WD-repeat Protein Research Articles

Control of trichome formation in Arabidopsis by poplar single-repeat R3 MYB transcription factors.

In Arabidopsis, trichome formation is regulated by the interplay of R3 MYBs and several others transcription factors including the WD40-repeat protein TRANSPARENT TESTA GLABRA1 (TTG1), the R2R3 MYB transcription factor GLABRA1 (GL1), the bHLH transcription factor GLABRA3 (GL3) or ENHANCER OF GLABRA3 (EGL3), and the homeodomain protein GLABRA2 (GL2). R3 MYBs including TRICHOMELESS1 (TCL1), TCL2, TRYPTICHON (TRY), CAPRICE (CPC), ENHANCER OF TRY AND CPC1 (ETC1), ETC2 and ETC3 negatively regulate trichome formation by competing with GL1 for binding GL3 or EGL3, thus blocking the formation of TTG1–GL3/EGL3–GL1, an activator complex required for the activation of the trichome positive regulator gene GL2. However, it is largely unknown if R3 MYBs in other plant species especially woody plants have similar functions. By BLASTing the Populus trichocarpa protein database using the entire amino acid sequence of TCL1, an Arabidopsis R3 MYB transcription factor, we identified a total of eight R3 MYB transcription factor genes in poplar, namely P. trichocarpa TRICHOMELESS1 through 8 (PtrTCL1–PtrTCL8). The amino acid signature required for interacting with bHLH transcription factors and the amino acids required for cell-to-cell movement of R3 MYBs are not fully conserved in all PtrTCLs. When tested in Arabidopsis protoplasts, however, all PtrTCLs interacted with GL3. Expressing each of the eight PtrTCL genes in Arabidopsis resulted in either glabrous phenotypes or plants with reduced trichome numbers, and expression levels of GL2 in all transgenic plants tested were greatly reduced. Expression of PtrTCL1 under the control of TCL1 native promoter almost completely complemented the mutant phenotype of tcl. In contrast, expression of PtrTCL1 under the control of TRY native promoter in the try mutant, or under the control of CPC native promoter in the cpc mutant resulted in glabrous phenotypes, suggesting that PtrTCL1 functions similarly to TCL1, but not TRY and CPC.

An overview of the gene regulatory network controlling trichome development in the model plant, Arabidopsis.

Trichomes are specialized epidermal cells located on aerial parts of plants and are associated with a wide array of biological processes. Trichomes protect plants from adverse conditions including UV light and herbivore attack and are also an important source of a number of phytochemicals. The simple unicellular trichomes of Arabidopsis serve as an excellent model to study molecular mechanism of cell differentiation and pattern formation in plants. The emerging picture suggests that the developmental process is controlled by a transcriptional network involving three major groups of transcription factors (TFs): the R2R3 MYB, basic helix-loop-helix (bHLH), and WD40 repeat (WDR) protein. These regulatory proteins form a trimeric activator complex that positively regulates trichome development. The single repeat R3 MYBs act as negative regulators of trichome development. They compete with the R2R3 MYBs to bind the bHLH factor and form a repressor complex. In addition to activator–repressor mechanism, a depletion mechanism may operate in parallel during trichome development. In this mechanism, the bHLH factor traps the WDR protein which results in depletion of WDR protein in neighboring cells. Consequently, the cells with high levels of bHLH and WDR proteins are developed into trichomes. A group of C2H2 zinc finger TFs has also been implicated in trichome development. Phytohormones, including gibberellins and jasmonic acid, play significant roles in this developmental process. Recently, microRNAs have been shown to be involved in trichome development. Furthermore, it has been demonstrated that the activities of the key regulatory proteins involved in trichome development are controlled by the 26S/ubiquitin proteasome system (UPS), highlighting the complexity of the regulatory network controlling this developmental process. To complement several excellent recent relevant reviews, this review focuses on the transcriptional network and hormonal interplay controlling trichome development in Arabidopsis.

Hat2p recognizes the histone H3 tail to specify the acetylation of the newly synthesized H3/H4 heterodimer by the Hat1p/Hat2p complex.

Post-translational modifications of histones are significant regulators of replication, transcription, and DNA repair. Particularly, newly synthesized histone H4 in H3/H4 heterodimers becomes acetylated on N-terminal lysine residues prior to its incorporation into chromatin. Previous studies have established that the histone acetyltransferase (HAT) complex Hat1p/Hat2p medicates this modification. However, the mechanism of how Hat1p/Hat2p recognizes and facilitates the enzymatic activities on the newly assembled H3/H4 heterodimer remains unknown. Furthermore, Hat2p is a WD40 repeat protein, which is found in many histone modifier complexes. However, how the WD40 repeat proteins facilitate enzymatic activities of histone modification enzymes is unclear. In this study, we first solved the high-resolution crystal structure of a Hat1p/Hat2p/CoA/H4 peptide complex and found that the H4 tail interacts with both Hat1p and Hat2p, by which substrate recruitment is facilitated. We further discovered that H3 N-terminal peptides can bind to the Hat2p WD40 domain and solved the structure of the Hat1p/Hat2p/CoA/H4/H3 peptide complex. Moreover, the interaction with Hat2p requires unmodified Arg2/Lys4 and Lys9 on the H3 tail, suggesting a novel model to specify the activity of Hat1p/Hat2p toward newly synthesized H3/H4 heterodimers. Together, our study demonstrated the substrate recognition mechanism by the Hat1p/Hat2p complex, which is critical for DNA replication and other chromatin remodeling processes.

Involvement of rRNA biosynthesis in the regulation of CUC1 gene expression and pre-meristematic cell mound formation during shoot regeneration.

At an early stage of shoot regeneration from calli of Arabidopsis, pre-meristematic cell mounds develop in association with localized strong expression of CUP-SHAPED COTYLEDON (CUC) genes. Previous characterization of root initiation-defective 3 (rid3), an Arabidopsis mutant originally isolated as being temperature-sensitive for adventitious root formation, with respect to shoot regeneration implicated RID3 in the negative regulation of CUC1 expression and the restriction of cell division in pre-meristematic cell mounds. Positional cloning has identified RID3 as a WD40 repeat protein gene whose molecular function was not investigated before. Here we performed in silico analysis of RID3 and found that RID3 is orthologous to IPI3, which mediates pre-rRNA processing in Saccharomyces cerevisiae. In the rid3 mutant, rRNA precursors accumulated to a very high level in a temperature-dependent manner. This result indicates that RID3 is required for pre-rRNA processing as is IPI3. We compared rid3 with rid2, a temperature-sensitive mutant that is mutated in a putative RNA methyltransferase gene and is impaired in pre-rRNA processing, for seedling morphology, shoot regeneration, and CUC1 expression. The rid2 and rid3 seedlings shared various developmental alterations, such as a pointed-leaf phenotype, which is often observed in ribosome-related mutants. In tissue culture for the induction of shoot regeneration, both rid2 and rid3 mutations perturbed cell-mound formation and elevated CUC1 expression. Together, our findings suggest that rRNA biosynthesis may be involved in the regulation of CUC1 gene expression and pre-meristematic cell-mound formation during shoot regeneration.

Targeting vertebrate intron-encoded box C/D 2'-O-methylation guide RNAs into the Cajal body.

Post-transcriptional pseudouridylation and 2′-O-methylation of splicesomal small nuclear ribonucleic acids (snRNAs) is mediated by box H/ACA and box C/D small Cajal body (CB)-specific ribonucleoproteins (scaRNPs), respectively. The WD-repeat protein 79 (WDR79) has been proposed to interact with both classes of modification scaRNPs and target them into the CB. The box H/ACA scaRNAs carry the common CAB box motif (consensus, ugAG) that is required for both WDR79 binding and CB-specific accumulation. Thus far, no cis-acting CB-localization element has been reported for vertebrate box C/D scaRNAs. In this study, systematic mutational analysis of the human U90 and another newly identified box C/D scaRNA, mgU2-47, demonstrated that the CB-specific accumulation of vertebrate intron-encoded box C/D scaRNAs relies on GU- or UG-dominated dinucleotide repeat sequences which are predicted to form the terminal stem-loop of the RNA apical hairpin. While the loop nucleotides are unimportant, the adjacent terminal helix that is composed mostly of consecutive G.U and U.G wobble base-pairs is essential for CB-specific localization of box C/D scaRNAs. Co-immunoprecipitation experiments confirmed that the newly identified CB localization element, called the G.U/U.G wobble stem, is crucial for in vivo association of box C/D scaRNPs with WDR79.

Dynamics of WD-repeat containing proteins in SSU processome components.

Nine WD-repeat containing proteins in human SSU processome components have been found in a HeLa cell nuclear matrix fraction. In these proteins, t-UTP sub-complex components, i.e., CIRH1A, UTP15, and WDR43, were shown to be immobilized in the fibrillar centers of nucleoli in living cells. In this study, the dynamics of the remaining six proteins fused with green fluorescent protein (GFP), i.e., PWP2-GFP, TBL3-GFP, GFP-UTP18, GFP-NOL10, GFP-WDR46, and GFP-WDSOF1, were examined in living cells. The findings were as follows. (i) The majority of UTP-B sub-complex components, i.e., PWP2-GFP, TBL3-GFP, and GFP-UTP18, are localized to the dense fibrillar component and granular component regions in nucleoli; (ii) When rRNA transcription is suppressed, the majority of GFP-fused UTP-B sub-complex components are localized in the cap and body regions of nucleoli. (iii) The mobility of these proteins except for GFP-WDSOF1, and half of GFP-UTP18 and GFP-WDR46, respectively, is very low in living cells. (iv) When rRNA transcription is suppressed, the mobility of these proteins except for GFP-WDSOF1 is accelerated but still slow. These findings and others suggest that these WD-repeat proteins other than GFP-WDSOF1 found in the nuclear matrix fraction bind tightly to some macro-protein complexes and act as a scaffold or a core for the complexes in nucleoli.

The WD40-repeat Proteins WDR-20 and WDR-48 Bind and Activate the Deubiquitinating Enzyme USP-46 to Promote the Abundance of the Glutamate Receptor GLR-1 in the Ventral Nerve Cord of Caenorhabditis elegans

Ubiquitin-mediated endocytosis and degradation of glutamate receptors controls their synaptic abundance and is implicated in modulating synaptic strength. The deubiquitinating enzymes (DUBs) that function in the nervous system are beginning to be defined, but the mechanisms that control DUB activity in vivo are understood poorly. We found previously that the DUB USP-46 deubiquitinates the Caenorhabditis elegans glutamate receptor GLR-1 and prevents its degradation in the lysosome. The WD40-repeat (WDR) proteins WDR20 and WDR48/UAF1 have been shown to bind to USP46 and stimulate its catalytic activity in other systems. Here we identify the C. elegans homologs of these WDR proteins and show that C. elegans WDR-20 and WDR-48 can bind and stimulate USP-46 catalytic activity in vitro. Overexpression of these activator proteins in vivo increases the abundance of GLR-1 in the ventral nerve cord, and this effect is further enhanced by coexpression of USP-46. Biochemical characterization indicates that this increase in GLR-1 abundance correlates with decreased levels of ubiquitin-GLR-1 conjugates, suggesting that WDR-20, WDR-48, and USP-46 function together to deubiquitinate and stabilize GLR-1 in neurons. Overexpression of WDR-20 and WDR-48 results in alterations in locomotion behavior consistent with increased glutamatergic signaling, and this effect is blocked in usp-46 loss-of-function mutants. Conversely, wdr-20 and wdr-48 loss-of-function mutants exhibit changes in locomotion behavior that are consistent with decreased glutamatergic signaling. We propose that WDR-20 and WDR-48 form a complex with USP-46 and stimulate the DUB to deubiquitinate and stabilize GLR-1 in vivo.

GIGANTUS1 (GTS1), a member of Transducin/WD40 protein superfamily, controls seed germination, growth and biomass accumulation through ribosome-biogenesis protein interactions in Arabidopsis thaliana

BackgroundWD40 domains have been found in a plethora of eukaryotic proteins, acting as scaffolding molecules assisting proper activity of other proteins, and are involved in multi-cellular processes. They comprise several stretches of 44-60 amino acid residues often terminating with a WD di-peptide. They act as a site of protein-protein interactions or multi-interacting platforms, driving the assembly of protein complexes or as mediators of transient interplay among other proteins. In Arabidopsis, members of WD40 protein superfamily are known as key regulators of plant-specific events, biologically playing important roles in development and also during stress signaling.ResultsUsing reverse genetic and protein modeling approaches, we characterize GIGANTUS1 (GTS1), a new member of WD40 repeat protein in Arabidopsis thaliana and provide evidence of its role in controlling plant growth development. GTS1 is highly expressed during embryo development and negatively regulates seed germination, biomass yield and growth improvement in plants. Structural modeling analysis suggests that GTS1 folds into a β-propeller with seven pseudo symmetrically arranged blades around a central axis. Molecular docking analysis shows that GTS1 physically interacts with two ribosomal protein partners, a component of ribosome Nop16, and a ribosome-biogenesis factor L19e through β-propeller blade 4 to regulate cell growth development.ConclusionsOur results indicate that GTS1 might function in plant developmental processes by regulating ribosomal structural features, activities and biogenesis in plant cells. Our results suggest that GIGANTUS1 might be a promising target to engineer transgenic plants with higher biomass and improved growth development for plant-based bioenergy production.

Enhanced Expression of WD Repeat-Containing Protein 35 via Nuclear Factor-Kappa B Activation in Bupivacaine-Treated Neuro2a Cells

The family of WD repeat proteins comprises a large number of proteins and is involved in a wide variety of cellular processes such as signal transduction, cell growth, proliferation, and apoptosis. Bupivacaine is a sodium channel blocker administered for local infiltration, nerve block, epidural, and intrathecal anesthesia. Recently, we reported that bupivacaine induces reactive oxygen species (ROS) generation and p38 mitogen-activated protein kinase (MAPK) activation, resulting in an increase in the expression of WD repeat-containing protein 35 (WDR35) in mouse neuroblastoma Neuro2a cells. It has been shown that ROS activate MAPK through phosphorylation, followed by activation of nuclear factor-kappa B (NF-κB) and activator protein 1 (AP-1). The present study was undertaken to test whether NF-κB and c-Jun/AP-1 are involved in bupivacaine-induced WDR35 expression in Neuro2a cells. Bupivacaine activated both NF-κB and c-Jun in Neuro2a cells. APDC, an NF-κB inhibitor, attenuated the increase in NF-κB activity and WDR35 protein expression in bupivacaine-treated Neuro2a cells. GW9662, a selective peroxisome proliferator-activated receptor-γ antagonist, enhanced the increase in NF-κB activity and WDR35 protein expression in bupivacaine-treated Neuro2a cells. In contrast, c-Jun siRNA did not inhibit the bupivacaine-induced increase in WDR35 mRNA expression. These results indicate that bupivacaine induces the activation of transcription factors NF-κB and c-Jun/AP-1 in Neuro2a cells, while activation of NF-κB is involved in bupivacaine-induced increases in WDR35 expression.

Isolation of a WD40-repeat gene regulating anthocyanin biosynthesis in storage roots of purple-fleshed sweet potato

Many transcriptional factors including the R2R3-MYB domain, basic helix-loop-helix (bHLH) domain and WD40 repeat proteins, which regulate flavonoid biosynthesis, have been identified in various plant species. However, there is little information on WD40 proteins in underground organs. In this study, a WD40-repeat protein gene was isolated from purple-fleshed sweet potato (Ipomoea batatas (L.) Lam. cv. Yamakawamurasaki) (IbWD40). The expression patterns of this gene were positively correlated with anthocyanin accumulation in different sweet potato cultivars. An IbWD40-GFP fusion protein was observed only in the nucleus of onion epidermal cells, which was consistent with its role as a transcriptional regulator. Stable transformation analysis revealed that IbWD40 was up-regulated in Arabidopsis thaliana seedlings, which accumulated anthocyanins, with possible additional effects on the formation of other flavonoid compounds in other tissues. These results suggest that in storage roots of purple-fleshed sweet potato the activity of IbWD40 plays a critical role in the regulation of anthocyanin biosynthesis.

The WD40 Repeat Protein Mutations: Genetics, Molecular Mechanisms and Therapeutic Implications

Eukaryotes contain numerous WD40 repeat proteins which perform diverse functions in signal transduction, gene transcriptional regulation, vesicular trafficking, cytoskeleton assembly, genome stability maintenance and cell cycle control. Importantly, mutations of WD40 repeat proteinsare genetically associated with diseases such as Lissencephaly, Cockayne syndrome, cancer, Parkinson’s disease, Chediak-Higashi syndrome, amyotrophic lateral sclerosis, cardiovascular diseases and colorectal neoplasms. Over the past years, great progresses have been made in understanding the molecular mechanisms and therapeutic strategies of these diseases. This reviewbriefly provides current perspectives onWD40 repeat protein functions, andmainly summarizes the most recent understandings onmolecular basis of WD40 repeat proteins involved in diseases and their therapeutic implications.

Cordyceps cicadae induces G2/M cell cycle arrest in MHCC97H human hepatocellular carcinoma cells: a proteomic study.

BackgroundCordyceps cicadae is a medicinal fungus that is often used for treating cancer. However, the anticancer mechanisms of C. cicadae are largely unknown. This study aims to investigate the anticancer mechanisms of C. cicadae against hepatocellular carcinoma cells in vitro using a proteomic approach.MethodsHuman hepatocellular carcinoma MHCC97H cells were treated with a water extract of C. cicadae (0, 100, 250, 500, and 1000 μg/mL) for 48 h and harvested for cell viability assays. The significant differences in protein expression between control and C. cicadae-treated cells were analyzed by two-dimensional gel-based proteomics coupled with matrix-assisted laser desorption ionization-time of flight mass spectrometry. Flow cytometry analysis was employed to investigate the cell cycle and cell death. The anticancer molecular mechanism was analyzed by whole proteome mapping.ResultsThe water extract of C. cicadae (0, 100, 250, 500, and 1000 μg/mL) inhibited the growth of MHCC97H cells in a dose-dependent manner via G2/M phase cell cycle arrest with no evidence of apoptosis. Among the identified proteins with upregulated expression were dynactin subunit 2, N-myc downstream-regulated gene 1, heat shock protein beta-1, alpha-enolase isoform 1, phosphatidylinositol transfer protein, and WD repeat-containing protein 1. Meanwhile, the proteins with downregulated expression were 14-3-3 gamma, BUB3, microtubule-associated protein RP/EB family member 1, thioredoxin-like protein, chloride intracellular channel protein 1, ectonucleoside triphosphate diphosphohydrolase 5, xaa-Pro dipeptidase, enoyl-CoA delta isomerase 1, protein-disulfide isomerase-related chaperone Erp29, hnRNP 2H9B, peroxiredoxin 1, WD-40 repeat protein, and serine/threonine kinase receptor-associated protein.ConclusionThe water extract of C. cicadae reduced the growth of human hepatocellular carcinoma MHCC97H cells via G2/M cell cycle arrest.

Among the three striatin family members SG2NA was first to arise during evolution

Striatin, SG2NA, and zinedin constitute a three-member subfamily of WD-40 repeat proteins. They are found only in metazoans and are likely to have scaffolding functions. Apart from WD-40 repeats, they also have a caveolin-binding motif, a coiled-coil structure, and a calmodulin-binding domain. This paper focuses on the analysis of their evolution as a paradigm of understanding the metazoan scaffolds. Each member of the family forms distinct phylogenetic clusters, wherein striatins, SG2NAs, and zinedins have 13, 10, and 9 conserved motifs, respectively. Furthermore, two of those motifs each in striatin and in zinedin and three in SG2NA are exclusive for the respective subfamily. Of those exclusive motifs for SG2NA, two encompass the caveolin-binding and coiled-coiled domains. Collectively, they show the presence of 11 conserved motifs, suggestive of convergence of individual motifs and creation of patterns. A prokaryotic WD-40 repeat motif pCM-I was found only in the corresponding domain of SG2NA but not in other family members. It is thus hypothesized that striatin family members have evolved from bacteria, and SG2NA was the first member to arise.

Opposing roles for JNK and Aurora A in regulating the association of WDR62 with spindle microtubules.

WD40-repeat protein 62 (WDR62) is a spindle pole protein required for normal cell division and neuroprogenitor differentiation during brain development. Microcephaly-associated mutations in WDR62 lead to mitotic mislocalization, highlighting a crucial requirement for precise WDR62 spatiotemporal distribution, although the regulatory mechanisms are unknown. Here, we demonstrate that the WD40-repeat region of WDR62 is required for microtubule association, whereas the disordered C-terminal region regulates cell-cycle-dependent compartmentalization. In agreement with a functional requirement for the WDR62–JNK1 complex during neurogenesis, WDR62 specifically recruits JNK1 (also known as MAPK8), but not JNK2 (also known as MAPK9), to the spindle pole. However, JNK-mediated phosphorylation of WDR62 T1053 negatively regulated microtubule association, and loss of JNK signaling resulted in constitutive WDR62 localization to microtubules irrespective of cell cycle stage. In contrast, we identified that Aurora A kinase (AURKA) and WDR62 were in complex and that AURKA-mediated phosphorylation was required for the spindle localization of WDR62 during mitosis. Our studies highlight complex regulation of WDR62 localization, with opposing roles for JNK and AURKA in determining its spindle association.

Microcephaly-Associated Protein WDR62 Regulates Neurogenesis through JNK1 in the Developing Neocortex

Mutations of WD40-repeat protein 62 (WDR62) have been identified recently to cause human MCPH (autosomal-recessive primary microcephaly), a neurodevelopmental disorder characterized by decreased brain size. However, the underlying mechanism is unclear. Here, we investigate the function of WDR62 in brain development and the pathological role of WDR62 mutations. We find that WDR62 knockdown leads to premature differentiation of neural progenitor cells (NPCs). The defect can be rescued by wild-type human WDR62, but not by the five MCPH-associated WDR62 mutants. We demonstrate that WDR62 acts upstream of JNK signaling in the control of neurogenesis. Depletion of JNK1 and WDR62 incurs very similar defects including abnormal spindle formation and mitotic division of NPCs as well as premature NPC differentiation during cortical development. Thus, our findings indicate that WDR62 is required for proper neurogenesis via JNK1 and provide an insight into the molecular mechanisms underlying MCPH pathogenesis.

Complexity and robustness of the flavonoid transcriptional regulatory network revealed by comprehensive analyses of MYB–bHLH–WDR complexes and their targets in Arabidopsis seed

In Arabidopsis thaliana, proanthocyanidins (PAs) accumulate in the innermost cell layer of the seed coat (i.e. endothelium, chalaza and micropyle). The expression of the biosynthetic genes involved relies on the transcriptional activity of R2R3-MYB and basic helix-loop-helix (bHLH) proteins which form ternary complexes ('MBW') with TRANSPARENT TESTA GLABRA1 (TTG1) (WD repeat protein). The identification of the direct targets and the determination of the nature and spatio-temporal activity of these MBW complexes are essential steps towards a comprehensive understanding of the transcriptional mechanisms that control flavonoid biosynthesis. In this study, various molecular, genetic and biochemical approaches were used. Here, we have demonstrated that, of the 12 studied genes of the pathway, only dihydroflavonol-4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX), BANYULS (BAN), TRANSPARENT TESTA 19 (TT19), TT12 and H(+) -ATPase isoform 10 (AHA10) are direct targets of the MBW complexes. Interestingly, although the TT2-TT8-TTG1 complex plays the major role in developing seeds, three additional MBW complexes (i.e. MYB5-TT8-TTG1, TT2-EGL3-TTG1 and TT2-GL3-TTG1) were also shown to be involved, in a tissue-specific manner. Finally, a minimal promoter was identified for each of the target genes of the MBW complexes. Altogether, by answering fundamental questions and by demonstrating or invalidating previously made hypotheses, this study provides a new and comprehensive view of the transcriptional regulatory mechanisms controlling PA and anthocyanin biosynthesis in Arabidopsis.

Genome-wide analysis of the WD-repeat protein family in cucumber and Arabidopsis

The WD-repeat (WDR) proteins comprise an astonishingly diverse superfamily of regulatory proteins. To date, genome-wide characterization of this family has only been conducted in Arabidopsis and little is known about WDR genes in cucumber (Cucumis sativus L.). This study identified 191 cucumber WDR genes in the latest cucumber genome and the CsWDR family contained a smaller number of identified genes compared to Arabidopsis. The results of this study were also supported by genome distribution and gene duplication analysis. Phylogenetic analysis showed that the WDR proteins could be classified into 21 subgroups. Moreover, an additional 12 AtWDR proteins were also identified and a complete overview of this gene family in Arabidopsis is presented, including the phylogeny, chromosome locations and duplication events. In addition, a comparative analysis between these genes in cucumber and Arabidopsis was performed and it suggested that there was strong gene conservation and that there was an expansion of particular functional genes during the evolution of the two species. The transcript abundance level analysis during abiotic stress (NaCl, ABA and low temperature treatments) identified six CsWDR genes that responded to one or more treatments. Tissue-specific expression profiles of these six genes were also analyzed. This study has produced a comparative genomics analysis of the WDR gene family in cucumber and Arabidopsis and provides the first steps towards the selection of CsWDR genes for cloning and functional dissection that can be used in further studies into their roles in cucumber stress resistance.

WDR34 Mutations that Cause Short-Rib Polydactyly Syndrome Type III/Severe Asphyxiating Thoracic Dysplasia Reveal a Role for the NF-κB Pathway in Cilia

Short-rib polydactyly (SRP) syndrome type III, or Verma-Naumoff syndrome, is an autosomal-recessive chondrodysplasia characterized by short ribs, a narrow thorax, short long bones, an abnormal acetabulum, and numerous extraskeletal malformations and is lethal in the perinatal period. Presently, mutations in two genes, IFT80 and DYNC2H1, have been identified as being responsible for SRP type III. Via homozygosity mapping in three affected siblings, a locus for the disease was identified on chromosome 9q34.11, and homozygosity for three missense mutations in WDR34 were found in three independent families, as well as compound heterozygosity for mutations in one family. WDR34 encodes a member of the WD repeat protein family with five WD40 domains, which acts as a TAK1-associated suppressor of the IL-1R/TLR3/TLR4-induced NF-κB activation pathway. We showed, through structural modeling, that two of the three mutations altered specific structural domains of WDR34. We found that primary cilia in WDR34 mutant fibroblasts were significantly shorter than normal and had a bulbous tip. This report expands on the pathogenesis of SRP type III and demonstrates that a regulator of the NF-κB activation pathway is involved in the pathogenesis of the skeletal ciliopathies.

Functional Analysis of Sporophytic Transcripts Repressed by the Female Gametophyte in the Ovule of Arabidopsis thaliana

To investigate the genetic and molecular regulation that the female gametophyte could exert over neighboring sporophytic regions of the ovule, we performed a quantitative comparison of global expression in wild-type and nozzle/sporocyteless (spl) ovules of Arabidopsis thaliana (Arabidopsis), using Massively Parallel Signature Sequencing (MPSS). This comparison resulted in 1517 genes showing at least 3-fold increased expression in ovules lacking a female gametophyte, including those encoding 89 transcription factors, 50 kinases, 25 proteins containing a RNA-recognition motif (RRM), and 20 WD40 repeat proteins. We confirmed that eleven of these genes are either preferentially expressed or exclusive of spl ovules lacking a female gametophyte as compared to wild-type, and showed that six are also upregulated in determinant infertile1 (dif1), a meiotic mutant affected in a REC8-like cohesin that is also devoided of female gametophytes. The sporophytic misexpression of IOREMPTE, a WD40/transducin repeat gene that is preferentially expressed in the L1 layer of spl ovules, caused the arrest of female gametogenesis after differentiation of a functional megaspore. Our results show that in Arabidopsis, the sporophytic-gametophytic cross talk includes a negative regulation of the female gametophyte over specific genes that are detrimental for its growth and development, demonstrating its potential to exert a repressive control over neighboring regions in the ovule.

Novel small Cajal-body-specific RNAs identified in Drosophila: probing guide RNA function.

The spliceosomal small nuclear RNAs (snRNAs) are modified post-transcriptionally by introduction of pseudouridines and 2'-O-methyl modifications, which are mediated by box H/ACA and box C/D guide RNAs, respectively. Because of their concentration in the nuclear Cajal body (CB), these guide RNAs are known as small CB-specific (sca) RNAs. In the cell, scaRNAs are associated with the WD-repeat protein WDR79. We used coimmunoprecipitation with WDR79 to recover seven new scaRNAs from Drosophila cell lysates. We demonstrated concentration of these new scaRNAs in the CB by in situ hybridization, and we verified experimentally that they can modify their putative target RNAs. Surprisingly, one of the new scaRNAs targets U6 snRNA, whose modification is generally assumed to occur in the nucleolus, not in the CB. Two other scaRNAs have dual guide functions, one for an snRNA and one for 28S rRNA. Again, the modification of 28S rRNA is assumed to take place in the nucleolus. These findings suggest that canonical scaRNAs may have functions in addition to their established role in modifying U1, U2, U4, and U5 snRNAs. We discuss the likelihood that processing by scaRNAs is not limited to the CB.