RNase L restricts the mobility of engineered retrotransposons in cultured human cells

Retrotransposons are mobile genetic elements, and their mobility can lead to genomic instability. Retrotransposon insertions are associated with a diverse range of sporadic diseases, including cancer. Thus, it is not a surprise that multiple host defense mechanisms suppress retrotransposition. The 2′,5′-oligoadenylate (2-5A) synthetase (OAS)-RNase L system is a mechanism for restricting viral infections during the interferon antiviral response. Here, we investigated a potential role for the OAS-RNase L system in the restriction of retrotransposons. Expression of wild type (WT) and a constitutively active form of RNase L (NΔ385), but not a catalytically inactive RNase L mutant (R667A), impaired the mobility of engineered human LINE-1 (L1) and mouse intracisternal A-type particle retrotransposons in cultured human cells. Furthermore, WT RNase L, but not an inactive RNase L mutant (R667A), reduced L1 RNA levels and subsequent expression of the L1-encoded proteins (ORF1p and ORF2p). Consistently, confocal immunofluorescent microscopy demonstrated that WT RNase L, but not RNase L R667A, prevented formation of L1 cytoplasmic foci. Finally, siRNA-mediated depletion of endogenous RNase L in a human ovarian cancer cell line (Hey1b) increased the levels of L1 retrotransposition by ∼2-fold. Together, these data suggest that RNase L might function as a suppressor of structurally distinct retrotransposons.     

Ty1-copia group retrotransposons and the evolution of retroelements in the eukaryotes

Ty1-copia group retrotransposons are among the best studied transposable elements in the eukaryotes. This review discusses the extent of these transposons in the eukaryote kingdoms and compares models for the evolution of these genetic elements in the light of recent phylogenetic data. These data show that the Ty1-copia group is widespread among invertebrate eukaryotes, especially in the higher plant kingdom, where these genetic elements are unusually common and heterogeneous in their sequence. The phylogenetic data also suggest that the present day spectrum of Ty1-copia group retrotransposons has been influenced both by divergence during vertical transmission down evolving lineages and by horizontal transmission between distantly related species. Lastly, the factors affecting Ty1-copia group retrotransposon copy number and sequence heterogeneity in eukaryotic genomes and the effects of transpositional quiescence and defective retrotransposons upon evolution of Ty1-copia group retrotransposons are discussed.     

Heterochromatin restricts the mobility of nuclear bodies

Nuclear bodies are relatively immobile organelles. Here, we investigated the mechanisms underlying their movement using experimentally induced interphase prenucleolar bodies (iPNBs). Most iPNBs demonstrated constrained diffusion, exhibiting infrequent fusions with other iPNBs and nucleoli. Fusion events were actin-independent and appeared to be the consequence of stochastic collisions between iPNBs. Most iPNBs were surrounded by condensed chromatin, while fusing iPNBs were usually found in a single heterochromatin-delimited compartment (“cage”). The experimentally induced over-condensation of chromatin significantly decreased the frequency of iPNB fusion. Thus, the data obtained indicate that the mobility of nuclear bodies is restricted by heterochromatin.     

A widespread occurrence of extra open reading frames in plant Ty3/gypsy retrotransposons

Long terminal repeat (LTR) retrotransposons make up substantial parts of most higher plant genomes where they accumulate due to their replicative mode of transposition. Although the transposition is facilitated by proteins encoded within the gag-pol region which is common to all autonomous elements, some LTR retrotransposons were found to potentially carry an additional protein coding capacity represented by extra open reading frames located upstream or downstream of gag-pol. In this study, we performed a comprehensive in silico survey and comparative analysis of these extra open reading frames (ORFs) in the group of Ty3/gypsy LTR retrotransposons as the first step towards our understanding of their origin and function. We found that extra ORFs occur in all three major lineages of plant Ty3/gypsy elements, being the most frequent in the Tat lineage where most (77?%) of identified elements contained extra ORFs. This lineage was also characterized by the highest diversity of extra ORF arrangement (position and orientation) within the elements. On the other hand, all of these ORFs could be classified into only two broad groups based on their mutual similarities or the presence of short conserved motifs in their inferred protein sequences. In the Athila lineage, the extra ORFs were confined to the element 3' regions but they displayed much higher sequence diversity compared to those found in Tat. In the lineage of Chromoviruses the extra ORFs were relatively rare, occurring only in 5' regions of a group of elements present in a single plant family (Poaceae). In all three lineages, most extra ORFs lacked sequence similarities to characterized gene sequences or functional protein domains, except for two Athila-like elements with similarities to LOGL4 gene and part of the Chromoviruses extra ORFs that displayed partial similarity to histone H3 gene. Thus, in these cases the extra ORFs most likely originated by transduction or recombination of cellular gene sequences. In addition, the protein domain which is otherwise associated with DNA transposons have been detected in part of the Tat-like extra ORFs, pointing to their origin from an insertion event of a mobile element.     

A complex of nuclear pore proteins required for pore function

A family of proteins bearing novel N-acetylglucosamine residues has previously been found to be required to form functional nuclear pores. To begin to determine which of the proteins in this family are essential for pore function, antisera were raised to each of three members of the family, p62, p58, and p54. With these antisera, it was possible to deplete nuclear reconstitution extracts of the proteins and to test the depleted nuclei for nuclear transport. In the course of the experiments, it was found that the three proteins exist as a complex; antisera to any one, while specific on a protein blot, coimmunoprecipitated all three proteins. This complex of pore proteins is stable to 2 M salt, 2 M urea, and the detergent Mega 10, indicating the presence of specific and tight protein-protein interactions. By gel filtration, the complex has a molecular mass of 550-600 kD. Nuclei containing pores depleted of the complex are found to be defective for nuclear transport; moreover, we observe a strict linear correlation between the amount of complex present in nuclei and the amount of nuclear transport of which those nuclei are capable. Thus, the p62-p58-p54 complex defines a group of proteins with strong protein-protein interactions that form a unit of pore structure essential for pore function.     

Modular evolution of the integrase domain in the Ty3/Gypsy class of LTR retrotransposons

A phylogenetic analysis of the Ty3/Gypsy group of retrotransposons identified a conserved domain (GPY/F) present in the integrases of several members of this group as well as of certain vertebrate retroviruses. The analysis suggested an evolutionary scheme for the acquisition and loss of the GPY/F domain as well as the acquisition of a chromodomain module in the integrase encoded by this group of elements that may direct targeting specificity in the host genome.     

Evolutionary dynamics of the Ty3/gypsy LTR retrotransposons in the genome of Anopheles gambiae

Ty3/gypsy elements represent one of the most abundant and diverse LTR-retrotransposon (LTRr) groups in the Anopheles gambiae genome, but their evolutionary dynamics have not been explored in detail. Here, we conduct an in silico analysis of the distribution and abundance of the full complement of 1045 copies in the updated AgamP3 assembly. Chromosomal distribution of Ty3/gypsy elements is inversely related to arm length, with densities being greatest on the X, and greater on the short versus long arms of both autosomes. Taking into account the different heterochromatic and euchromatic compartments of the genome, our data suggest that the relative abundance of Ty3/gypsy LTRrs along each chromosome arm is determined mainly by the different proportions of heterochromatin, particularly pericentric heterochromatin, relative to total arm length. Additionally, the breakpoint regions of chromosomal inversion 2La appears to be a haven for LTRrs. These elements are underrepresented more than 7-fold in euchromatin, where 33% of the Ty3/gypsy copies are associated with genes. The euchromatin on chromosome 3R shows a faster turnover rate of Ty3/gypsy elements, characterized by a deficit of proviral sequences and the lowest average sequence divergence of any autosomal region analyzed in this study. This probably reflects a principal role of purifying selection against insertion for the preservation of longer conserved syntenyc blocks with adaptive importance located in 3R. Although some Ty3/gypsy LTRrs show evidence of recent activity, an important fraction are inactive remnants of relatively ancient insertions apparently subject to genetic drift. Consistent with these computational predictions, an analysis of the occupancy rate of putatively older insertions in natural populations suggested that the degenerate copies have been fixed across the species range in this mosquito, and also are shared with the sibling species Anopheles arabiensis.     

Lnx-2b restricts gsc expression to the dorsal mesoderm by limiting Nodal and Bozozok activity

Coordinated Nodal-related signals and Bozozok (Boz) activity are critical for the initial specification of dorsal mesoderm and anterior neuroectoderm during zebrafish embryogenesis. Overexpression of Boz expands gsc expression into the ventro-lateral marginal blastomeres where Nodal signaling is active, but is insufficient to induce ectopic gsc expression in the animal region. We found that overexpression of Boz together with depletion of Lnx-2b (previously named Lnx-like, Lnx-l), but not each manipulation alone, causes robust gsc expression in all blastomeres. Furthermore, nodal-related signals are required for gsc expression in embryos with elevated Boz activity. Through targeted injection into single cells at the 128-cell stage we illustrate the role of maternally deposited Lnx-2b to restrict the expansion of gsc expression into the presumptive ectodermal region. This report provides a novel mechanism for limiting dorsal organizer specification to a defined region of the early zebrafish embryo.     

Isolation and characterization of two novel retrotransposons of the Ty1-copia group in oat genomes

Two repetitive sequences, As32 and As22, of 826 and 742 bp, respectively, were isolated from Avena strigosa (As genome). Databank searches revealed their high homology to different segments of the family of Ty1-copia retrotransposons. Southern hybridization showed them to be present in diploid and polyploid oat species. Polymerase chain reaction with primers designed to amplify the segment between them showed that As32 and As22 sequences are composed of two different Ty1-copia retrotransposons. The segment amplified from the pAs32 insert was 2,264 bp long and contained the entire GAG and AP domains, and more than half of the IN domain. This new element has been designated TAS-1 (transposon, A. strigosa, 1) and appears to contain a long open reading frame that encodes a polypeptide of 625 amino acids. Slot-blot and fluorescence in situ hybridization analyses revealed it to be a component of both A- and D-genome chromosomes. Further, the chromosomes involved in one C-A intergenomic translocation in A. murphyi (AC genomes), one C-D intergenomic translocation in A. byzantina cv. Kanota (ACD genomes), and two C-D intergenomic translocations in A. sativa cv. Extra Klock, were identified. Based on its physical distribution and Southern hybridization pattern, a parental retro-transposon represented by TAS-1 appears to have been active at least twice during the evolution of the genomes in species of Avena.