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The norovirus genome consists of a single positive-stranded RNA. The mechanism by which this single-stranded RNA genome is replicated is not well understood. To reveal the mechanism underlying the initiation of the norovirus genomic RNA synthesis by its RNA-dependent RNA polymerase (RdRp), we used an in vitro assay to detect the complementary RNA synthesis activity. Results showed that the purified recombinant RdRp was able to synthesize the complementary positive-sense RNA from a 100-nt template corresponding to the 3'-end of the viral antisense genome sequence, but that the RdRp could not synthesize the antisense genomic RNA from the template corresponding to the 5'-end of the positive-sense genome sequence. We also predicted that the 31 nt region at the 3'-end of the RNA antisense template forms a stem-loop structure. Deletion of this sequence resulted in the loss of complementary RNA synthesis by the RdRp, and connection of the 31 nt to the 3'-end of the inactive positive-sense RNA template resulted in the gain of complementary RNA synthesis by the RdRp. Similarly, an electrophoretic mobility shift assay further revealed that the RdRp bound to the antisense RNA specifically, but was dependent on the 31 nt at the 3'-end. Therefore, based on this observation and further deletion and mutation analyses, we concluded that the predicted stem-loop structure in the 31 nt end and the region close to the antisense viral genomic stem sequences are both important for initiating the positive-sense human norovirus genomic RNA synthesis by its RdRp., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The endonuclease Argonaute2 (Ago2) mediates the degradation of the target mRNA within the RNA-induced silencing complex. We determined the binding and cleavage properties of recombinant human Ago2. Human Ago2 was unable to cleave preformed RNA duplexes and exhibited weaker binding affinity for RNA duplexes compared with the single strand RNA. The enzyme exhibited greater RNase H activity in the presence of Mn2+ compared with Mg2+. Human Ago2 exhibited weaker binding affinities and reduced cleavage activities for antisense RNAs with either a 5'-terminal hydroxyl or abasic nucleotide. Binding kinetics suggest that the 5'-terminal heterocycle base nucleates the interaction between the enzyme and the antisense RNA, and the 5'-phosphate stabilizes the interaction. Mn2+ ameliorated the effects of the 5'-terminal hydroxyl or abasic nucleotide on Ago2 cleavage activity and binding affinity. Nucleotide substitutions at the 3' terminus of the antisense RNA had no effect on human Ago2 cleavage activity, whereas 2'-methoxyethyl substitutions at position 2 reduced binding and cleavage activity and 12-14 reduced the cleavage activity. RNase protection assays indicated that human Ago2 interacts with the first 14 nucleotides at the 5'-pole of the antisense RNA. Human Ago2 preloaded with the antisense RNA exhibited greater binding affinities for longer sense RNAs suggesting that the enzyme interacts with regions in the sense RNA outside the site for antisense hybridization. Finally, transiently expressed human Ago2 immunoprecipitated from HeLa cells contained the double strand RNA-binding protein human immunodeficiency virus, type 1, trans-activating response RNA-binding protein, and deletion mutants of Ago2 showed that trans-activating response RNA-binding protein interacts with the PIWI domain of the enzyme.

Neurofibrillary tangles containing filaments of the microtubule-associated protein tau are found in a variety of neurodegenerative diseases. Mutations in the tau gene itself cause frontotemporal dementia with parkinsonism, demonstrating the critical role of tau in pathogenesis. Many of these mutations in tau are silent, are found at the 5'-splice site of exon 10, and lead to increased inclusion of exon 10. These silent mutations are predicted to destabilize a stem loop structure at the exon 10 5'-splice site; however, the existence of this stem loop under physiological conditions and its role in splice regulation are controversial. Here we show that base changes that stabilize this stem loop in vitro substantially decrease exon 10 inclusion in a wild type tau minigene and rescue the increase in exon 10 splicing caused by a dementia-causing point mutation. Moreover, we probed the intracellular structure of the tau stem loop with antisense RNA and demonstrate that the stability of the stem loop dictates antisense effectiveness. Together these results validate the stem loop as a bona fide structure regulating tau exon 10 splicing.

The stem cell leukemia (SCL) transcription factor is essential for vertebrate hematopoiesis. Using the powerful zebrafish model for embryonic analysis, we compared the effects of either reducing or ablating Scl using morpholino-modified antisense RNAs. Ablation of Scl resulted in the loss of primitive and definitive hematopoiesis, consistent with its essential role in these processes. Interestingly, in embryos with severely reduced Scl levels, erythroid progenitors expressing gata1 and embryonic globin developed. Erythroid maturation was deficient in these Scl hypomorphs, supporting that Scl was required both for the erythroid specification and for the maturation steps, with maturation requiring higher Scl levels than specification. Although all hematopoietic functions were rescued by wild-type Scl mRNA, an Scl DNA binding mutant rescued primitive and definitive hematopoiesis but did not rescue primitive erythroid maturation. Together, we showed that there is a distinct Scl hypomorphic phenotype and demonstrated that distinct functions are required for the roles of Scl in the specification and differentiation of primitive and definitive hematopoietic lineages. Our results revealed that Scl participates in multiple processes requiring different levels and functions. Further, we identified an Scl hypomorphic phenotype distinct from the null state.

Cancer cells are more susceptible to chemotherapeutic agent-induced apoptosis than their normal counterparts. Although it has been demonstrated that the increased sensitivity results from deregulation of oncoproteins during cancer development (Evan, G. I., and Vousden, K. H. (2001) Nature 411, 342-348; Green, D. R., and Evan, G. I. (2002) Cancer Cell 1, 19-30), little is known about the signaling pathways leading to changes in the apoptotic threshold in cancer cells. Here we show that low RKIP expression levels in tumorigenic human prostate and breast cancer cells are rapidly induced upon chemotherapeutic drug treatment, sensitizing the cells to apoptosis. We show that the maximal RKIP expression correlates perfectly with the onset of apoptosis. In cancer cells resistant to DNA-damaging agents, treatment with the drugs does not up-regulate RKIP expression. However, ectopic expression of RKIP resensitizes DNA-damaging agent-resistant cells to undergo apoptosis. This sensitization can be reversed by up-regulation of survival pathways. Down-regulation of endogenous RKIP by expression of antisense and small interfering RNA (siRNA) confers resistance on sensitive cancer cells to anticancer drug-induced apoptosis. Our studies suggest that RKIP may represent a novel effector of signal transduction pathways leading to apoptosis and a prognostic marker of the pathogenesis of human cancer cells and tumors after treatment with clinically relevant chemotherapeutic drugs.

Staphylococcus aureus cause infections by producing toxins, a process regulated by cell-cell communication (quorum sensing) through the histidine-phosphorylation of the target of RNAIII-activating protein (TRAP). We show here that TRAP is highly conserved in staphylococci and contains three completely conserved histidine residues (His-66, His-79, His-154) that are phosphorylated and essential for its activity. This was tested by constructing a TRAP(-) strain with each of the conserved histidine residues changed to alanine by site-directed mutagenesis. All mutants were tested for pathogenesis in vitro (expression of RNAIII and hemolytic activity) and in vivo (murine cellulitis model). Results show that RNAIII is not expressed in the TRAP(-) strain, that it is non hemolytic, and that it does not cause disease in vivo. These pathogenic phenotypes could be rescued in the strain containing the recovered traP, confirming the importance of TRAP in S. aureus pathogenesis. The phosphorylation of TRAP mutated in any of the conserved histidine residues was significantly reduced, and mutants defective in any one of these residues were non-pathogenic in vitro or in vivo, whereas those mutated in a non-conserved histidine residue (His-124) were as pathogenic as the wild type. These results confirm the importance of the three conserved histidine residues in TRAP activity. The phosphorylation pattern, structure, and gene organization of TRAP deviates from signaling molecules known to date, suggesting that TRAP belongs to a novel class of signal transducers.

The binding pathway of the natural antisense RNA CopA to its target CopT proceeds through a hierarchical order of steps. It initiates by reversible loop-loop contacts followed by unidirectional helix progression into the upper stems. This involves extensive breakage of intramolecular base pairs and the subsequent formation of two intermolecular helices, B and B'. Based on the known tRNA anticodon loop structure and on results from the Sok/Hok antisense/target RNA system, it had been suggested that a U-turn (or pi-turn) in the loop of CopT might determine the directionality of helix progression. Data presented here show that the putative U-turn is one of the structural elements of antisense/target RNA pairs required to achieve fast binding kinetics. Swapping of the hypothetical U-turn structure from the target RNA to the antisense RNA retained regulatory performance in vivo and binding rates in vitro but altered the binding pathway by changing the direction in which the initiating helix was extended. In addition, our data indicate that a helical stem immediately adjacent to the target loop sequence is required to provide a scaffold for the U-turn.

We previously have described the use of an antisense RNA strategy termed random homozygous knock-out (RHKO) to identify negative regulators of cell proliferation. Here we report the discovery that RHKO-mediated deficiency of the nCL-4 calpain protease results in cellular transformation of and tumorigenesis by murine NIH3T3 fibroblasts. We isolated cell clones able to form colonies on 0.5% soft agar and found that these cells generated tumors when injected subcutaneously into nude mice. The gene inactivated by RHKO was identified as nCL-4 by genomic library screening, transcript analysis, and DNA sequencing. Anchorage-independent growth, as indicated by colony formation on soft agar, was reversed by reversal of antisense-mediated homozygous inactivation, but continued haplo-insufficiency of nCL-4 resulting from insertional mutagenesis of one nCL-4 allele was associated with persistent tumorigenesis. nCL-4 cDNA expressed in naive 3T3 cells in the antisense, but not sense, direction under control of the cytomegalovirus early promoter reproduced the anchorage-independent growth effects of RHKO. Our results implicate deficiency of the nCL-4 calpain protease in neoplastic transformation.

The RNase III family of endoribonucleases participates in maturation and decay of cellular and viral transcripts by processing of double-stranded RNA. RNase III degradation is inherent to most antisense RNA-regulated gene systems in Escherichia coli. In the hok/sok system from plasmid R1, Sok antisense RNA targets the hok mRNA for RNase III-mediated degradation. An intermediate in the pairing reaction between Sok RNA and hok mRNA forms a three-way junction. A complex between a chimeric antisense RNA and hok mRNA that mimics the three-way junction was cleaved by RNase III both in vivo and in vitro. Footprinting using E117A RNase III binding to partially complementary RNAs showed protection of the 13 base pairs of interstrand duplex and of the bottom part of the transcriptional terminator hairpin of the antisense RNA. This suggests that the 13 base pairs of RNA duplex are coaxially stacked on the antisense RNA terminator stem-loop and that each stem forms a monomer half-site, allowing symmetrical binding of the RNase III dimer. This processing scheme shows an unanticipated diversity in RNase III substrates and may have a more general implication for RNA metabolism.

Angiogenesis promotes growth and metastasis of tumor cells. In this study, we have developed two peptide antagonists of human angiogenin by deducing the codes from the antisense RNA sequence corresponding to the receptor-binding site of angiogenin in either 5' --> 3' (chANG) or 3' --> 5' (chGNA) direction. chANG and chGNA peptides bind to angiogenin with specificity and high affinity (Kd approximately 44 nM) and inhibit the interaction of angiogenin with actin, which is regarded as the angiogenin-binding protein on the surface of endothelial cells. The peptides inhibit the neovascularization induced by angiogenin in the chick chorioallantoic membrane assay. The anti-angiogenic activity of the peptides is specific for angiogenin, and the peptides do not have any apparent effect on embryonic angiogenesis or the preexisting blood vessels. chANG and chGNA also inhibit the angiogenesis induced by the angiogenin-secreting PC 3 human prostate adenocarcinoma cells and have no direct effect on the proliferation as well as the adhesion of PC 3 cells to angiogenin. Therefore, the inhibition of the tumor-induced angiogenesis by the peptides is most likely caused by neutralization of the extracellular angiogenin secreted by PC 3 cells. Based on our results, chANG and chGNA peptides may be effective for treatment of various human tumors which secrete angiogenin.