行政院國家科學委員會補助專題研究計畫成果報告
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※ Hz-1 病毒潛伏關連基因之轉錄及其轉錄體和 ※
※ 核糖蛋白結合之探討 ※
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計畫類別：□個別型計畫 □整合型計畫
計畫編號：NSC89－2311－B－006－003
執行期間：88 年 8 月 1 日至 89 年 7 月 31 日
計畫主持人：陳虹樺
共同主持人：吳定峰
執行單位：國立成功大學生物科技研究所
中 華 民 國 89 年 10 月 31 日
行政院國家科學委員會專題研究計畫成果報告
國科會專題研究計畫成果報告撰寫格式說明
Pr epar ation of NSC Pr oject Repor ts
計畫編號：NSC 89-2311-B-006-004
執行期限：88 年 8 月 1 日至 89 年 7 月 31 日
主持人：陳虹樺 執行機構及單位名稱：成大生物科技所
共同主持人：吳定峰 執行機構及單位名稱：嘉南藥理科技大學
一、中文摘要 PAT1 潛伏感染轉錄體是 Hz-1 病毒在潛伏感 染大量表現的轉錄體。PAT1 大小為 2.9kb，主要位 於細胞核內。本計畫主要證明 PAT1 是由核酸聚合 酵素 III (RNA polymerase III)所轉錄，且證明 PAT1 會和細胞內的 snRNP 及 nonsnRNP 核糖蛋白結 合，且 PAT1 的序列和 U2 snRNA 有互補的現象。 推測由核酸聚合酵素 III 所轉錄的 PAT1，可能藉由 和的 U2 snRNA 互補而和核糖蛋白結合，進而可能 干擾正常細胞內的剪接過程。 關鍵詞：Hz-1 病毒、潛伏感染轉錄體、核酸聚合酵 素、核糖蛋白 Abstr actPersistence-associated transcript 1 (PAT1) is the major transcript expressed during Hz-1 viral persistence, while the rest of the viral genome is shut off. PAT1 is an abundant, 2.9-kb noncoding, uncapped and unpolyadenylated RNA. It displays a speckled distribution in the nuclei of persistently infected cells. Evidence is presented here from immunoprecipitation assay using monoclonal antibodies against a snRNP splicing factor and a non-snRNP splicing factor SC35 that PAT1 is associated with ribonucleoprotein (RNP) complexes of the pre-mRNA splicing machinery.The association was probably mediated by U2 snRNA since a stretch of complementary sequence between PAT1 and the branch point recognition site of U2 snRNA was revealed by computer analysis. Furthermore, like U6 snRNA, synthesis of PAT1 was insensitive to α-amanitin at a relatively low concentration (2 µg/ml), while significantly impaired by the specific inhibitor of pol III tagetitoxin in both in vitro runoff and runon experiments. In conclusion, PAT1 is most likely transcribed by RNA polymerase III, and associated with the RNPs to form speckles in the nucleus and thus may affect the cellular RNA splicing process.
Keywor ds: Hz-1 virus, PAT1, pol III, nuclear
speckles, small ribonucleoproteins, splicing factor SC35, complementary
二、緣由與目的
Hz-1 virus is rod-shaped virus containing a circular double-stranded DNA genome of 228 Kb (1, 2). It was identified as a persistent viral infection in the
Heliothis zea cell line, IMC-1 (3-5). Originally, Hz-1 virus was classified as a member of the
Nudibaculoviridae, but is currently an unclassified invertebrate virus due to not producing occlusion bodies (6).
Hz-1 virus a first virus infecting invertebrates that has been studied for temporal gene expression during persistent infection (7). It replicates in the nuclei of infected cells and produces numerous virions during productive infection in most infected cells. Persistent infection is established in only a very small portion (0.01-0.05%) of the infected cells (8, 9). During a productive infection, Hz-1 virus produces more than 100 different transcripts, but only one transcript, the persistence-associated transcript 1 (PAT1) (7) is detected in the persistent infection. PAT1 is expressed as early as 2 hours post infection (hpi), and its level of expression remains constant up to 12 hpi (7). It has been reported that PAT1 may be involved in the establishment of Hz-1 viral persistence (10).
PAT1 is a non-coding RNA since no significant open reading frames can be identified in it (10). It is not associated with polysomes (10) and no protein products can be detected by its in vitro translation (11). Both direct and inverted repeats are abundant in the PAT1 sequence. The direct repeats form three clusters with unknown significance (10). The characteristics of both RNA polymerase II (pol II) and pol III-transcribed genes are present in the gene structure of PAT1. Similar to the pol II-transcribed genes, the putative promoter of PAT1 contains a TATA box at nucleotide (nt) -30 and a CAAT box at nt -80 (10). A "CAGT" motif is present at nt +3 to +6 of PAT1, which is known to be essential for the expression of immediate early genes of AcMNPV (12). PAT1 lacks both 5’-capping and 3’-polyadenylation, and does not contain any apparent introns (11). The vast majority of PAT1 is localized in the nuclei of persistently infected cells and displays a speckled distribution in the nucleus (10). We noticed that a stretch of U-residues is present at its 3' end that could serve as the termination signal for pol III. Although an AAUAAA sequence is located at 8-nt downstream of the stretch of U-residues,
no GT-rich sequence nearby is observed nor poly (A) tail is detected, suggesting that PAT1 is probably transcribed by pol III.
α-amanitin is a specific inhibitor of pol II by binding to the largest subunit of pol II with high affinity (Kd=10-9 M) and presenting it to the cellular
proteolytic machinery (13). Tagetitoxin, a bacterial phytotoxin is produced by Pseudomonas syringae pv.
Tagetis, is a selective inhibitor of pol III. The action of tagetitoxin against RNA pol III protmoter-directed transcription extends across a broad phylogenic range, including vertebrates, insects and yeast (14). It is well documented that mammalian pol II is sensitive to α -amanitin at a low concentration of 2 µg/ml, while no significant inhibition of this enzyme by tagetitoxin was observed below 25 units of the latter. The transcription by pol III is inhibited by α-amanitin only at a level higher than 100 µg/ml, while it is highly sensitive to tagetitoxin. The available information on the sensitivities of insect pol II and pol III to tagetitoxin came mainly from the studies of silkworm (Bombyx mori) and Drosophila melanogaster (14). It has been reported that synthesis of U6 snRNA by insect pol III is resistant to relatively high (100 µg/ml) level of α-amanitin, and that tagetitoxin is a potent inhibitor of B. mori RNA pol III. In insect Spodoptera frugiperda
SF9 cells, transcription of a pol II gene (gp64) was strongly inhibited by 1 µg/ml of α-amanitin (15).
Nuclear splicing of eukaryotic precursor mRNA takes place in the spliceosome that is formed by an ordered assembly of snRNPs including U1, U2, U4/U6 and U5 (16-18). The snRNPs display a speckled or punctate intranuclear distribution in the form of aggregates (19-21). In HeLa cells, at least eight Sm proteins B’ (29 kD), b (28 kD), D1 (16 kD), D2 (16.5 kD), D3 (18 kD), E (12 kD), F (11 kD), and G (9 kD) form a core of the snRNPs (22). Many non-snRNP splicing factors are members of an evolutionarily conserved family of proteins that share a serine/arginine dipeptide-rich (SR) motif and known as the SR proteins (23, 24). Among them, SC35 is involved in the 5’ splice site selection of alternatively spliced pre-mRNAs (25), and colocalize with snRNPs in the nuclear speckled domains (26, 27). .
In this study, we determined the transcription machinery for PAT1 by recruiting inhibitors of either pol II or pol III to in vitro RNA runoff and runon experiments. PAT1 transcription showed similar sensitivities toward α-amanitin and tagetitoxin similar to that of U6 snRNA transcription, suggesting that PAT1 is transcribed by pol III. Furthermore, PAT1 is associated with the splicing factors by co-immunoprecipitation experiment using monoclonal antibodies (MAb) against snRNP splicing factor Sm and SC35. The loop region of PAT1 from nucleotide 1910 to 1916 was found complementary to the branch site recognition sequence of U2 snRNA.
三、研究成果
Characterization of the transcription of PAT1
-To understand how PAT1 is trasncribed in insect cells,
in vitro runoff transcription was performed using nuclear extracts prepared from TN368 cells. For inhibition study, α-amanitin or tagetitoxin was added to the runoff reaction mixture. The results showed that α-amanitin had no effect on the transcription of PAT1 at a low concentration (2 µg/ml) though an inhibitory effect was registered at a higher concentration (100 µg/ml) (Fig. 1A, lanes 2, 3). Tagetitoxin, however, reduced transcription of PAT1 dramatically at 10 U/reaction (Fig. 1A, lanes 4-7). A 502-bp fragment of the Drosophila U6 gene containing the U6 promoter was used as a control for RNA pol III-transcribed gene in insect cells. It is resistant to α-amanitin up to 100 µg/ml (Fig. 1B, lanes 1-3), but sensitive to tagetitoxin at 10 U/reaction. Synthesis of actin RNA was used as a control for pol II-transcribed genes which is sensitive to α-amanitin, but not affected by the presence of 10 U of tagetitoxin. These results suggest that the synthesis of PAT1 is most likely catalyzed by pol III.
PAT1 is transcribed by pol III in the nuclei of persistently infected cells - To confirm the above observation, PAT1 transcription was examined in intact nuclei. In vitro runon experiment was carried out using isolated nuclei from TNPC3 cells that express PAT1 at a high abundance (7). The isolated nuclei were preincubated with different concentrations of α-amanintin, and allowed to complete the already initiated transcription. The synthesized RNAs were then extracted and hybridized with various DNA fragments immobilized on the filters. The in vitro
runon synthesis of PAT1 had a similar lack of sensitivity to α-amanitin to that of U6 by that their syntheses were reduced at both low and high concentrations of α-amanitin. In vitro runon synthesis of actin RNA in these cells was very sensitive to α-amanitin. Thus these results support that PAT1 is synthesized by RNA pol III though its length (2.9 Kb) is much longer than the usual pol III-transcribed RNA species.
Interestingly, differential sensitivities of pol III catalyzed transcription toward α-amanitin were observed in the in vitro runoff and runon transcription assays in Fig. 2. This could be due to either the different cell lines examined between TN368 and TNPC3 cells, or the different assays used between the
in vitro runoff versus the in vitro runon assays. To clarify this, the in vitro runon synthesis of U6 was carried out using the nuclei isolated from mock infected TN368 cells. Similar to the in vitro runoff synthesis of U6 with nuclear extracts of TN368 cells, the in vitro runon synthesis of U6 with isolated TN368 nuclei was also resistant to α-amanitin up to 10 µg/ml. Thus, the elevated sensitivity of pol III activity of persistently infected TNPC3 cells toward α-amanitin observed above was likely due to the persistently infected cells.
Among the pol III-transcribed RNAs, various functions have been identified. Previously, it has been shown that PAT1 is localized in the nucleus and forms a speckle distribution that is similar to the U snRNAs
involved in the splicing events. We then analyzed whether PAT1 is associated with snRNPs splicing factors and/or non-snRNP splicing factor SC35.
PAT1 is associated with the ribonucleoprotein (RNP) - It has been shown that the snRNPs and non-snRNPs splicing factors are conserved through evolution (35, 36). To confirm whether the MAb anti-Sm and MAb SC35 that are monoclonal antibodies against human proteins can also recognize the nuclearspeckles in insect cells, immunostaining experiments were carried out with both TN368 and TNPC3 cells. Results showed that the MAb against human Sm and SC35 can react to the insect counterpart proteins and localized the nuclear speckles in insect cells.
To analyze whether PAT1 is associated with the snRNP complexes of the pre-mRNA splicing machinery, immunoprecipitation experiments were carried out. The results from RT-PCR show that PAT1 is coprecipitated with the snRNPs by MAb anti-Sm and MAb SC35in the persistently infected TNPC2 and TNPC3 cells. The signals in TNPC3 are stronger than that of TNPC2 reflecting that the PAT1 is more abundant in the TNPC3 cells. To serve as negative controls, no amplified DNA fragments were detected from IP with the protein G beads, and no amplified DNA fragments were detected when MAb anti-TNP was added. Half of the total RNA samples from TNPC2 and TNPC3 cells were used as templates for RT-PCR and run as a positive control. Furthermore, U6 snRNA is also detectable in the precipitate with both MAb anti-Sm) and MAb SC35. These results suggest that PAT1 interacts with the RNP complex of splicing factors containing U snRNAs, snRNPs and non-snRNP splicing factor SC35.
Complementarity between PAT1 and U2 snRNA - It is possible that the association of PAT1 with RNP complex is mediated by the complementary sequences between PAT1 and U snRNAs. To analyze this, the sequences of the U class snRNAs were examined by computer-assisted analysis to see whether there is a complementary region to PAT1. It is found that PAT1 is complementary to a region from nt 33 to nt 39 in U2 RNA responsible for binding the branch site of introns. The complementary region of PAT1 resides between nt 1910 to nt 1916 region forms a loop domain favoring the formation of hydrogen bond with the U2 snRNA).
四、討論
We show here that the noncoding, 2.9 kb PAT1 of Hz-1 virus is most likely transcribed by pol III despite that it has the hallmarks of both pol II and pol III (10). Among the pol III-transcribed genes, various types of promoters have been identified. Previously, it has been shown that the PAT1 gene contains both extragenic and intragenic promoters (10). The extragenic promoter of PAT1 is a typical promoter of pol II-transcribed genes including a TATA box and a CAAT box. The intragenic promoter of PAT1 is at the
nucleotide +9 to +29 region (10). However, our recent results of the site-directed mutagenesis of the intragenic promoter indicated that the intragenic promoter of PAT1 is dispensable for its transcription (unpublished data).
The promoters of RNA pol III-transcribed genes are categorized into three different types (37). Type-1 genes have an intragenic promoter with both box A and box C, such as the 5S rRNA coding gene (38). Type-2 genes have also an intragenic promoter but with both box A and box B, such as tRNA coding gene (39). Type-3 genes lack intragenic promoter, and their transcription are dependent on an extragenic promoter (40). U6, a typical example of type III genes, has a TATA sequence near position -30 and a proximal sequence element (PSE) at around position –60 (41). Thus, the promoter of PAT1 is more similar to that of U6 in which the nucleotide sequences required for its transcription lie in the region upstream from the transcription start site.
As more and more pol III-transcribed genes have been reported, variations in the promoter sequences required for pol III transcription are observed. The promoter of the Epstein-Barr-virus (EBV)-encoded RNA gene (EBER) contains both pol II and pol III hallmarks and yet it is transcribed by pol III (42). Maximal expression of EBER requires the internal A and B elements as well as an external TATA box at position -30, and binding sites for RNA pol II transcription factor SP1 and activating transcription factor (ATF) (31). In contrast, c-myc is an example of a gene with a promoter of pol II-transcribed genes, and yet its transcription can be initiated by both pol II and pol III (43). Transcription of c-myc by RNA pol III terminates near the end of exon 1 in which short stretches of T-residues are present (43). These results suggest that pol III can recognize various promoters for its transcriptionBesides PAT1, several viral noncoding RNAs have also been reported. They include EBERs of EBV, VA RNAs of adenovirus, and leader RNA of Varicella-zoster virus (VZV). EBER1 and EBER2 are small RNAs of 166 and 172 nucleotides respectively, and both are located in the nucleus of EBV latently infected cells (44, 45), and transcribed in large amounts (5 x 106 per cell each) (46, 47). EBERs are pol III-transcribed, uncapped but ended with a poly U tract, that is specifically recognized by the La antigen (48). Other viral small noncoding RNA including adenovirus VA RNA, HVP-1 and HVP-2 of herpesvirus papio (49), and the leader RNA of VZV can also bind to La (50). However, EBER is associated with a 15-kD ribosomal L22 protein within the nucleus (51).
Previously, it has been postulated that PAT1 functions in the establishing Hz-1 viral persistence (10). It is possible that PAT1 affects the cellular mRNA splicing process by association with the snRNPs. The disruption of cellular RNA splicing by association of viral RNA with RNPs has been observed for nut-1 of Kaposi’s sarcoma-associated herpesviers (KSHV, human herpesvirus 8) (34) and
HSURs of herpesvirus saimiri (HVS) (52, 53). The nut-1 of KSHV interacts with Sm antigen-containing RNPs. Besides, a high complementarity between nt 18 to nt 25 of U12 and nt 899 to nt 913 of nut-1 was identified, and that may affect the splicing of intron containing the AU/AC splicing sites (34). The U-like RNAs are synthesized during the infection of monkey lymphocytes by HVS. They contain the 5’-trimethylguanosine cap and are associated with the host Sm proteins (52, 53). It is likely that association between viral RNA and RNP could be a common approach adopted by viral persistent/latent RNA species to regulate host gene expression by affecting the cellular mRNA splicing events. In conclusion, the implication of these results is that the high abundance of pol III-transcribed PAT1 in the Hz-1 virus persistently infected cells may interfere with the cellular splicing process and this may affect the protein synthesis.
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