Functional and Structural Divergence of an Unusual LTR Retrotransposon Family in Plants

Retrotransposons with long terminal repeats (LTRs) more than 3 kb are not frequent in most eukaryotic genomes. Rice LTR retrotransposon, Retrosat2, has LTRs greater than 3.2 kb and two open reading frames (ORF): ORF1 encodes enzymes for retrotransposition whereas no function can be assigned to ORF0 as it is not found in any other organism. A variety of experimental and in silico approaches were used to determine the origin of Retrosat2 and putative function of ORF0. Our data show that not only is Retrosat2 highly abundant in the Oryza genus, it may yet be active in rice. Homologs of Retrosat2 were identified in maize, sorghum, Arabidopsis and other plant genomes suggesting that the Retrosat2 family is of ancient origin. Several putatively cis-acting elements, some multicopy, that regulate retrotransposon replication or responsiveness to environmental factors were found in the LTRs of Retrosat2. Unlike the ORF1, the ORF0 sequences from Retrosat2 and homologs are divergent at the sequence level, 3D-structures and predicted biological functions. In contrast to other retrotransposon families, Retrosat2 and its homologs are dispersed throughout genomes and not concentrated in the specific chromosomal regions, such as centromeres. The genomic distribution of Retrosat2 homologs varies across species which likely reflects the differing evolutionary trajectories of this retrotransposon family across diverse species.     

Arabidopsis AtRRP44A Is the Functional Homolog of Rrp44/Dis3, an Exosome Component,Is Essential for Viability and Is Required for RNA Processing and Degradation

The RNA exosome is a multi-subunit complex that is responsible for 3ʹ to 5ʹ degradation and processing of cellular RNA. Rrp44/Dis3 is the catalytic center of the exosome in yeast and humans. However, the role of Rrp44/Dis3 homologs in plants is still unidentified. Here, we show that Arabidopsis AtRRP44A is the functional homolog of Rrp44/Dis3, is essential for plant viability and is required for RNA processing and degradation. We characterized AtRRP44A and AtRRP44B/SOV, two predicted Arabidopsis Rrp44/Dis3 homologs. AtRRP44A could functionally replace S. cerevisiae Rrp44/Dis3, but AtRRP44B/SOV could not. rrp44a knock-down mutants showed typical phenotypes of exosome function deficiency, 5.8S rRNA 3ʹ extension and rRNA maturation by-product over-accumulation, but rrp44b mutants did not. Conversely, AtRRP44B/SOV mutants showed elevated levels of a selected mRNA, on which rrp44a did not have detectable effects. Although T-DNA insertion mutants of AtRRP44B/SOV had no obvious phenotype, those of AtRRP44A showed defects in female gametophyte development and early embryogenesis. These results indicate that AtRRP44A and AtRRP44B/SOV have independent roles for RNA turnover in plants.     

Genetic and Epigenetic Dynamics of a Retrotransposon After Allopolyploidization of Wheat

Allopolyploidy, or the combination of two or more distinct genomes in one nucleus, is usually accompanied by radical genomic changes involving transposable elements (TEs). The dynamics of TEs after an allopolyploidization event are poorly understood. In this study, we analyzed the methylation state and genetic rearrangements of a high copied, newly amplified terminal-repeat retrotransposon in miniature (TRIM) family in wheat termed Veju. We found that Veju insertion sites underwent massive methylation changes in the first four generations of a newly formed wheat allohexaploid. Hypomethylation or hypermethylation occurred in ∼43% of the tested insertion sites; while hypomethylation was significantly predominant in the first three generations of the newly formed allohexaploid, hypermethylation became predominant in the subsequent generation. In addition, we determined that the methylation state of Veju long terminal repeats (LTRs) might be correlated with the deletion and/or insertion of the TE. While most of the methylation changes and deletions of Veju occurred in the first generation of the newly formed allohexaploid, most Veju insertions were seen in the second generation. Finally, using quantitative PCR, we quantitatively assessed the genome composition of Veju in the newly formed allohexaploid and found that up to 50% of Veju LTRs were deleted in the first generation. Retrotransposition bursts in subsequent generations, however, led to increases in Veju elements. In light of these findings, the underlying mechanisms of TRIM rearrangements are discussed.TRANSPOSABLE elements (TEs) are DNA sequences that range in size from several hundred base pairs to >15 kb and that have the ability to move to different locations within the genome. TE movement occurs through either a copy-and-paste mechanism involving RNA intermediates (class 1) or a cut-and-paste mechanism involving DNA intermediates (class 2). Class 1 elements are also called retrotransposons, or retroelements, and comprise two main types: (1) long terminal repeat (LTR) retrotransposons, flanked by LTRs, and (2) non-LTR elements (such as long interspersed nuclear elements and short interspersed nuclear elements).LTR retrotransposons are the most abundant mobile elements in plant genomes (Feschotte et al. 2002), as the replicative mode of retroelement transposition enables the LTR retrotransposon to accrue in high copy number. Indeed, in some grasses, LTR retrotransposons represent up to 90% of the genome (Bennetzen and Kellogg 1997; Feschotte et al. 2002). As such, retrotransposon sequences function well as substrates for illegitimate and unequal recombinations that can lead to a variety of mutations, such as deletions, insertions, translocations, and others (Parisod et al. 2009).The replicative nature of TEs seems to be stimulated by a variety of specific stress conditions (reviewed by Wessler 1996; Capy et al. 2000; Grandbastien et al. 2005), including challenges to the genome such as interspecific hybridization, an idea first proposed by Barbara McClintock 26 years ago (McClintock 1984). Accordingly, allopolyploidization is usually coupled with rapid and reproducible genomic changes, including the elimination of DNA sequences (Liu et al. 1998a,b; Ozkan et al. 2001; Shaked et al. 2001; Adams and Wendel 2005b; Skalicka et al. 2005), gene silencing (Chen and Pikaard 1997; Comai et al. 2000; Kashkush et al. 2002; Simons et al. 2006), alteration of cytosine methylation (Shaked et al. 2001; Madlung et al. 2002; Salmon et al. 2005; Beaulieu et al. 2009; Xu et al. 2009), activation of genes and retrotransposons (Kashkush et al. 2002, 2003; O''Neill et al. 2002), massively altered gene expression patterns (Kashkush et al. 2002; Wang et al. 2006), and organ-specific subfunctionalization, i.e., differential expression of homeoalleles in different tissues and at different developmental stages (Adams et al. 2003; Adams and Wendel 2004). These and other studies (Levy and Feldman 2002; Osborn et al. 2003; Adams and Wendel 2005a; Rapp and Wendel 2005; Chen and Ni 2006; Chen 2007) demonstrate the dynamic nature of allopolyploid plant genomes.Although allopolyploidization has generally been assumed to induce large bursts of TE activity (Matzke and Matzke 1998), several studies that focused on different allopolyploid systems failed to provide any evidence for a transposition burst and offered only limited evidence for the transposition of specific TEs (Madlung et al. 2005; Ainouche et al. 2009; Beaulieu et al. 2009). In newly formed Arabidopsis allopolyploids, no evidence for transposition bursts was reported (Beaulieu et al. 2009), although limited evidence suggested that transposition events occurred in a specific TE called Sunfish (Madlung et al. 2005). Little evidence of TE transposition was found in a natural population of the 150-year-old allopolyploid, Spartina anglica (Ainouche et al. 2009), and no evidence of transposition of Wis 2-1A retrotransposons in a newly formed wheat allotetraploid was present (Kashkush et al. 2003). The results of these works and others indicate that, in the short term, TE proliferation after allopolyploidization may be restricted to a few specific TEs in particular allopolyploidy systems (Parisod et al. 2009).This study entailed a detailed investigation of the methylation patterns and rearrangements of a one terminal-repeat retrotransposon in miniature (TRIM) family in wheat termed Veju. TRIM elements possess the classical structure of LTR retrotransposons, but they are distinguished by their small overall sizes (0.4 to ∼2.5 kb). A nonautonomous retrotransposon, Veju is 2520 bp long with 374 bp of identical LTRs, yet does not contain the proteins required for retrotransposition (Sanmiguel et al. 2002). However, because Veju elements contain polypurine tracts (PPTs) and primer binding sites (PBSs), they are capable of transposing if the retrotransposition proteins are available from another source. In addition, the identical sequences of the Veju 5′ and 3′ LTRs indicate that some members of the Veju family retain retrotransposition activity.In silico analysis of Veju sequences revealed them to be one of the most active and most recently inserted sequences in the wheat genome (Sanmiguel et al. 2002; Sabot et al. 2005a). As such, we have determined and compared the methylation patterns of >880 Veju insertion sites in the first four generations of a newly formed wheat allohexaploid, as well as in the parental lines. We then tested the correlation between the cytosine methylation and genetic rearrangements (i.e., deletions and insertions) of Veju and addressed the precise developmental timing of these rearrangements. Finally, we successfully tested overall changes in the copy numbers of Veju in the newly formed allohexaploid using real-time quantitative PCR.     

TRIM56 Is an Essential Component of the TLR3 Antiviral Signaling Pathway

Members of the tripartite motif (TRIM) proteins are being recognized as important regulators of host innate immunity. However, specific TRIMs that contribute to TLR3-mediated antiviral defense have not been identified. We show here that TRIM56 is a positive regulator of TLR3 signaling. Overexpression of TRIM56 substantially potentiated extracellular dsRNA-induced expression of interferon (IFN)-β and interferon-stimulated genes (ISGs), while knockdown of TRIM56 greatly impaired activation of IRF3, induction of IFN-β and ISGs, and establishment of an antiviral state by TLR3 ligand and severely compromised TLR3-mediated chemokine induction following infection by hepatitis C virus. The ability to promote TLR3 signaling was independent of the E3 ubiquitin ligase activity of TRIM56. Rather, it correlated with a physical interaction between TRIM56 and TRIF. Deletion of the C-terminal portion of TRIM56 abrogated the TRIM56-TRIF interaction as well as the augmentation of TLR3-mediated IFN response. Together, our data demonstrate TRIM56 is an essential component of the TLR3 antiviral signaling pathway and reveal a novel role for TRIM56 in innate antiviral immunity.     

Structure Prediction and Analysis of DNA Transposon and LINE Retrotransposon Proteins

Despite the considerable amount of research on transposable elements, no large-scale structural analyses of the TE proteome have been performed so far. We predicted the structures of hundreds of proteins from a representative set of DNA and LINE transposable elements and used the obtained structural data to provide the first general structural characterization of TE proteins and to estimate the frequency of TE domestication and horizontal transfer events. We show that 1) ORF1 and Gag proteins of retrotransposons contain high amounts of structural disorder; thus, despite their very low conservation, the presence of disordered regions and probably their chaperone function is conserved. 2) The distribution of SCOP classes in DNA transposons and LINEs indicates that the proteins of DNA transposons are more ancient, containing folds that already existed when the first cellular organisms appeared. 3) DNA transposon proteins have lower contact order than randomly selected reference proteins, indicating rapid folding, most likely to avoid protein aggregation. 4) Structure-based searches for TE homologs indicate that the overall frequency of TE domestication events is low, whereas we found a relatively high number of cases where horizontal transfer, frequently involving parasites, is the most likely explanation for the observed homology.     

Hcn1 Is a Tremorgenic Genetic Component in a Rat Model of Essential Tremor

Genetic factors are thought to play a major role in the etiology of essential tremor (ET); however, few genetic changes that induce ET have been identified to date. In the present study, to find genes responsible for the development of ET, we employed a rat model system consisting of a tremulous mutant strain, TRM/Kyo (TRM), and its substrain TRMR/Kyo (TRMR). The TRM rat is homozygous for the tremor (tm) mutation and shows spontaneous tremors resembling human ET. The TRMR rat also carries a homozygous tm mutation but shows no tremor, leading us to hypothesize that TRM rats carry one or more genes implicated in the development of ET in addition to the tm mutation. We used a positional cloning approach and found a missense mutation (c. 1061 C>T, p. A354V) in the hyperpolarization-activated cyclic nucleotide-gated 1 channel (Hcn1) gene. The A354V HCN1 failed to conduct hyperpolarization-activated currents in vitro, implicating it as a loss-of-function mutation. Blocking HCN1 channels with ZD7288 in vivo evoked kinetic tremors in nontremulous TRMR rats. We also found neuronal activation of the inferior olive (IO) in both ZD7288-treated TRMR and non-treated TRM rats and a reduced incidence of tremor in the IO-lesioned TRM rats, suggesting a critical role of the IO in tremorgenesis. A rat strain carrying the A354V mutation alone on a genetic background identical to that of the TRM rats showed no tremor. Together, these data indicate that body tremors emerge when the two mutant loci, tm and Hcn1^A354V, are combined in a rat model of ET. In this model, HCN1 channels play an important role in the tremorgenesis of ET. We propose that oligogenic, most probably digenic, inheritance is responsible for the genetic heterogeneity of ET.     

Structural and Functional Characterization of NikO, an Enolpyruvyl Transferase Essential in Nikkomycin Biosynthesis

Nikkomycins are peptide-nucleoside compounds with fungicidal, acaricidal, and insecticidal properties because of their strong inhibition of chitin synthase. Thus, they are potential antibiotics especially for the treatment of immunosuppressed patients, for those undergoing chemotherapy, or after organ transplants. Although their chemical structure has been known for more than 30 years, only little is known about their complex biosynthesis. The genes encoding for proteins involved in the biosynthesis of the nucleoside moiety of nikkomycins are co-transcribed in the same operon, comprising the genes nikIJKLMNO. The gene product NikO was shown to belong to the family of enolpyruvyl transferases and to catalyze the transfer of an enolpyruvyl moiety from phosphoenolpyruvate to the 3'-hydroxyl group of UMP. Here, we report activity and inhibition studies of the wild-type enzyme and the variants C130A and D342A. The x-ray crystal structure revealed differences between NikO and its homologs. Furthermore, our studies led to conclusions concerning substrate binding and preference as well as to conclusions about inhibition/alkylation by the antibiotic fosfomycin.     

Notch Is a Critical Component of the Mouse Somitogenesis Oscillator and Is Essential for the Formation of the Somites

Segmentation of the vertebrate body axis is initiated through somitogenesis, whereby epithelial somites bud off in pairs periodically from the rostral end of the unsegmented presomitic mesoderm (PSM). The periodicity of somitogenesis is governed by a molecular oscillator that drives periodic waves of clock gene expression caudo-rostrally through the PSM with a periodicity that matches somite formation. To date the clock genes comprise components of the Notch, Wnt, and FGF pathways. The literature contains controversial reports as to the absolute role(s) of Notch signalling during the process of somite formation. Recent data in the zebrafish have suggested that the only role of Notch signalling is to synchronise clock gene oscillations across the PSM and that somite formation can continue in the absence of Notch activity. However, it is not clear in the mouse if an FGF/Wnt-based oscillator is sufficient to generate segmented structures, such as the somites, in the absence of all Notch activity. We have investigated the requirement for Notch signalling in the mouse somitogenesis clock by analysing embryos carrying a mutation in different components of the Notch pathway, such as Lunatic fringe (Lfng), Hes7, Rbpj, and presenilin1/presenilin2 (Psen1/Psen2), and by pharmacological blocking of the Notch pathway. In contrast to the fish studies, we show that mouse embryos lacking all Notch activity do not show oscillatory activity, as evidenced by the absence of waves of clock gene expression across the PSM, and they do not develop somites. We propose that, at least in the mouse embryo, Notch activity is absolutely essential for the formation of a segmented body axis.     

Human Condensin Function Is Essential for Centromeric Chromatin Assembly and Proper Sister Kinetochore Orientation

Condensins I and II in vertebrates are essential ATP-dependent complexes necessary for chromosome condensation in mitosis. Condensins depletion is known to perturb structure and function of centromeres, however the mechanism of this functional link remains elusive. Depletion of condensin activity is now shown to result in a significant loss of loading of CENP-A, the histone H3 variant found at active centromeres and the proposed epigenetic mark of centromere identity. Absence of condensins and/or CENP-A insufficiency produced a specific kinetochore defect, such that a functional mitotic checkpoint cannot prevent chromosome missegregation resulting from improper attachment of sister kinetochores to spindle microtubules. Spindle microtubule-dependent deformation of both inner kinetochores and the HEC1/Ndc80 microtubule-capturing module, then results in kinetochore separation from the Aurora B pool and ensuing reduced kinase activity at centromeres. Moreover, recovery from mitosis-inhibition by monastrol revealed a high incidence of merotelic attachment that was nearly identical with condensin depletion, Aurora B inactivation, or both, indicating that the Aurora B dysfunction is the key defect leading to chromosome missegregation in condensin-depleted cells. Thus, beyond a requirement for global chromosome condensation, condensins play a pivotal role in centromere assembly, proper spatial positioning of microtubule-capturing modules and positioning complexes of the inner centromere versus kinetochore plates.     

The p53-induced Gene Ei24 Is an Essential Component of the Basal Autophagy Pathway

Ei24 is a DNA damage response gene involved in growth suppression and apoptosis. The physiological function of Ei24, however, is poorly understood. Here we generated conditional knock-out mice of Ei24 and demonstrated that EI24 is an essential component of the basal autophagy pathway. Mice with neural-specific Ei24 deficiency develop age-dependent neurological abnormalities caused by massive axon degeneration and extensive neuron loss in brain and spinal cord. Notably, ablation of Ei24 leads to vacuolated oligodendroglial cells and demyelination of axons. Liver-specific depletion of Ei24 causes severe hepatomegaly with hepatocyte hypertrophy. Ei24 deficiency impairs autophagic flux, leading to accumulation of LC3, p62 aggregates, and ubiquitin-positive inclusions. Our study indicates that Ei24 is an essential autophagy gene and plays an important role in clearance of aggregate-prone proteins in neurons and hepatocytes.