Su Yan, Testaverde James R, Davis Candice N, Hayajneh Wail A, Adair Richard, Colberg-Poley Anamaris M
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.
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.
J Gen Virol. 2003 Jan;84(Pt 1):29-39. doi: 10.1099/vir.0.18700-0.
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.
人巨细胞病毒（HCMV）UL36 - 38立即早期（IE）基因座编码病毒生长所需的蛋白质。UL37 IE启动子驱动差异剪接和未剪接RNA的产生。为了研究它们的转录后加工过程，我们构建了编码每个UL37 RNA剪接底物的靶标小基因。靶标1 RNA跨越UL37外显子1（x1）供体和2（x2）受体以及相邻的内含子序列，但不包括UL38基因，在转染的允许细胞中准确地重现了UL37 x1/x2 RNA剪接。令人惊讶的是，从UL37x2受体中删除远端内含子序列nt -82至-143会导致异常剪接至上游非共识外显子供体。靶标1 RNA携带UL37x1聚腺苷酸化（PA）信号和位点以及下游SV40早期PA信号。UL37x1和SV40 PA信号在野生型靶标1 RNA中均被使用，但在UL37x1 PA信号突变体中受到抑制。在HCMV成熟的UL37和UL36剪接RNA中观察到的UL37外显子2至3或3A以及外显子3至4的可变RNA剪接，在用携带相应UL37外显子和内含子序列的靶标小基因RNA中准确重现。此外，在靶标和HCMV感染细胞的RNA中发现了使用两个新的UL37外显子3共有剪接供体（di和dii）的可变剪接。这些结果表明：（i）靶标小基因RNA准确地概括了HCMV感染细胞中UL37 IE RNA的加工过程；（ii）精确的UL37x1供体选择受3' - 远端UL37内含子序列调节；（iii）UL37外显子3包含多个可变共有剪接供体。
