Eukaryotic RNases H1 act processively by interactions through the duplex RNA-binding domain

Ribonucleases H have mostly been implicated in eliminating short RNA primers used for initiation of lagging strand DNA synthesis. Escherichia coli RNase HI cleaves these RNA–DNA hybrids in a distributive manner. We report here that eukaryotic RNases H1 have evolved to be processive enzymes by attaching a duplex RNA-binding domain to the RNase H region. Highly conserved amino acids of the duplex RNA-binding domain are required for processivity and nucleic acid binding, which leads to dimerization of the protein. The need for a processive enzyme underscores the importance in eukaryotic cells of processing long hybrids, most of which remain to be identified. However, long RNA–DNA hybrids formed during immunoglobulin class-switch recombination are potential targets for RNase H1 in the nucleus. In mitochondria, where RNase H1 is essential for DNA formation during embryogenesis, long hybrids may be involved in DNA replication

Selective inhibition of HIV-1 reverse transcriptase-associated ribonuclease H activity by hydroxylated tropolones

High-throughput screening of a National Cancer Institute library of pure natural products identified the hydroxylated tropolone derivatives β-thujaplicinol (2,7-dihydroxy-4-1(methylethyl)-2,4,6-cycloheptatrien-1-one) and manicol (1,2,3,4-tetrahydro-5-7-dihydroxy-9-methyl-2-(1-methylethenyl)-6H-benzocyclohepten-6-one) as potent and selective inhibitors of the ribonuclease H (RNase H) activity of human immunodeficiency virus-type 1 reverse transcriptase (HIV-1 RT). β-Thujaplicinol inhibited HIV-1 RNase H in vitro with an IC(50) of 0.2 μM, while the IC(50) for Escherichia coli and human RNases H was 50 μM and 5.7 μM, respectively. In contrast, the related tropolone analog β-thujaplicin (2-hydroxy-4-(methylethyl)-2,4,6-cycloheptatrien-1-one), which lacks the 7-OH group of the heptatriene ring, was inactive, while manicol, which possesses a 7-OH group, inhibited HIV-1 and E.coli RNases H with IC(50) = 1.5 μM and 40 μM, respectively. Such a result highlights the importance of the 2,7-dihydroxy function of these tropolone analogs, possibly through a role in metal chelation at the RNase H active site. Inhibition of HIV-2 RT-associated RNase H indirectly indicates that these compounds do not occupy the nonnucleoside inhibitor-binding pocket in the vicinity of the DNA polymerase domain. Both β-thujaplicinol and manicol failed to inhibit DNA-dependent DNA polymerase activity of HIV-1 RT at a concentration of 50 μM, suggesting that they are specific for the C-terminal RNase H domain, while surface plasmon resonance studies indicated that the inhibition was not due to intercalation of the analog into the nucleic acid substrate. Finally, we have demonstrated synergy between β-thujaplicinol and calanolide A, a nonnucleoside inhibitor of HIV-1 RT, raising the possibility that both enzymatic activities of HIV-1 RT can be simultaneously targeted

Selective inhibitory DNA aptamers of the human RNase H1

Human RNase H1 binds double-stranded RNA via its N-terminal domain and RNA–DNA hybrid via its C-terminal RNase H domain, the latter being closely related to Escherichia coli RNase HI. Using SELEX, we have generated a set of DNA sequences that can bind efficiently (K(d) values ranging from 10 to 80 nM) to the human RNase H1. None of them could fold into a simple perfect double-stranded DNA hairpin confirming that double-stranded DNA does not constitute a trivial ligand for the enzyme. Only two of the 37 DNA aptamers selected were inhibitors of human RNase H1 activity. The two inhibitory oligomers, V-2 and VI-2, were quite different in structure with V-2 folding into a large, imperfect but stable hairpin loop. The VI-2 structure consists of a central region unimolecular quadruplex formed by stacking of two guanine quartets flanked by the 5′ and 3′ tails that form a stem of six base pairs. Base pairing between the 5′ and 3′ tails appears crucial for conferring the inhibitory properties to the aptamer. Finally, the inhibitory aptamers were capable of completely abolishing the action of an antisense oligonucleotide in a rabbit reticulocyte lysate supplemented with human RNase H1, with IC(50) ranging from 50 to 100 nM

La-Related Protein 4 Binds Poly(A), Interacts with the Poly(A)-Binding Protein MLLE Domain via a Variant PAM2w Motif, and Can Promote mRNA Stability▿†

The conserved RNA binding protein La recognizes UUU-3′OH on its small nuclear RNA ligands and stabilizes them against 3′-end-mediated decay. We report that newly described La-related protein 4 (LARP4) is a factor that can bind poly(A) RNA and interact with poly(A) binding protein (PABP). Yeast two-hybrid analysis and reciprocal immunoprecipitations (IPs) from HeLa cells revealed that LARP4 interacts with RACK1, a 40S ribosome- and mRNA-associated protein. LARP4 cosediments with 40S ribosome subunits and polyribosomes, and its knockdown decreases translation. Mutagenesis of the RNA binding or PABP interaction motifs decrease LARP4 association with polysomes. Several translation and mRNA metabolism-related proteins use a PAM2 sequence containing a critical invariant phenylalanine to make direct contact with the MLLE domain of PABP, and their competition for the MLLE is thought to regulate mRNA homeostasis. Unlike all ∼150 previously analyzed PAM2 sequences, LARP4 contains a variant PAM2 (PAM2w) with tryptophan in place of the phenylalanine. Binding and nuclear magnetic resonance (NMR) studies have shown that a peptide representing LARP4 PAM2w interacts with the MLLE of PABP within the affinity range measured for other PAM2 motif peptides. A cocrystal of PABC bound to LARP4 PAM2w shows tryptophan in the pocket in PABC-MLLE otherwise occupied by phenylalanine. We present evidence that LARP4 expression stimulates luciferase reporter activity by promoting mRNA stability, as shown by mRNA decay analysis of luciferase and cellular mRNAs. We propose that LARP4 activity is integrated with other PAM2 protein activities by PABP as part of mRNA homeostasis

RNases H1 surface plasmon resonance analysis () RNases H1 of mouse, human, and sequence alignment are shown

<p><b>Copyright information:</b></p><p>Taken from "Eukaryotic RNases H1 act processively by interactions through the duplex RNA-binding domain"</p><p>Nucleic Acids Research 2005;33(7):2166-2175.</p><p>Published online 14 Apr 2005</p><p>PMCID:PMC1079969.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Numbering refers to the mouse protein. Mouse and human RNases H1 mitochondrial localization signals are underlined. The methionine at the start of the protein used in these studies is marked by an inverted solid triangle and amino acids W43, K59 and K60, marked by an asterisk, are the amino acids that were changed to A for RNase H1, RNase H1 and RNase H1. RNase H (RNase H domain, gold background) starts at amino acid 137 and ends at amino acid 285. The dsRHbd sequences are on a green background with the positions of the α helices (red boxes) and β strands (blue boxes) of RNase H1 first dsRHbd noted below the dsRHbd sequences. For the sequence, amino acids in α helices are red and β strands are blue. Increase in protein bound to duplex RNAs as a function of protein concentration. Some of the sensograms from which these data were obtained are presented in Supplementary Figure S1. Wild-type mouse RNase H1 data are represented by a solid black circle, RNase HI by a solid black square, RNase H1 by an inverted solid black triangle, RNase H1 by an open circle, RNase H1 by an inverted open triangle and RNase H1 by a solid black diamond. () and () are from data collected with the 12 bp RNA–DNA hairpin hybrid while the surface in () is a 12 bp RNA–RNA hairpin duplex RNA of the same sequence as the 12 bp RNA–DNA hybrid. The lack of binding of mouse RNase H1 to the dsRNA at 10 mM MgCl is indicated in (d) by the open square. Nucleic acid sequences of the hairpin duplexes are shown above in (b–d) with RNA in lower case and DNA in upper case letters. Bio-T indicates the biotin modified dT to which the nucleic acid was attached to the streptavidin on the chip surface

Yonetani–Theorell plot for the inhibition of HIV-1 RT in the presence of the NNRTI calanolide A and β-thujaplicinol

<p><b>Copyright information:</b></p><p>Taken from "Selective inhibition of HIV-1 reverse transcriptase-associated ribonuclease H activity by hydroxylated tropolones"</p><p>Nucleic Acids Research 2005;33(4):1249-1256.</p><p>Published online 1 Mar 2005</p><p>PMCID:PMC552956.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> The inverse of the rate of RNase H cleavage was plotted as a function of β-thujaplicinol concentration at calanolide A concentrations of 12.5 μM (open square), 0.78 μM (filled square), 0.39 μM (open circle) and DMSO (filled circle). The convergent best-fit lines indicate mutually exclusive binding sites for calanolide A and β-thujaplicinol

Specific recognition of RNA/DNA hybrid and enhancement of human RNase H1 activity by HBD

Human RNase H1 contains an N-terminal domain known as dsRHbd for binding both dsRNA and RNA/DNA hybrid. We find that dsRHbd binds preferentially to RNA/DNA hybrids by over 25-fold and rename it as hybrid binding domain (HBD). The crystal structure of HBD complexed with a 12 bp RNA/DNA hybrid reveals that the RNA strand is recognized by a protein loop, which forms hydrogen bonds with the 2′-OH groups. The DNA interface is highly specific and contains polar residues that interact with the phosphate groups and an aromatic patch that appears selective for binding deoxyriboses. HBD is unique relative to non-sequence-specific dsDNA- and dsRNA-binding domains because it does not use positive dipoles of α-helices for nucleic acid binding. Characterization of full-length enzymes with defective HBDs indicates that this domain dramatically enhances both the specific activity and processivity of RNase H1. Similar activity enhancement by small substrate-binding domains linked to the catalytic domain likely occurs in other nucleic acid enzymes