PolIVb influences RNA-directed DNA methylation independently of its role in siRNA biogenesis

DNA-dependent RNA polymerase (Pol)IV in Arabidopsis exists in two isoforms (PolIVa and PolIVb), with NRPD1a and NRPD1b as their respective largest subunits. Both isoforms are implicated in production and activity of siRNAs and in RNA-directed DNA methylation (RdDM). Deep sequence analysis of siRNAs in WT Arabidopsis flowers and in nrpd1a and nrpd1b mutants identified >4,200 loci producing siRNAs in a PolIV-dependent manner, with PolIVb reinforcing siRNA production by PolIVa. Transposable element identity and pericentromeric localization are both features that predispose a locus for siRNA production via PolIV proteins and determine the extent to which siRNA production relies on PolIVb. Detailed analysis of DNA methylation at PolIV-dependent loci revealed unexpected deviations from the previously noted association of PolIVb-dependent siRNA production and RdDM. Notably, PolIVb functions independently in DNA methylation and siRNA generation. Additionally, we have uncovered siRNA-directed loss of DNA methylation, a process requiring both PolIV isoforms. From these findings, we infer that the role of PolIVb in siRNA production is secondary to a role in chromatin modification and is influenced by chromatin context.     

Antisilencing role of the RNA-directed DNA methylation pathway and a histone acetyltransferase in Arabidopsis

REPRESSOR OF SILENCING 1 (ROS1) is a DNA demethylation enzyme that was previously identified during a genetic screen for the silencing of both RD29A-LUC and 35S-NPTII transgenes on a T-DNA construct. Here we performed a genetic screen to identify additional mutants in which the 35S-NPTII transgene is silenced. We identified several alleles of ros1 and of the following components of the RNA-directed DNA methylation (RdDM) pathway: NRPD1 (the largest subunit of polymerase IV), RDR2, NRPE1 (the largest subunit of polymerase V), NRPD2, AGO4, and DMS3. Our results show that the silencing of 35S-NPTII in the RdDM pathway mutants is due to the reduced expression of ROS1 in the mutants. We also identified a putative histone acetyltransferase (ROS4) from the genetic screen. The acetyltransferase contains a PHD-finger domain that binds to unmethylated histone H3K4. The mutation in ROS4 led to reduction of H3K18 and H3K23 acetylation levels. We show that the silencing of 35S-NPTII and some transposable element genes was released by the ddm1 mutation but that this also required ROS4. Our study identifies a unique antisilencing factor, and reveals that the RdDM pathway has an antisilencing function due to its role in maintaining ROS1 expression.     

Translationally controlled tumor protein is a conserved mitotic growth integrator in animals and plants

The growth of an organism and its size determination require the tight regulation of cell proliferation and cell growth. However, the mechanisms and regulatory networks that control and integrate these processes remain poorly understood. Here, we address the biological role of Arabidopsis translationally controlled tumor protein (AtTCTP) and test its shared functions in animals and plants. The data support a role of plant AtTCTP as a positive regulator of mitotic growth by specifically controlling the duration of the cell cycle. We show that, in contrast to animal TCTP, plant AtTCTP is not implicated in regulating postmitotic growth. Consistent with this finding, plant AtTCTP can fully rescue cell proliferation defects in Drosophila loss of function for dTCTP. Furthermore, Drosophila dTCTP is able to fully rescue cell proliferation defects in Arabidopsis tctp knockouts. Our data provide evidence that TCTP function in regulating cell division is part of a conserved growth regulatory pathway shared between plants and animals. The study also suggests that, although the cell division machinery is shared in all multicellular organisms to control growth, cell expansion can be uncoupled from cell division in plants but not in animals.     

Vitamin B1 biosynthesis in plants requires the essential iron sulfur cluster protein, THIC

Vitamin B1 (thiamin) is an essential compound in all organisms acting as a cofactor in key metabolic reactions and has furthermore been implicated in responses to DNA damage and pathogen attack in plants. Despite the fact that it was discovered almost a century ago and deficiency is a widespread health problem, much remains to be deciphered about its biosynthesis. The vitamin is composed of a thiazole and pyrimidine heterocycle, which can be synthesized by prokaryotes, fungi, and plants. Plants are the major source of the vitamin in the human diet, yet little is known about the biosynthesis of the compound therein. In particular, it has never been verified whether the pyrimidine heterocycle is derived from purine biosynthesis through the action of the THIC protein as in bacteria, rather than vitamin B6 and histidine as demonstrated for fungi. Here, we identify a homolog of THIC in Arabidopsis and demonstrate its essentiality not only for vitamin B1 biosynthesis, but also plant viability. This step takes place in the chloroplast and appears to be regulated at several levels, including through the presence of a riboswitch in the 3'-untranslated region of THIC. Strong evidence is provided for the involvement of an iron-sulfur cluster in the remarkable chemical rearrangement reaction catalyzed by the THIC protein for which there is no chemical precedent. The results suggest that vitamin B1 biosynthesis in plants is in fact more similar to prokaryotic counterparts and that the THIC protein is likely to be the key regulatory protein in the pathway.     

Conformational coupling between the active site and residues within the KC-channel of the Vibrio cholerae cbb3-type (C-family) oxygen reductase

The respiratory chains of nearly all aerobic organisms are terminated by proton-pumping heme-copper oxygen reductases (HCOs). Previous studies have established that C-family HCOs contain a single channel for uptake from the bacterial cytoplasm of all chemical and pumped protons, and that the entrance of the K^C-channel is a conserved glutamate in subunit III. However, the majority of the K^C-channel is within subunit I, and the pathway from this conserved glutamate to subunit I is not evident. In the present study, molecular dynamics simulations were used to characterize a chain of water molecules leading from the cytoplasmic solution, passing the conserved glutamate in subunit III and extending into subunit I. Formation of the water chain, which controls the delivery of protons to the K^C-channel, was found to depend on the conformation of Y241^Vc, located in subunit I at the interface with subunit III. Mutations of Y241^Vc (to A/F/H/S) in the Vibrio cholerae cbb₃ eliminate catalytic activity, but also cause perturbations that propagate over a 28-Å distance to the active site heme b₃. The data suggest a linkage between residues lining the K^C-channel and the active site of the enzyme, possibly mediated by transmembrane helix α7, which contains both Y241^Vc and the active site cross-linked Y255^Vc, as well as two Cu_B histidine ligands. Other mutations of residues within or near helix α7 also perturb the active site, indicating that this helix is involved in modulation of the active site of the enzyme.Nearly all aerobic organisms rely on one or more heme-copper oxygen reductases (HCOs) as the terminal enzyme in their respiratory chains. These enzymes are virtually all proton pumps, coupling the generation of a proton motive force to the chemistry of reducing O₂ to water (1–3). Critical components of these enzymes include proton-conducting channels, which are conserved structures within each major family of HCOs (A, B, and C families) (4–6). These proton channels are defined by conserved polar residues, as well as internal water molecules that provide a hydrogen-bonded pathway for proton diffusion within the protein by a Grotthus-type mechanism (7–9). The channels are required to provide pathways both for chemical protons, which are consumed at the enzyme active site to form water, and for pumped protons, which are transported across the membrane. For prokaryotic A-family HCOs, there are two well-defined proton-input channels leading from the bacterial cytoplasm to the vicinity of the active site, designated the D-channel and the K-channel, respectively (10, 11).For each O₂ consumed, four chemical protons are taken from the bacterial cytoplasm and an additional four protons are taken from the cytoplasm and delivered to the periplasm, for Gram-negative organisms. Previous studies have demonstrated that all four of the pumped protons along with two chemical protons are taken up by the D-channel, whereas the K-channel is required for delivering two chemical protons to the active site during the portion of the catalytic cycle before the binding of O₂ (11). An H-channel has also been proposed for the mammalian cytochrome c oxidase (3), but with no equivalent in the prokaryotic or fungal HCOs.Surprisingly, the B- and C-families of oxygen reductases, which are found only in prokaryotes, each uses only one proton-input channel, located in the homologous part of the protein as the K-channel of the A-family oxygen reductases (6, 12, 13). We refer to these proton channels as the K^B- and K^C-channels for the B- and C-family enzymes, respectively. The patterns of conserved polar residues that define the K^B- and K^C-channels are unique to each family of enzymes, and both differ from the polar residues conserved within the K-channel of the A-family oxygen reductases. Because there is no equivalent of the D-channel in the B- and C-family oxygen reductases, all four of the chemical protons, as well as all pumped protons, must use the K^B- and K^C-channels. The present study explored the properties of mutations within the K^C-channel of cytochrome cbb₃ from Vibrio cholerae.The X-ray structure of the cytochrome cbb₃ (C-family oxygen reductase) from Pseudomonas stutzeri has confirmed the presence of only a single proton-conducting channel, located mostly within subunit I (CcoN) (13). Although the resolution is insufficient to define internal water molecules within the K^C-channel, molecular dynamics (MD) simulations have demonstrated that highly dynamic water wires can form within the K^C-channel and provide a pathway for proton translocation (14). Furthermore, these simulations suggest that the chemical and pumped protons share the same track but diverge after Y317^Ps (P. stutzeri)^*, at which point two pathways are possible, one for chemical protons leading to the active site and another for pumped protons leading to the propionates of heme b₃, which is near the periplasmic bulk aqueous phase.Although the bulk of the K^C-channel is located in subunit I (CcoN), mutagenesis studies of the cytochrome cbb₃ from Rhodobacter sphaeroides have demonstrated that the channel entrance is a highly conserved glutamate located on the cytoplasmic surface of subunit III (CcoP; E49^IIIPs) (15) (Fig. 1). The proton pathway from E49^IIIPs to the portion of the channel within subunit I (CcoN) is not evident. Here we used MD simulations to identify a hydrated pathway from the cytoplasmic solution and mediating direct contacts between E49^III and CcoN portion of the K^C-channel, supporting the role of E49^IIIPs in proton transfer. Site-directed mutagenesis studies of cytochrome cbb₃ from V. cholerae confirm the importance of the residues implicated as being part of the K^C-channel. An important finding is that a number of mutations within the K^C-channel also perturb the active site of the enzyme, suggesting a conformational coupling between the channel and the active site of the enzyme.Open in a separate windowFig. 1.Structure of cytochrome cbb₃ from P. stutzeri. (A) Ribbon structure with the CcoN in pink, CcoO in green, and CcoP in blue. Heme b and heme b₃ are highlighted along with the residues in the K^C-channel. (B) Enlargement of the region showing residues in the K^C-channel examined in the present study. The structure is that of PDB ID code 3MK7 from P. stutzeri (13), but the residue numbering is that from V. cholerae. Displayed water molecules were modeled using the DOWSER program (22).     

Recognition and repair of UV lesions in loop structures of duplex DNA by DASH-type cryptochrome

DNA photolyases and cryptochromes (cry) form a family of flavoproteins that use light energy in the blue/UV-A region for the repair of UV-induced DNA lesions or for signaling, respectively. Very recently, it was shown that members of the DASH cryptochrome subclade repair specifically cyclobutane pyrimidine dimers (CPDs) in UV-damaged single-stranded DNA. Here, we report the crystal structure of Arabidopsis cryptochrome 3 with an in-situ-repaired CPD substrate in single-stranded DNA. The structure shows a binding mode similar to that of conventional DNA photolyases. Furthermore, CPD lesions in double-stranded DNA are bound and repaired with similar efficiency as in single-stranded DNA if the CPD lesion is present in a loop structure. Together, these data reveal that DASH cryptochromes catalyze light-driven DNA repair like conventional photolyases but lack an efficient flipping mechanism for interaction with CPD lesions within duplex DNA.     

An evolutionary conserved function of the JAK-STAT pathway in anti-dengue defense

Here, we show that the major mosquito vector for dengue virus uses the JAK-STAT pathway to control virus infection. Dengue virus infection in Aedes aegypti mosquitoes activates the JAK-STAT immune signaling pathway. The mosquito''s susceptibility to dengue virus infection increases when the JAK-STAT pathway is suppressed through RNAi depletion of its receptor Domeless (Dome) and the Janus kinase (Hop), whereas mosquitoes become more resistant to the virus when the negative regulator of the JAK-STAT pathway, PIAS, is silenced. The JAK-STAT pathway exerts its anti-dengue activity presumably through one or several STAT-regulated effectors. We have identified, and partially characterized, two JAK-STAT pathway-regulated and infection-responsive dengue virus restriction factors (DVRFs) that contain putative STAT-binding sites in their promoter regions. Our data suggest that the JAK-STAT pathway is part of the A. aegypti mosquito''s anti-dengue defense and may act independently of the Toll pathway and the RNAi-mediated antiviral defenses.     

Extranuclear protection of chromosomal DNA from oxidative stress

Eukaryotic organisms evolved under aerobic conditions subjecting nuclear DNA to damage provoked by reactive oxygen species (ROS). Although ROS are thought to be a major cause of DNA damage, little is known about the molecular mechanisms protecting nuclear DNA from oxidative stress. Here we show that protection of nuclear DNA in plants requires a coordinated function of ROS-scavenging pathways residing in the cytosol and peroxisomes, demonstrating that nuclear ROS scavengers such as peroxiredoxin and glutathione are insufficient to safeguard DNA integrity. Both catalase (CAT2) and cytosolic ascorbate peroxidase (APX1) play a key role in protecting the plant genome against photorespiratory-dependent H(2)O(2)-induced DNA damage. In apx1/cat2 double-mutant plants, a DNA damage response is activated, suppressing growth via a WEE1 kinase-dependent cell-cycle checkpoint. This response is correlated with enhanced tolerance to oxidative stress, DNA stress-causing agents, and inhibited programmed cell death.     

The Mo-Se active site of nicotinate dehydrogenase

Nicotinate dehydrogenase (NDH) from Eubacterium barkeri is a molybdoenzyme catalyzing the hydroxylation of nicotinate to 6-hydroxynicotinate. Reactivity of NDH critically depends on the presence of labile (nonselenocysteine) selenium with an as-yet-unidentified form and function. We have determined the crystal structure of NDH and analyzed its active site by multiple wavelengths anomalous dispersion methods. We show that selenium is bound as a terminal MoSe ligand to molybdenum and that it occupies the position of the terminal sulfido ligand in other molybdenum hydroxylases. The role of selenium in catalysis has been assessed by model calculations, which indicate an acceleration of the critical hydride transfer from the substrate to the selenido ligand in the course of substrate hydroxylation when compared with an active site containing a sulfido ligand. The MoO(OH)Se active site of NDH shows a novel type of utilization and reactivity of selenium in nature.