共查询到20条相似文献,搜索用时 15 毫秒
1.
Human adenoviruses (HAds) encode for one or two highly abundant virus-associated RNAs, designated VA RNAI and VA RNAII, which fold into stable hairpin structures resembling miRNA precursors. Here we show that the terminal stem of the VA RNAs originating from Ad4, Ad5, Ad11 and Ad37, all undergo Dicer dependent processing into virus-specific miRNAs (so-called mivaRNAs). We further show that the mivaRNA duplex is subjected to a highly asymmetric RISC loading with the 3′-strand from all VA RNAs being the favored strand, except for the Ad37 VA RNAII, where the 5′-mivaRNAII strand was preferentially assembled into RISC. Although the mivaRNA seed sequences are not fully conserved between the HAds a bioinformatics prediction approach suggests that a large fraction of the VA RNAII-, but not the VA RNAI-derived mivaRNAs still are able to target the same cellular genes. Using small RNA deep sequencing we demonstrate that the Dicer processing event in the terminal stem of the VA RNAs is not unique and generates 3′-mivaRNAs with a slight variation of the position of the 5′ terminal nucleotide in the RISC loaded guide strand. Also, we show that all analyzed VA RNAs, except Ad37 VA RNAI and Ad5 VA RNAII, utilize an alternative upstream A start site in addition to the classical +1 G start site. Further, the 5′-mivaRNAs with an A start appears to be preferentially incorporated into RISC. Although the majority of mivaRNA research has been done using Ad5 as the model system our analysis demonstrates that the mivaRNAs expressed in Ad11- and Ad37-infected cells are the most abundant mivaRNAs associated with Ago2-containing RISC. Collectively, our results show an unexpected variability in Dicer processing of the VA RNAs and a serotype-specific loading of mivaRNAs into Ago2-based RISC. 相似文献
2.
Adenovirus virus-associated RNAII-derived small RNAs are efficiently incorporated into the rna-induced silencing complex and associate with polyribosomes 总被引:1,自引:1,他引:0
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Adenovirus type 5 encodes two highly structured short RNAs, the virus-associated (VA) RNAI and RNAII. Both are processed by Dicer into small RNAs that are incorporated into the RNA-induced silencing complex (RISC). We show here, by cloning of small RNAs, that approximately 80% of Ago2-containing RISC immunopurified from late-infected cells is associated with VA RNA-derived small RNAs (mivaRNAs). Most surprisingly, VA RNAII, which is expressed at 20-fold lower levels compared to that of VA RNAI, appears to be the preferred substrate for Dicer and accounts for approximately 60% of all small RNAs in RISC. The mivaRNAs are derived from the 3' strand of the terminal stems of the VA RNAs, with the major fraction of VA RNAII starting at position 138. The small RNAs derived from VA RNAI were more heterogeneous in size, with the two predominant small RNAs starting at positions 137 and 138. Collectively, our results suggest that the mivaRNAs are efficiently used for RISC assembly in late-infected cells. Potentially, they function as miRNAs, regulating translation of cellular mRNAs. In support of this hypothesis, we detected a fraction of the VA RNAII-derived mivaRNAs on polyribosomes. 相似文献
3.
4.
Andersson MG Haasnoot PC Xu N Berenjian S Berkhout B Akusjärvi G 《Journal of virology》2005,79(15):9556-9565
We show that human adenovirus inhibits RNA interference (RNAi) at late times of infection by suppressing the activity of two key enzyme systems involved, Dicer and RNA-induced silencing complex (RISC). To define the mechanisms by which adenovirus blocks RNAi, we used a panel of mutant adenoviruses defective in virus-associated (VA) RNA expression. The results show that the virus-associated RNAs, VA RNAI and VA RNAII, function as suppressors of RNAi by interfering with the activity of Dicer. The VA RNAs bind Dicer and function as competitive substrates squelching Dicer. Further, we show that VA RNAI and VA RNAII are processed by Dicer, both in vitro and during a lytic infection, and that the resulting short interfering RNAs (siRNAs) are incorporated into active RISC. Dicer cleaves the terminal stem of both VA RNAI and VA RNAII. However, whereas both strands of the VA RNAI-specific siRNA are incorporated into RISC, the 3' strand of the VA RNAII-specific siRNA is selectively incorporated during a lytic infection. In summary, our work shows that adenovirus suppresses RNAi during a lytic infection and gives insight into the mechanisms of RNAi suppression by VA RNA. 相似文献
5.
Characterization of a low-molecular-weight virus-associated (VA) RNA encoded by simian adenovirus type 7 which functionally can substitute for adenovirus type 5 VA RNAI. 总被引:6,自引:4,他引:2
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Human adenoviruses (Ads), like Ad type 2 (Ad2) and Ad5, encode a low-molecular-weight RNA (designated virus-associated [VA] RNAI) which is required for the efficient translation of viral mRNAs late after infection. We cloned and characterized a VA RNA gene from simian adenovirus type 7 (SA7) which appears to have biological activity analogous to that of Ad2 VA RNAI. Thus, SA7 VA RNA stimulates protein synthesis in a transient expression assay and can also functionally substitute for VA RNAI during lytic growth of human Ad5. The SA7 genome encodes only one VA RNA species, in contrast to human Ad2, which encodes two distinct species. This RNA is transcribed by RNA polymerase III in the rightward direction from a gene located at about coordinate 30 on the viral genome, like its Ad2 counterparts. SA7 VA RNA shows only a limited primary sequence homology with the Ad2 VA RNAs (approximately 55%); the flanking sequences, in fact, are better conserved than the VA RNA gene itself. The predicted secondary structure of SA7 VA RNA is, however, very similar to that of Ad2 VA RNAI, inferring that the double-stranded nature rather than the primary sequence of VA RNA is important for its biological activity. 相似文献
6.
7.
8.
9.
G Akusjrvi 《Journal of virology》1985,56(3):879-886
10.
Control of ColE1 replication: low affinity specific binding of Rop (Rom) to RNAI and RNAII. 总被引:9,自引:3,他引:6
下载免费PDF全文
![点击此处可从《The EMBO journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
We have studied the interactions between the three molecules Rop, RNAI and RNAII that are involved in the regulatory mechanism controlling the replication of ColE1 plasmids. We show that it is possible to purify the two RNA molecules by passing an RNA mixture through an affinity column containing Rop immobilized to a solid support. The dissociation constants of the Rop-RNAI and Rop-RNAII complexes are of the order of 10(-4) M, several orders of magnitude higher than dissociation constants of stable protein-nucleic acid complexes (10(-10) M in the lambda repressor system). Although complete RNAI molecules have higher affinity, stem-and-loop I alone can also bind Rop, suggesting that this structure plays an important role in the interaction. Rop protects the stems of RNAI and RNAII from digestion by RNases while the sensitivity of the loops to digestion by RNase T1 is not affected by high concentrations of Rop. We propose a model for Rop-RNAI/RNAII interaction in which the dimeric protein acts as an adaptor between stem structures to position the two RNAs in the correct position for loop interaction. 相似文献
11.
12.
Removal of double-stranded contaminants from RNA transcripts: synthesis of adenovirus VA RNAI from a T7 vector 总被引:12,自引:4,他引:8
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
K H Mellits T Pe''''ery L Manche H D Robertson M B Mathews 《Nucleic acids research》1990,18(18):5401-5406
Bacteriophage RNA polymerases are widely used to synthesize defined RNAs on a large scale in vitro. Unfortunately, the RNA product contains a small proportion of contaminating RNAs, including complementary species, which can lead to errors of interpretation. We cloned the gene encoding Ad2 VA RNAI into a vector containing a T7 RNA polymerase promoter in order to generate large quantities of VA RNA for the study of its interaction with the dsRNA-dependent protein kinase DAI. Exact copies of VA RNAI were synthesized efficiently, but were contaminated with small amounts of dsRNA which activated DAI and confounded interpretation of kinase assays. We therefore developed a method to remove the dsRNA contaminants, allowing VA RNAI and mutants to be tested for their ability to activate or inhibit DAI. This method appears to be generally applicable. 相似文献
13.
The rate of processing and degradation of antisense RNAI regulates the replication of ColE1-type plasmids in vivo. 总被引:16,自引:0,他引:16
We show that the rate of degradation of RNAI, an anti-sense repressor of the replication primer RNAII, is a key element of control in the replication of ColE1-type plasmids in vivo. Cleavage of RNAI by RNAase E, a ribosomal RNA-processing enzyme encoded or controlled by the rne (also known as ams) locus, relieves repression by endonucleolytically converting RNAI to a very rapidly decaying product, pRNAI-5. A 5' triphosphate-terminated homolog of pRNAI-5 is degraded slowly and consequently inhibits replication. Nucleotide substitutions within the RNAase E cleavage sequence alter RNAI half-life and plasmid copy number, changing also the incompatibility phenotype. RNAI variants lacking the sequence cleaved by RNAase E are eliminated by growth rate-dependent degradation, resulting in growth-responsive control of plasmid replication and copy number. 相似文献
14.
La antigen recognizes and binds to the 3''-oligouridylate tail of a small RNA. 总被引:30,自引:8,他引:22
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The La antigen is a cellular protein which interacts with many RNA species that are products of RNA polymerase III, including the adenovirus virus-associated (VA) RNAs. We demonstrate that the efficiency of antigen binding in vitro is determined by the number of U residues at the RNA 3' terminus. Forms of VA RNAI with more than two terminal U residues are fully bound, forms with two U residues are partially bound, and forms with fewer than two U residues are not bound at all. The antigen can be covalently linked to VA RNA by UV irradiation, and the site of cross-linking is shown to contain the 3' terminus of the RNA. We conclude that the antigen recognizes the U-rich 3' tail of VA RNA, and presumably that of other polymerase III products, and that it binds at or close to this site. 相似文献
15.
16.
Replication of ColE1-type plasmids is regulated by RNAI, an antisense RNA that interacts with the replication pre-primer, RNAII. Exonucleolytic attack at the 3' end of RNAI is impeded in pcnB mutant bacteria, which lack poly(A) polymerase I-the principal RNA polyadenylase of E. coli; this leads to accumulation of an RNAI decay intermediate (RNAI(-5)) and dramatic reduction of the plasmid copy number. Here, we report that polyadenylation can also affect RNAI-mediated control of plasmid DNA replication by inhibiting interaction of RNAI(-5) with RNAII. We show that mutation of the host pcnB gene profoundly affects the plasmid copy number, even under experimental conditions that limit the effects of polyadenylation on RNAI(-5) decay. Moreover, poly(A) tails interfere with RNAI/RNAII interaction in vitro without producing any detectable alteration of RNAI secondary structure. Our results establish the existence of a previously undetected mechanism by which RNA polyadenylation can control plasmid copy number. 相似文献
17.
18.
A mechanism for the control of protein synthesis by adenovirus VA RNAI 总被引:55,自引:0,他引:55
19.