt -30 and at nt -115). We tested whether the proximal AG dinucleotide cleavage site, the PPT and the BP consensus site were sufficient for authentic UL37x1/x2 RNA splicing using target 1S RNA (Fig. 3a). RT-PCR analysis detected the presence of the full-length unspliced product (Fig. 3a, lane 3). However, the RT-PCR fragment from target 1S spliced RNA was markedly smaller (Fig. 3a, lane 3) than that from authentically spliced UL37x1/x2 RNA species observed in HCMV-infected cells (Fig. 3a, lane 2). A heteroduplex fragment (~315 bp) was observed in target 1S- and in target 1S PA Mut-transfected cell RNA (lanes 3 and 4). Conclusive evidence for the identity of the heteroduplex was reproduction of the band by denaturation and renaturation of spliced and unspliced RNA in vitro. The heteroduplex band did not form when the RNAs were mixed but not heated. We concluded that target 1S RNA had been cryptically spliced and that UL37 intron 1 sequences nt -82 to -143 upstream of the UL37x2 acceptor are required for accurate UL37x1/x2 RNA splicing.



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Fig. 3. (a) Deletion of intronic sequences upstream (nt -82 to -143) of the UL37x2 acceptor results in cryptic splicing of target 1S RNA. (b) Accurate polyadenylation of target 1S RNA at the UL37x1 PA site. HFF cells were transfected with vectors expressing target 1S (1S) (lane 3), target 1S PA Mut (1S PA Mut) (lane 4) or with an irrelevant control target 2A (2A) (lane5). Control cells were uninfected (Un) (lane 1) or HCMV-infected (HCMV) (lane 2). All cultures were treated with anisomycin 16 h prior to harvesting RNA. Total RNA (5 ng) was analysed by RT-PCR using primers 176 and 179 (a) or primers 176 and 213 (b). The positions of the DNA size markers and unspliced and spliced (a) and polyadenylated (b) RT-PCR products are indicated. The asterisk marks the position of cryptically spliced target 1S and 1S PA Mut RNAs. Primers 176, 179 and 213 are represented in Fig. 2.

 
To determine the identity of the cryptically spliced junction, we sequenced the target 1S spliced cDNA (Table 2). Target 1S RNA was spliced from nt 52251 to nt 50989. These results established that an abnormal upstream UL37x1 donor was spliced to the authentic UL37x2 acceptor. We concluded that intronic sequences (nt -82 to -143 to UL37x2) affect UL37x1 5' splice site selection during UL37 RNA splicing.

To determine whether these intronic sequences also affect the accuracy of target 1 RNA polyadenylation at nt 50998, we further examined target 1S RNA processing (Fig. 3b). RT-PCR amplification using primers 176 and 213 amplified a fragment of 205 bp in transfected cell RNA corresponding to target 1S RNA polyadenylated at nt 50998 (Fig. 3b, lane 3). To determine whether the PA signal at nt 51015–51020 is required for this processing, target 1S PA Mut was examined. Mutation of the UL37x1 PA signal resulted in absence of the 205 bp fragment (Fig. 3b, lane 4). Thus, similar to target 1 RNA, polyadenylation of target 1S RNA at nt 50998 is determined by the PA signal at nt 51015–51020 (Table 2).

We also examined target 1S PA Mut to determine whether cryptic splicing, previously observed in target 1S RNA, was affected by the PA signal at nt 51015–51020 (Fig. 3a). The RT-PCR fragments corresponding to unspliced (249 bp) and cryptically spliced target 1S PA Mut RNA were observed (Fig. 3a, lane 4). These were indistinguishable from the parental target 1S RNA products (Fig. 3a, lane 3) and smaller than HCMV-infected UL37x1/x2 spliced product (Fig. 3a, lane 2). Thus, the UL37x1 PA signal mutation did not alter selection of the UL37x1 5' splice site.

Accurate UL37x2/x3 splicing of target 2 RNA
Target 2 RNA includes 41 nucleotides upstream of the UL37x2 donor, the complete intron 2 and 55 nucleotides downstream of the UL37x3 acceptor. To determine whether it contains the cis elements necessary for correct UL37x2/x3 RNA splicing, we examined the processing of target 2 RNA by RT-PCR using primers 180 and 181 (Fig. 4a). RT-PCR fragments corresponding to target 2 unspliced (209 bp) and spliced RNA (105 bp) were observed in transfected cell RNA (Fig. 4a, lane 1) and in HCMV-infected cell RNA (Fig. 4a, lane 5). The sizes of the RT-PCR fragments were indistinguishable, suggesting RNA splicing of target 2 RNA at the authentic UL37x2/x3 junction. The identity of the x2/x3 spliced cDNA was verified by sequencing (Table 2). A heteroduplex fragment (~360 bp) was observed in both HCMV-infected cell RNA (lane 5) and in target 2-transfected cell RNA (Fig. 4a, lane 1). These results demonstrated that target 2 spanning upstream of the UL37x2 donor, the complete intron 2 and downstream UL37x3 sequences carries the necessary cis elements for authentic UL37x2/x3 RNA splicing.



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Fig. 4. (a) Accurate UL37 exon 2/3 splicing of target 2 RNA. HFF cells were lipofected with vectors expressing target 2 (2) (lanes 1 and 2) or target 3 (3) (lanes 3 and 4). Control cells were HCMV-infected (HCMV) (lanes 5 and 6) or uninfected (Un) (lanes 7 and 8). Total RNA (10 ng) was analysed by RT-PCR (+) using primers 180 and 181. Control reactions lacked RT (-) (lanes 2, 4, 6 and 8). The positions of size markers and unspliced and spliced RT-PCR products are indicated. Target 2 RNA and primers 180 and 181 are represented below. (b) Accurate UL37 exon 2/3 and exon 2/3A splicing of target 2A RNA. HFF cells were lipofected with expression vectors of target 2A (2A) (lanes 5 and 6) or irrelevant control target 1 (1) (lanes 7 and 8). Control cells were uninfected (Un) (lanes 1 and 2) or HCMV-infected (HCMV) (lanes 3 and 4). Total RNA (10 ng) was analysed by RT-PCR (+) using primers 180 and 196. Control reactions (-) lacked RT (lanes 2, 4, 6 and 8). The positions of DNA size markers and unspliced and differentially spliced cDNAs are indicated. The differentially spliced x2/x3di/x3A and x2/x3dii/x3A RNAs are indicated with the arrowhead. Target 2A RNA and primers 180 and 196 are represented below.

 
Alternative splicing of target 2A RNA
In addition to splicing to the UL37x3 acceptor (nt 50842), UL37x2 is alternatively spliced to the downstream exon 3A acceptor (nt 50587) in transfected cells (Goldmacher et al., 1999). To determine whether alternative RNA splicing occurs when both UL37 acceptors – exon 3 and exon 3A – are present and whether UL37x2/x3A is detected in HCMV-infected cells, we examined splicing of target 2A and of HCMV-infected cell RNA (Fig. 4b). The RT-PCR fragments corresponding to unspliced RNA (480 bp) as well as UL37x2/x3 (376 bp) and UL37x2/x3A (121 bp) spliced RNAs were observed in target 2A RNA (Fig. 4b, lane 5) and in HCMV-infected cell RNA (Fig. 4b, lane 3). Surprisingly, unanticipated species (195 and 182 bp) corresponding to the use of two new UL37 exons were observed in both HCMV-infected and target 2A RNA (marked by an arrowhead). The identities of the unspliced and spliced as well as the novel cDNAs were determined by sequencing (Table 2). The novel UL37 cDNAs resulted from splicing of the UL37x2 donor to the known UL37x3 acceptor, and of a novel UL37x3 donor at either nt 50782 (dii) or nt 50770 (di) to the known UL37x3A acceptor. Thus, alternative UL37 RNA splicing at the known UL37x2/x3 and UL37x2/x3A junctions as well as at the new UL37x2/x3di/x3A and UL37x2/x3dii/x3A junctions was observed in HCMV-infected HFF cells and in target 2A-transfected cells. The predicted products of the alternatively spliced UL37di and UL37dii RNAs span the UL37x1 and x2 ORFs but lack the UL37x3 encoded N-glycosylation, transmembrane and cytosolic tail present in gpUL37 and gpUL37M. Because of their unique C-terminal sequences, these pUL37 isoforms are predicted to act differentially from other UL37 proteins.

Accurate UL37x3/x4 splicing of target 3 RNA
The last spliced junction present in UL37, UL37M and UL36 RNA joins exon 3 to exon 4. Target 3 includes the UL37x3 donor, the complete intron 3 and the UL37x4 acceptor. To determine whether target 3 RNA carries the cis elements sufficient for accurate splicing of UL37x3 to x4, we examined its processing in transfected HFF cells (Fig. 5). RT-PCR of target 3 RNA using primers 182 and 183 detected cDNAs corresponding to unspliced (202 bp) and spliced (99 bp) RNAs and a heteroduplex fragment (~315 bp) in transfected cells (Fig. 5, lane 5). The sizes of the unspliced and spliced RT-PCR fragments were indistinguishable from those obtained in HCMV-infected HFF cells (Fig. 5, lane 3). The spliced junction of UL37x3/x4 was verified by sequencing (Table 2). Taken together, these results suggest that UL37 exon 3 to exon 4 splicing is accurately reproduced by the target 3 RNA substrate.



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Fig. 5. Accurate UL37 exon 3/4 splicing of target 3 RNA. HFF cells were lipofected with vectors expressing target 3 (3) (lanes 5 and 6) or control irrelevant target 1 (1) (lanes 7 and 8) RNA. Control cells were uninfected (Un) (lanes 1 and 2) or HCMV-infected (HCMV) (lanes 3 and 4). Total RNA (5 ng) was analysed by RT-PCR (+) using primers 182 and 183. Control reactions (-) (lanes 2, 4, 6 and 8) lacked RT. The positions of DNA sizemarkers and spliced and unspliced RT-PCR products areindicated. Target 3 RNA and primers 182 and 183 are represented below.

 

   Discussion
Top
ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
Post-transcriptional RNA splicing and polyadenylation of HCMV UL37 IE RNAs has been accurately reproduced using minigene RNAs (190-467 nucleotides). The use of authentic and novel splice sites by the target RNAs was verified by RT-PCR, RNase protection assays (J. R. Testaverde & A. M. Colberg-Poley, unpublished results) and sequencing of all spliced target cDNAs. Three major signals, the 5' splice site, the BP and the 3' splice site, direct accurate splicing in higher eukaryotes (Wu & Green, 1997). We incorporated UL37 sequences based on established requirements for accurate RNA splicing. To facilitate interpretation of the splicing products, each UL37 donor, known to be used in HCMV-infected cells, was separately cloned into a target minigene vector. We incorporated 36–53 nucleotides upstream of the UL37 5' splice site, since spliceosomes are known to deposit a protein complex 20–24 nucleotides upstream of the 5' splice site (Le Hir et al., 2000). We observed accurate splicing with targets 1, 2, 2A and 3 RNAs validating that the necessary elements upstream of the 5' splice site were present. Moreover, the use of two new consensus donors (nt 50770 and nt 50782) in UL37x3 was observed.

The second element required for splicing is the BP, an intronic adenosine, which is generally located at nt -10 to -40 of the 3' splice site and initiates the nucleophilic attack on the exonic donor (Burge et al., 1998; Krämer, 1996). As introns 2, 2A and 3 are relatively small (103–359 nucleotides), we incorporated the complete introns into targets 2, 2A and 3. Consequently, these target RNAs carry and predictably use the same BP as HCMV UL37 RNA. Indeed, targets 2, 2A and 3 RNAs were accurately spliced at sites corresponding to those used in UL37 and UL37M RNAs in HCMV-infected HFF cells. Nonetheless, we will make use of the target RNAs to verify the use of the authentic BP in target RNAs, as in HCMV-infected cells.

Because of its large size (1·23 kb), UL37 intron 1 required a partial deletion. Based on the predicted location of the BP close to the 3' splice site, intron 1 sequences were minimized (from nt -143) in target 1. These intronic sequences were sufficient to enable the cellular splicing machinery to splice target 1 RNA accurately at the same site used in HCMV UL37x1/x2 RNA.

Based on the BP consensus YURAY sequence (Burge et al., 1998), we predicted that target 1 intron 1 had two potential BPs (at nt -30 and nt -115). Only the proximal predicted site (nt -30) is consensus and the distal BP (nt -115) has one non-consensus nucleotide. Nonetheless, the distal predicted BP might be favoured because the proximal consensus BP is part of the UL37x1 PA signal used throughout HCMV infection. We note, however, that the distal BP sequence (nt -115) lies upstream of five AG dinucleotide/PPT elements prior to the AG used in authentic UL37x1/x2 splicing. Cleavage of the downstream AG dinucleotide, essential for the second step of splicing, generally occurs at the first AG downstream of the BP (Smith et al., 1989; Zhuang & Weiner, 1990). Therefore, we consider it highly unlikely that the distal BP non-consensus site, which has multiple adjacent AG dinucleotide/PPT elements, would be used for splicing at the authentic UL37x2 acceptor.

To determine the cis elements required for UL37x1/x2 splicing, we further reduced target 1S RNA 3' sequences to include the known UL37x2 cleavage site and its proximal PPT and potential BP. Surprisingly, target 1S RNA was cryptically spliced. Deletion of UL37 distal 3' intronic sequences (nt -82 to -143 from the UL37x2 acceptor) altered selection of the UL37x1 5' splice site to an upstream non-consensus donor. Selection of 5' splice sites can be modulated by 3' splice site complexes and promoted by SR proteins, which provide a bridging function (Tarn & Steitz, 1995) or by splicing enhancers (Hastings & Krainer, 2001). Selection of the abnormal 5' splice site in target 1S RNA may result from use of the proximal BP sequence, in the absence of the deleted distal BP sequence. Base-pairing interactions between the 5' splice site and the BP sequences can affect 5' splice site selection (Cote & Chabot, 1997). SR proteins might alter UL37x1 5' splice site selection to the cryptic site through stabilization of U2 snRNP binding to the available proximal BP in target 1S RNA. SR proteins are known to recruit U2 snRNP to the BP and stabilize the complex assembled on the 3' splice site in the presence or absence of functional U1 snRNPs (Tarn & Steitz, 1995). This 3' complex can subsequently communicate with the 5' splice site bound by a U1 snRNP.

Alternatively, the UL37 intron 1 deleted sequence (nt -82 to -143 from the UL37x2 acceptor) may contain an intronic enhancer element that modulates UL37x1 5' splice site selection. Intronic enhancers are known to modulate 5' splice site selection in plant nuclei (McCullough & Schuler, 1997). At least two intronic splicing enhancers are known to affect alternative splicing of Protein 4·1R during erythroid differentiation in mouse cells (Deguillien et al., 2001). UL37 intron 1 might contain a splicing enhancer for the suboptimal UL37x1 normal donor.

The third major feature recognized during RNA splicing is the 3' splice site. In mammals, a PPT is highly conserved and is an essential 3' element (Reed & Maniatis, 1985; Ruskin & Green, 1985). Targets 1 and 1S RNAs both carry the adjacent PPT (nt -3 to -12) upstream of the UL37x2 acceptor. The intron 1 PPT contains a potential core binding site (UCUU) for PTB, a potent splicing suppressor (Lin & Patton, 1995; Singh et al., 1995), close to its 3' splice site (Chee et al., 1990). Targets 2, 2A and 3 all contain adjacent PPT elements upstream of the AG dinucleotides at which cleavage normally occurs within their corresponding intron/exon boundaries.

Finally, the 3' splice site can either be AG-dependent or AG-independent (Reed, 1989). The known UL37 acceptors appear to be AG-dependent as they conform better to the AG-dependent consensus 3' splice site (YYYYYYNYAG/GU; Wu et al., 1999) than to AG-independent sites. The UL37 exon 3 and 4 acceptors conform more closely to consensus acceptor sequences whereas the exon 2 and 3A acceptors conform less well and are predicted to be weaker.

Interestingly, UL37 intron 1 contains a strong PA signal, which is used virtually throughout HCMV infection (Kouzarides et al., 1988, Tenney & Colberg-Poley, 1991a, b). We examined whether this PA signal was used, even though the SV40 early PA signal had been included downstream of the inserts in the expression vectors. Messenger RNA polyadenylation requires the essentially invariant signal AAUAAA and downstream elements of more diffuse sequences that are generally rich in U or GU residues (Colgan & Manley, 1997; Wahle & Keller, 1996). In addition, some RNAs contain an auxiliary U-rich signal upstream of the PA signal, which act as enhancers of polyadenylation.

Target RNAs were found to be efficiently cleaved and polyadenylated downstream of the SV40 early PA signal based on reverse transcription using oligo(dT) primers and PCR amplification of full-length unspliced products. In addition, cleavage and polyadenylation occurred at the authentic UL37x1 PA site (nt -9 from exon 2) in target 1 and 1S RNAs. Target 1 and 1S RNA polyadenylation was, as predicted, dependent on the UL37x1 PA signal at nt -26 to -31 from exon 2. The UL37x1 RNA cleavage site maps within a potential core PPT binding protein site (Lin & Patton, 1995; Singh et al., 1995). Finally, target 1 and 1S include exon 2 sequences (nt 50975–51000) downstream of the 3' splice site. These sequences, which are G/U rich, may act to increase the efficiency of polyadenylation downstream of the UL37x1 PA signal by binding CstF (MacDonald et al., 1994).

We note that UL37 intron 2 and intron 2A also contain a PA signal just 24 nucleotides upstream of the exon 3 acceptor. However, this PA signal is not known to be used during IE times of HCMV infection. We will examine whether this PA signal is recognized in HCMV-infected cells or in target 2 RNA.

Use of target RNAs has allowed us to define UL37 RNA sequences sufficient for accurate RNA splicing and polyadenylation as well as the UL37 distal intronic sequences that modulate UL37x1 5' splice site selection. We are now using these defined RNA substrates to define further the processing of UL37 RNAs and to examine regulation of their processing.


   ACKNOWLEDGEMENTS
 
The authors thank Drs Dan Tenney and Nancy DiFronzo for their critical comments on the manuscript. This work was supported by Public Health Service Grant AI-46459 from the National Institute of Allergy and Infectious Diseases, American Heart Association Grant-in-Aid 150307N and Children's Research Institute Discovery Funds to A. C. P.


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Received 9 July 2002; accepted 6 September 2002.