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Efficient and productive virus infection often requires viral countermeasures that block innate immunity. The IFN-inducible 2',5'-oligoadenylate (2-5A) synthetases (OASs) and ribonuclease (RNase) L are components of a potent host antiviral pathway. We previously showed that murine coronavirus (MHV) accessory protein ns2, a 2H phosphoesterase superfamily member, is a phosphodiesterase (PDE) that cleaves 2-5A, thereby preventing activation of RNase L. The PDE activity of ns2 is required for MHV replication in macrophages and for hepatitis. Here, we show that group A rotavirus (RVA), an important cause of acute gastroenteritis in children worldwide, encodes a similar PDE. The RVA PDE forms the carboxy-terminal domain of the minor core protein VP3 (VP3-CTD) and shares sequence and predicted structural homology with ns2, including two catalytic HxT/S motifs. Bacterially expressed VP3-CTD exhibited 2',5'-PDE activity, which cleaved 2-5A in vitro. In addition, VP3-CTD expressed transiently in mammalian cells depleted 2-5A levels induced by OAS activation with poly(rI):poly(rC), preventing RNase L activation. In the context of recombinant chimeric MHV expressing inactive ns2, VP3-CTD restored the ability of the virus to replicate efficiently in macrophages or in the livers of infected mice, whereas mutant viruses expressing inactive VP3-CTD (H718A or H798R) were attenuated. In addition, chimeric viruses expressing either active ns2 or VP3-CTD, but not nonfunctional equivalents, were able to protect ribosomal RNA from RNase L-mediated degradation. Thus, VP3-CTD is a 2',5'-PDE able to functionally substitute for ns2 in MHV infection. Remarkably, therefore, two disparate RNA viruses encode proteins with homologous 2',5'-PDEs that antagonize activation of innate immunity.
Many tumor cells express globally reduced levels of microRNAs (miRNA), suggesting that decreased miRNA expression in premalignant cells contributes to their tumorigenic phenotype. In support of this, Dicer, an RNase III-like enzyme that controls the maturation of miRNA, was recently shown to function as a haploinsufficient tumor suppressor in nonhematopoietic cells. Because the Myc oncoprotein, a critical inducer of B-cell lymphomas, was reported to suppress the expression of multiple miRNAs in lymphoma cells, it was presumed that a deficiency of Dicer and subsequent loss of miRNA maturation would accelerate Myc-induced lymphoma development. We report here that, surprisingly, a haploinsufficiency of Dicer in B cells failed to promote B-cell malignancy or accelerate Myc-induced B-cell lymphomagenesis in mice. Moreover, deletion of Dicer in B cells of CD19-cre(+)/Emicro-myc mice significantly inhibited lymphomagenesis, and all lymphomas that did arise in these mice lacked functional Cre expression and retained at least one functional Dicer allele. Uncharacteristically, the lymphomas that frequently developed in the CD19-cre(+)/Dicer(fl/fl)/Emicro-myc mice were of very early precursor B-cell origin, a stage of B-cell development prior to Cre expression. Therefore, loss of Dicer function was not advantageous for lymphomagenesis, but rather, Dicer ablation was strongly selected against during Myc-induced B-cell lymphoma development. Moreover, deletion of Dicer in established B-cell lymphomas resulted in apoptosis, revealing that Dicer is required for B-cell lymphoma survival. Thus, Dicer does not function as a haploinsufficient tumor suppressor in B cells and is required for B-cell lymphoma development and survival.
MicroRNAs are endogenous, noncoding, small RNAs that regulate expression and function of genes, but little is known about regulation of microRNA in the kidneys under normal or pathologic states. Here, we generated a mouse model in which the proximal tubular cells lack Dicer, a key enzyme for microRNA production. These mice had normal renal function and histology under control conditions despite a global downregulation of microRNAs in the renal cortex; however, these animals were remarkably resistant to renal ischemia-reperfusion injury (IRI), showing significantly better renal function, less tissue damage, lower tubular apoptosis, and improved survival compared with their wild-type littermates. Microarray analysis showed altered expression of specific microRNAs during renal IRI. Taken together, these results demonstrate evidence for a pathogenic role of Dicer and associated microRNAs in renal IRI.
Dicer initiates RNA interference by generating small RNAs involved in various silencing pathways. Dicer participates in centromeric silencing, but its role in the epigenetic regulation of other chromatin domains has not been explored. Here we show that Dicer1 deficiency in Mus musculus leads to decreased DNA methylation, concomitant with increased telomere recombination and telomere elongation. These DNA-methylation defects correlate with decreased expression of Dnmt1, Dnmt3a and Dnmt3b DNA methyltransferases (Dnmts), and methylation levels can be recovered by their overexpression. We identify the retinoblastoma-like 2 protein (Rbl2) as responsible for decreased Dnmt expression in Dicer1-null cells, suggesting the existence of Dicer-dependent small RNAs that target Rbl2. We identify the miR-290 cluster as being downregulated in Dicer1-deficient cells and show that it silences Rbl2, thereby controlling Dnmt expression. These results identify a pathway by which miR-290 directly regulates Rbl2-dependent Dnmt expression, indirectly affecting telomere-length homeostasis.
We have used the nuclease alpha-sarcin to map the binding sites of the 19-kDa and the 68/72-kDa proteins of signal recognition particle (SRP) on SRP RNA. We found that the regions of protection to nuclease afforded by the two proteins were distinct. p19 protected primarily the two tips in the RNA secondary structure. p68/72 protected a large region extending across the center of the particle and altered the nuclease pattern in the regions that p19 would bind, suggesting that these two proteins may be in close proximity in the particle. The protection afforded by the two proteins in combination was equal to the sum of the individual protections. We have not observed cooperativity in the binding of these two proteins as assessed by the protection assay; nor do we have any evidence that the structure becomes more compact as it assembles. The map derived from this "footprint" analysis places the signal recognition domain (p54 bound to the RNA via the 19-kDa protein) and the elongation arrest domain (associated with the Alu end of the particle) on opposite ends of the particle. Thus, it is possible that SRP recognizes signals by the direct interaction of p54 with the signal sequence at the nascent chain exit site and simultaneously blocks elongation by the entrance of p9/14 into the aminoacyl tRNA site 16 nm away.
Using subtractive hybridization to identify genes that are androgen regulated in the mouse epididymis, a number of cDNAs were identified that represented mitochondrial genes including cytochrome oxidase c subunits I, II, and III, cytochrome b, NADH dehydrogenase subunit 5, a region of the displacement loop, and the 16S rRNA. Northern blot analysis of RNA from intact, castrate, or testosterone-replaced epididymides confirmed that these mitochondrial mRNAs as well as the rRNA were androgen regulated with a 2- to 5-fold reduction in expression observed after 4 weeks castration with partial to full recovery to precastrate levels upon 4 weeks of testosterone replacement. In contrast to the mitochondrial genes, the expression of the RNA component of the mitochondrial RNA-processing endoribonuclease (RNAase MRP), a nuclear factor which is thought to be involved in the regulation of mitochondrial DNA synthesis, increased in the epididymis upon castration and then returned to precastrate levels after testosterone replacement. An examination of other androgen-responsive tissues showed that mitochondrial gene expression was also regulated by androgens in the kidney. The RNAase MRP RNA levels, however, showed an increase after castration only in the reproductive tissues (epididymis, vas deferens, and seminal vesicle) and not in the kidney. No correlative increase in mitochondrial DNA levels was observed for any of the tissues. Finally, an analysis of various mouse tissues as well as the different regions of the epididymis revealed large differences in mitochondrial mRNA levels. While for most tissues the mRNA levels correlated with the mitochondrial DNA content, the levels of the RNAase MRP RNA did not. Taken together, these findings not only show the large variations in mitochondrial gene expression between tissues but also demonstrate that the expression of mitochondrial genes and ultimately mitochondrial function are androgen regulated in the epididymis and kidney.