Structural improvement of LidNA: delta-type LidNA is a potent miRNA inhibitor constructed with unmodified DNA

ABSTRACT

Many miRNA inhibitors have been developed, including chemically modified oligonucleotides, such as 2′-O-methylated RNA and locked nucleic acid (LNA). Unmodified DNA has not yet been reported as a miRNA inhibitor due to relatively low DNA/miRNA binding affinity. We designed a structured DNA, LidNA, which was constructed with unmodified DNA, consisting of a complementary sequence to the target miRNA flanked by two structured DNA regions, such as double-stranded DNA. LidNA inhibited miRNA activity more potently than 2′-O-methylated RNA or LNA. To optimize LidNA, two double-stranded regions were joined, causing the molecule to assume a delta-like shape, which we termed delta-type LidNA. Delta-type LidNAs were developed to target endogenous and exogenous miRNAs, and exhibited potent miRNA inhibitory effects with a duration of at least 10 days. Delta-type LidNA-21, which targeted miR-21, inhibited the growth of cancer cell lines. This newly developed LidNA could contribute to miRNA studies across multiple fields.

Abbreviations: LidNA: DNA that puts a lid on miRNA function; LNA: locked nucleic acid; 3′-UTR: 3′-untranslated regions; RISC: RNA-induced silencing complex; MBL: Molecular beacon-like LidNA; YMBL: Y-type molecular beacon-like LidNA; TDMD: target-directed microRNA degradation.     

The RdgC protein employs a novel mechanism involving a finger domain to bind to circular DNA

The DNA-binding protein RdgC has been identified as an inhibitor of RecA-mediated homologous recombination in Escherichia coli. In Neisseria species, RdgC also has a role in virulence-associated antigenic variation. We have previously solved the crystal structure of the E. coli RdgC protein and shown it to form a toroidal dimer. In this study, we have conducted a mutational analysis of residues proposed to mediate interactions at the dimer interfaces. We demonstrate that destabilizing either interface has a serious effect on in vivo function, even though a stable complex with circular DNA was still observed. We conclude that tight binding is required for inhibition of RecA activity. We also investigated the role of the RdgC finger domain, and demonstrate that it plays a crucial role in the binding of circular DNA. Together, these data allow us to propose a model for how RdgC loads onto DNA. We discuss how RdgC might inhibit RecA-mediated strand exchange, and how RdgC might be displaced by other DNA metabolism enzymes such as polymerases and helicases.     

Cooperative interplay of let-7 mimic and HuR with MYC RNA

Both RNA-binding proteins (RBP) and miRNA play important roles in the regulation of mRNA expression, often acting together to regulate a target mRNA. In some cases the RBP and miRNA have been reported to act competitively, but in other instances they function cooperatively. Here, we investigated HuR function as an enhancer of let-7-mediated translational repression of c-Myc despite the separation of their binding sites. Using an in vitro system, we determined that a let-7 mimic, consisting of single-stranded (ss)DNA complementary to the let-7 binding site, enhanced the affinity of HuR for a 122-nt MYC RNA encompassing both binding sites. This finding supports the biophysical principle of cooperative binding by an RBP and miRNA purely through interactions at distal mRNA binding sites.     

Inhibiting plant microRNA activity: molecular SPONGEs,target MIMICs and STTMs all display variable efficacies against target microRNAs

Elucidation of microRNA (miRNA) function through a loss‐of‐function approach has proven difficult due to extensive genetic redundancy among most plant and animal miRNA families. Consequently, miRNA decoy technologies such as target MIMICs (MIMs) and short tandem target MIMICs (STTMs) in plants or molecular SPONGEs (SPs) in animals have been developed to generate loss‐of‐function phenotypes by perturbing endogenous miRNA activity. To test whether SPs can inhibit plant miRNA activity, synthetic SP transgenes containing multiple miRNA binding sites targeting different Arabidopsis miRNA families were generated. Additionally, their silencing efficacies were compared to the corresponding MIM and STTM transgenes via scoring the frequency and severity of phenotypic abnormalities elicited by each transgene. While SPs with wild‐type miRNA binding sites have no apparent impact, SPs containing miRNA binding sites with two central mismatches (cmSPs) can generate strong loss‐of‐function phenotypes. However, their efficacy varied dramatically, from inducing strong loss‐of‐function phenotypes to failing to produce any phenotypic impact. Variability was also observed when MIMs and STTMs were compared to cmSPs. While cmSP165/166 and STTM165/166 showed a stronger efficacy than MIM165/166, MIM159 was stronger than cmSP159 and STTM159. Although increasing the number of miRNA binding sites or strengthening the free energy of the miRNA binding site interaction can improve decoy efficacy, clearly additional unknown overriding factors are at play. In conclusion, we demonstrate that no one approach guarantees the strongest miRNA inhibition, but rather distinct miRNA families respond differently to the various approaches, suggesting that multiple approaches may need to be taken to generate the desired loss‐of‐function outcome.     

A PCR-Based Method to Construct Lentiviral Vector Expressing Double Tough Decoy for miRNA Inhibition

DNA vector-encoded Tough Decoy (TuD) miRNA inhibitor is attracting increased attention due to its high efficiency in miRNA suppression. The current methods used to construct TuD vectors are based on synthesizing long oligonucleotides (~90 mer), which have been costly and problematic because of mutations during synthesis. In this study, we report a PCR-based method for the generation of double Tough Decoy (dTuD) vector in which only two sets of shorter oligonucleotides (< 60 mer) were used. Different approaches were employed to test the inhibitory potency of dTuDs. We demonstrated that dTuD is the most efficient method in miRNA inhibition in vitro and in vivo. Using this method, a mini dTuD library against 88 human miRNAs was constructed and used for a high-throughput screening (HTS) of AP-1 pathway-related miRNAs. Seven miRNAs (miR-18b-5p, -101-3p, -148b-3p, -130b-3p, -186-3p, -187-3p and -1324) were identified as candidates involved in AP-1 pathway regulation. This novel method allows for an accurate and cost-effective generation of dTuD miRNA inhibitor, providing a powerful tool for efficient miRNA suppression in vitro and in vivo.     

Staphylococcus aureus protein SAUGI acts as a uracil-DNA glycosylase inhibitor

DNA mimic proteins are unique factors that control the DNA binding activity of target proteins by directly occupying their DNA binding sites. The extremely divergent amino acid sequences of the DNA mimics make these proteins hard to predict, and although they are likely to be ubiquitous, to date, only a few have been reported and functionally analyzed. Here we used a bioinformatic approach to look for potential DNA mimic proteins among previously reported protein structures. From ∼14 candidates, we selected the Staphylococcus conserved hypothetical protein SSP0047, and used proteomic and structural approaches to show that it is a novel DNA mimic protein. In Staphylococcus aureus, we found that this protein acts as a uracil-DNA glycosylase inhibitor, and therefore named it S. aureus uracil-DNA glycosylase inhibitor (SAUGI). We also determined and analyzed the complex structure of SAUGI and S. aureus uracil-DNA glycosylase (SAUDG). Subsequent BIAcore studies further showed that SAUGI has a high binding affinity to both S. aureus and human UDG. The two uracil-DNA glycosylase inhibitors (UGI and p56) previously known to science were both found in Bacillus phages, and this is the first report of a bacterial DNA mimic that may regulate SAUDG’s functional roles in DNA repair and host defense.     

Contributions of ubiquitin- and PCNA-binding domains to the activity of Polymerase eta in Saccharomyces cerevisiae

Bypassing of DNA lesions by damage-tolerant DNA polymerases depends on the interaction of these enzymes with the monoubiquitylated form of the replicative clamp protein, PCNA. We have analyzed the contributions of ubiquitin and PCNA binding to damage bypass and damage-induced mutagenesis in Polymerase η (encoded by RAD30) from the budding yeast Saccharomyces cerevisiae. We report here that a ubiquitin-binding domain provides enhanced affinity for the ubiquitylated form of PCNA and is essential for in vivo function of the polymerase, but only in conjunction with a basal affinity for the unmodified clamp, mediated by a conserved PCNA interaction motif. We show that enhancement of the interaction and function in damage tolerance does not depend on the ubiquitin attachment site within PCNA. Like its mammalian homolog, budding yeast Polymerase η itself is ubiquitylated in a manner dependent on its ubiquitin-binding domain.     

The Upregulation of Genomic Imprinted DLK1-Dio3 miRNAs in Murine Lupus Is Associated with Global DNA Hypomethylation

Epigenetic factors such as DNA methylation and microRNAs (miRNAs) are now increasingly recognized as vital contributors to lupus etiology. In this study, we investigated the potential interaction of these two epigenetic factors in lupus-prone MRL-lpr mice. We recently reported dysregulated expression of miRNAs in splenocytes of MRL-lpr mice. Here, we report that a majority of the upregulated miRNAs in MRL-lpr mice is located at the genomic imprinted DLK1-Dio3 domain. Further, we show a differential magnitude of upregulation of DLK1-Dio3 miRNA cluster in purified splenic CD4⁺ T, CD19⁺ B, and splenic CD4^-CD19^- cells from MRL-lpr lupus mice when compared to control MRL mice. MRL-lpr splenocytes (especially CD19⁺ and CD4^-CD19^- subsets) were hypomethylated compared to cells from control, MRL mice. We further show that deliberate demethylation of splenocytes from control MRL mice, but not from MRL-lpr lupus mice, with specific DNA methylation inhibitor 5-Aza-2’-deoxycytidine significantly augmented DLK1-Dio3 miRNAs expression. These findings strongly indicate that the upregulation of DLK1-Dio3 miRNAs in lupus splenic cell subsets is associated with reduced global DNA methylation levels in lupus cells. There was a differential upregulation of DLK-Dio3 miRNAs among various demethylated splenic cell subsets, which implies varied sensitivity of DLK1-Dio3 miRNA cluster in these cell subsets to DNA hypomethylation. Finally, inhibition of select DLK1-Dio3 miRNA such as miR-154, miR-379 and miR-300 with specific antagomirs significantly reduced the production of lupus-relevant IFNγ, IL-1β, IL-6, and IL-10 in lipopolysaccharide (LPS) activated splenocytes from MRL-lpr mice. Our study is the first to show that DNA methylation regulates genomic imprinted DLK1-Dio3 miRNAs in autoimmune lupus, which suggests a connection of DNA methylation, miRNA and genomic imprinting in lupus pathogenesis.     

The C-Terminal Part of Microcin B Is Crucial for DNA Gyrase Inhibition and Antibiotic Uptake by Sensitive Cells

Microcin B (McB) is a ribosomally synthesized antibacterial peptide. It contains up to nine oxazole and thiazole heterocycles that are introduced posttranslationally and are required for activity. McB inhibits the DNA gyrase, a validated drug target. Previous structure-activity analyses indicated that two fused heterocycles located in the central part of McB are important for antibacterial action and gyrase inhibition. Here, we used site-specific mutagenesis of the McB precursor gene to assess the functional significance of the C-terminal part of McB that is located past the second fused heterocycle and contains two single heterocycles as well as an unmodified four-amino-acid C-terminal tail. We found that removal of unmodified C-terminal amino acids of McB, while having no effect on fused heterocycles, has a very strong negative effect on activity in vivo and in vitro. In fact, even nonconservative point substitutions in the last McB amino acid have a very strong effect by simultaneously decreasing uptake and ability to inhibit the gyrase. The results highlight the importance of unmodified McB amino acids for function and open the way for creation of recombinant McB derivatives with an altered or expanded spectrum of antibacterial action.     

Mouse apolipoprotein B editing complex 3 (APOBEC3) is expressed in germ cells and interacts with dead-end (DND1)

Background

The dead-end (Dnd1) gene is essential for maintaining the viability of germ cells. Inactivation of Dnd1 results in sterility and testicular tumors. The Dnd1 encoded protein, DND1, is able to bind to the 3′-untranslated region (UTR) of messenger RNAs (mRNAs) to displace micro-RNA (miRNA) interaction with mRNA. Thus, one function of DND1 is to prevent miRNA mediated repression of mRNA. We report that DND1 interacts specifically with APOBEC3. APOBEC3 is a multi-functional protein. It inhibits retroviral replication. In addition, recent studies show that APOBEC3 interacts with cellular RNA-binding proteins and to mRNA to inhibit miRNA-mediated repression of mRNA.

Methodology/Principal Findings

Here we show that DND1 specifically interacts with another cellular protein, APOBEC3. We present our data which shows that DND1 co-immunoprecipitates APOBEC3 from mammalian cells and also endogenous APOBEC3 from mouse gonads. Whether the two proteins interact directly remains to be elucidated. We show that both DND1 and APOBEC3 are expressed in germ cells and in the early gonads of mouse embryo. Expression of fluorescently-tagged DND1 and APOBEC3 indicate they localize to the cytoplasm and when DND1 and APOBEC3 are expressed together in cells, they sequester near peri-nuclear sites.

Conclusions/Significance

The 3′-UTR of mRNAs generally encode multiple miRNA binding sites as well as binding sites for a variety of RNA binding proteins. In light of our findings of DND1-APOBEC3 interaction and taking into consideration reports that DND1 and APOBEC3 bind to mRNA to inhibit miRNA mediated repression, our studies implicate a possible role of DND1-APOBEC3 interaction in modulating miRNA-mediated mRNA repression. The interaction of DND1 and APOBEC3 could be one mechanism for maintaining viability of germ cells and for preventing germ cell tumor development.     

A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro

Mechanisms that allow replicative DNA polymerases to attain high processivity are often specific to a given polymerase and cannot be generalized to others. Here we report a protein engineering-based approach to significantly improve the processivity of DNA polymerases by covalently linking the polymerase domain to a sequence non-specific dsDNA binding protein. Using Sso7d from Sulfolobus solfataricus as the DNA binding protein, we demonstrate that the processivity of both family A and family B polymerases can be significantly enhanced. By introducing point mutations in Sso7d, we show that the dsDNA binding property of Sso7d is essential for the enhancement. We present evidence supporting two novel conclusions. First, the fusion of a heterologous dsDNA binding protein to a polymerase can increase processivity without compromising catalytic activity and enzyme stability. Second, polymerase processivity is limiting for the efficiency of PCR, such that the fusion enzymes exhibit profound advantages over unmodified enzymes in PCR applications. This technology has the potential to broadly improve the performance of nucleic acid modifying enzymes.