Structures, functions and adaptations of the human LINE-1 ORF2 protein.

The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)1. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer2,3, autoimmunity4,5 and ageing6,7, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p 'core' (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production6-8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1.

Nectar bacteria stimulate pollen germination and bursting to enhance microbial fitness.

Many organisms consume pollen, yet mechanisms of its digestion remain a fundamental enigma in pollination biology,1-3 as pollen is protected by a recalcitrant outer shell.4-8 Pollen is commonly found in floral nectar,9,10 as are nectar microbes, which are nearly ubiquitous among flowers.11-13 Nectar specialist bacteria, like Acinetobacter, can reach high densities (up to 109 cells/mL), despite the fact that floral nectar is nitrogen poor.14-17 Here, we show evidence that the genus Acinetobacter, prevalent nectar- and bee-associated bacteria,12,18-20 can induce pollen germination and bursting, gain access to protoplasm nutrients, and thereby grow to higher densities. Although induced germination had been suggested as a potential method in macroscopic pollen consumers,2,21-23 and fungal inhibition of pollen germination has been shown,24-27 direct biological induction of germination has not been empirically documented outside of plants.28-32Acinetobacter pollinis SCC47719 induced over 5× greater pollen germination and 20× greater pollen bursting than that of uninoculated pollen by 45 min. When provided with germinable pollen, A. pollinis stimulates protein release and grows to nearly twice the density compared to growth with ungerminable pollen, indicating that stimulation of germination benefits bacterial fitness. In contrast, a common nectar-inhabiting yeast (Metschnikowia)33 neither induced nor benefited from pollen germination. We conclude that Acinetobacter both specifically causes and benefits from inducing pollen germination and bursting. Further study of microbe-pollen interactions may inform many aspects of pollination ecology, including floral microbial ecology,34,35 pollinator nutrient acquisition from pollen,2,3,21,36 and cues of pollen germination for plant reproduction.37-39.

The linker region of LINEs modulates DNA cleavage and DNA polymerization.

Long interspersed elements (LINEs) replicate by target primed reverse transcription (TPRT). Insertion involves two half reactions. Each half reaction involves DNA cleavage followed by DNA synthesis. The linker region, located just beyond the reverse transcriptase in the LINE open reading frame, contains a set of predicted helices that may form an α-finger, followed by a gag-like zinc-knuckle. Point mutations of moderately conserved amino-acid residues in the presumptive α-finger severely impair the DNA endonuclease and reverse transcriptase activities of the integration reaction during both half reactions. Mutations in the gag-like zinc-knuckle also impair DNA cleavage and DNA synthesis in some instances. Mutations in core residues that presumably disrupt the protein structure of the presumptive α-finger and the gag-like zinc-knuckle lead to a promiscuous DNA endonuclease and protein-nucleic acid complexes that get stuck in the well during analysis. The linker region appears to function as a protein, DNA, and RNA conformational switching area. The linker is used to properly position nucleic acid substrates into the active sites of the reverse transcriptase and of the DNA endonuclease.

Telomere-Specialized Retroelements in Drosophila: Adaptive Symbionts of the Genome, Neutral, or in Conflict?

Linear chromosomes shorten in every round of replication. In Drosophila, telomere-specialized long interspersed retrotransposable elements (LINEs) belonging to the jockey clade offset this shortening by forming head-to-tail arrays at Drosophila telomere ends. As such, these telomeric LINEs have been considered adaptive symbionts of the genome, protecting it from premature decay, particularly as Drosophila lacks a conventional telomerase holoenzyme. However, as reviewed here, recent work reveals a high degree of variation and turnover in the telomere-specialized LINE lineages across Drosophila. There appears to be no absolute requirement for LINE activity to maintain telomeres in flies, hence the suggestion that the telomere-specialized LINEs may instead be neutral or in conflict with the host, rather than adaptive.

Completion of LINE integration involves an open '4-way' branched DNA intermediate.

Long Interspersed Elements (LINEs), also known as non-LTR retrotransposons, encode a multifunctional protein that reverse transcribes its mRNA into DNA at the site of insertion by target primed reverse transcription. The second half of the integration reaction remains very poorly understood. Second-strand DNA cleavage and second-strand DNA synthesis were investigated in vitro using purified components from a site-specific restriction-like endonuclease (RLE) bearing LINE. DNA structure was shown to be a critical component of second-strand DNA cleavage. A hitherto unknown and unexplored integration intermediate, an open '4-way' DNA junction, was recognized by the element protein and cleaved in a Holliday junction resolvase-like reaction. Cleavage of the 4-way junction resulted in a natural primer-template pairing used for second-strand DNA synthesis. A new model for RLE LINE integration is presented.

Globular domain structure and function of restriction-like-endonuclease LINEs: similarities to eukaryotic splicing factor Prp8.

BACKGROUND: R2 elements are a clade of early branching Long Interspersed Elements (LINEs). LINEs are retrotransposable elements whose replication can have profound effects on the genomes in which they reside. No crystal or EM structures exist for the reverse transcriptase (RT) and linker regions of LINEs. RESULTS: Using limited proteolysis as a probe for globular domain structure, we show that the protein encoded by the Bombyx mori R2 element has two major globular domains: (1) a small globular domain consisting of the N-terminal zinc finger and Myb motifs, and (2) a large globular domain consisting of the RT, linker, and type II restriction-like endonuclease (RLE). Further digestion of the large globular domain occurred within the RT. Mapping these RT cleavages onto an updated model of the R2Bm RT indicated that the thumb of the RT was largely protected from proteolytic cleavage. The crystal structure of the large globular domain of Prp8, a eukaryotic splicing factor, was a major template used in building the R2Bm RT model, particularly the thumb region. The large fragment of Prp8 consists not only of a RT similar to R2Bm, but also an RLE and a linker connecting the two regions. The linker sequences adjacent to the RLE in LINEs and Prp8 share a set of two important α-helices and a (presumptive) knuckle/ßßα structural motif that are closely associated with the thumb. The RLEs of LINEs and Prp8 share a unique catalytic core residue spacing as well as other key residues. CONCLUSIONS: The protein encoded by RLE LINEs consists of two major globular domains. The larger of the two globular domain contains the RT, linker, and RLE and is similar to the large fragment of the spliceosomal protein Prp8. The similarities are suggestive of possible common ancestry.

Endonuclease domain of non-LTR retrotransposons: loss-of-function mutants and modeling of the R2Bm endonuclease.

Non-LTR retrotransposons are an important class of mobile elements that insert into host DNA by target-primed reverse transcription (TPRT). Non-LTR retrotransposons must bind to their mRNA, recognize and cleave their target DNA, and perform TPRT at the site of DNA cleavage. As DNA binding and cleavage are such central parts of the integration reaction, a better understanding of the endonuclease encoded by non-LTR retrotransposons is needed. This paper explores the R2 endonuclease domain from Bombyx mori using in vitro studies and in silico modeling. Mutations in conserved sequences located across the putative PD-(D/E)XK endonuclease domain reduced DNA cleavage, DNA binding and TPRT. A mutation at the beginning of the first α-helix of the modeled endonuclease obliterated DNA cleavage and greatly reduced DNA binding. It also reduced TPRT when tested on pre-cleaved DNA substrates. The catalytic K was located to a non-canonical position within the second α-helix. A mutation located after the fourth ß-strand reduced DNA binding and cleavage. The motifs that showed impaired activity form an extensive basic region. The R2 biochemical and structural data are compared and contrasted with that of two other well characterized PD-(D/E)XK endonucleases, restriction endonucleases and archaeal Holliday junction resolvases.

Independently derived targeting of 28S rDNA by A- and D-clade R2 retrotransposons: Plasticity of integration mechanism.

Restriction-like endonuclease (RLE) bearing non-LTR retrotransposons are site-specific elements that integrate into the genome through a target primed reverse transcription mechanism (TPRT). R2 elements have been used as a model system for investigating non-LTR retrotransposon integration. We previously demonstrated that R2 retrotransposons require two subunits of the element-encoded multifunctional protein to integrate-one subunit bound upstream of the insertion site and one bound downstream. R2 elements have been phylogenetically categorized into four clades: R2-A, B, C and D, that diverged from a common ancestor more than 850 million years ago. All R2 elements target the same sequence within 28S rDNA. The amino-terminal domain of R2Bm, an R2-D clade element, contains a single zinc finger and a Myb motif that are responsible for binding R2 protein downstream of the insertion site. Target site recognition is of interest as it is the first step in the integration reaction and may help elucidate evolutionary history and integration mechanism. The amino-terminal domain of R2-A clade members contains three zinc fingers and a Myb motif. We show here that R2Lp, an R2-A clade member, uses its amino-terminal DNA binding motifs to bind upstream of the insertion site. Because the R2-A and R2-D clade elements recognize 28S rDNA differently, we conclude the A- and D-clades represent independent targeting events to the 28S site. Our results also indicate a certain plasticity of insertional mechanics exists between the two clades.

Targeting novel sites: The N-terminal DNA binding domain of non-LTR retrotransposons is an adaptable module that is implicated in changing site specificities.

Restriction-like endonuclease (RLE) bearing non-LTR retrotransposons are site-specific elements that integrate into the genome through target primed reverse transcription (TPRT). RLE-bearing elements have been used as a model system for investigating non-LTR retrotransposon integration. R2 elements target a specific site in the 28S rDNA gene. We previously demonstrated that the two major sub-classes of R2 (R2-A and R2-D) target the R2 insertion site in an opposing manner with regard to the pairing of known DNA binding domains and bound sequences-indicating that the A- and D-clades represent independently derived modes of targeting that site. Elements have been discovered that group phylogenetically with R2 but do not target the canonical R2 site. Here we extend our earlier studies to show that a separate R2-A clade element, which targets a site other than the canonical R2 site, does so by using the N-terminal zinc fingers and Myb motifs. We further extend our targeting studies beyond R2 clade elements by investigating the ability of the N-terminal zinc fingers from the nematode NeSL-1 element to target its integration site. Our data are consistent with the use of an N-terminal DNA binding domain as one of the major targeting determinants used by RLE-bearing non-LTR retrotransposons to secure a protein subunit near the insertion site. This N-terminal DNA binding domain can undergo modifications, allowing the element to target novel sites. The binding orientation of the N-terminal domain relative to the insertion site is quite variable.

Convergently recruited nuclear transport retrogenes are male biased in expression and evolving under positive selection in Drosophila.

The analyses of gene duplications by retroposition have revealed an excess of male-biased duplicates generated from X chromosome to autosomes in flies and mammals. Investigating these genes is of primary importance in understanding sexual dimorphism and genome evolution. In a particular instance in Drosophila, X-linked nuclear transport genes (Ntf-2 and ran) have given rise to autosomal retroposed copies three independent times (along the lineages leading to Drosophila melanogaster, D. ananassae, and D. grimshawi). Here we explore in further detail the expression and the mode of evolution of these Drosophila Ntf-2- and ran-derived retrogenes. Five of the six retrogenes show male-biased expression. The ran-like gene of D. melanogaster and D. simulans has undergone recurrent positive selection. Similarly, in D. ananassae and D. atripex, the Ntf-2 and ran retrogenes show evidence of past positive selection. The data suggest that strong selection is acting on the origin and evolution of these retrogenes. Avoiding male meiotic X inactivation, increasing level of expression of X-linked genes in male testes, and/or sexual antagonism might explain the recurrent duplication of retrogenes from X to autosomes. Interestingly, the ran-like in D. yakuba has mostly pseudogenized alleles. Disablement of the ran-like gene in D. yakuba indicates turnover of these duplicates. We discuss the possibility that Dntf-2r and ran-like might be involved in genomic conflicts during spermatogenesis.

Secondary structures for 5' regions of R2 retrotransposon RNAs reveal a novel conserved pseudoknot and regions that evolve under different constraints.

Sequences from the 5' region of R2 retrotransposons of four species of silk moth are reported. In Bombyx mori, this region of the R2 messenger RNA contains a binding site for R2 protein and mediates interactions critical to R2 element insertion into the host genome. A model of secondary structure for a segment of this RNA is proposed on the basis of binding to oligonucleotide microarrays, chemical mapping, and comparative sequence analysis. Five conserved secondary structures are identified, including a novel pseudoknot. There is an apparent transition from an entirely RNA structure coding function in most of the 5' segment to a protein coding function near the 3' end. This suggests that local regions evolved under separate functional constraints (structural, coding, or both).

Isoenergetic penta- and hexanucleotide microarray probing and chemical mapping provide a secondary structure model for an RNA element orchestrating R2 retrotransposon protein function.

LNA (locked nucleic acids, i.e. oligonucleotides with a methyl bridge between the 2' oxygen and 4' carbon of ribose) and 2,6-diaminopurine were incorporated into 2'-O-methyl RNA pentamer and hexamer probes to make a microarray that binds unpaired RNA approximately isoenergetically. That is, binding is roughly independent of target sequence if target is unfolded. The isoenergetic binding and short probe length simplify interpretation of binding to a structured RNA to provide insight into target RNA secondary structure. Microarray binding and chemical mapping were used to probe the secondary structure of a 323 nt segment of the 5' coding region of the R2 retrotransposon from Bombyx mori (R2Bm 5' RNA). This R2Bm 5' RNA orchestrates functioning of the R2 protein responsible for cleaving the second strand of DNA during insertion of the R2 sequence into the genome. The experimental results were used as constraints in a free energy minimization algorithm to provide an initial model for the secondary structure of the R2Bm 5' RNA.

RNA from the 5' end of the R2 retrotransposon controls R2 protein binding to and cleavage of its DNA target site.

Non-LTR retrotransposons insert into eukaryotic genomes by target-primed reverse transcription (TPRT), a process in which cleaved DNA targets are used to prime reverse transcription of the element's RNA transcript. Many of the steps in the integration pathway of these elements can be characterized in vitro for the R2 element because of the rigid sequence specificity of R2 for both its DNA target and its RNA template. R2 retrotransposition involves identical subunits of the R2 protein bound to different DNA sequences upstream and downstream of the insertion site. The key determinant regulating which DNA-binding conformation the protein adopts was found to be a 320-nt RNA sequence from near the 5' end of the R2 element. In the absence of this 5' RNA the R2 protein binds DNA sequences upstream of the insertion site, cleaves the first DNA strand, and conducts TPRT when RNA containing the 3' untranslated region of the R2 transcript is present. In the presence of the 320-nt 5' RNA, the R2 protein binds DNA sequences downstream of the insertion site. Cleavage of the second DNA strand by the downstream subunit does not appear to occur until after the 5' RNA is removed from this subunit. We postulate that the removal of the 5' RNA normally occurs during reverse transcription, and thus provides a critical temporal link to first- and second-strand DNA cleavage in the R2 retrotransposition reaction.

Role of the Bombyx mori R2 element N-terminal domain in the target-primed reverse transcription (TPRT) reaction.

R2 is a site-specific non-long terminal repeat (non-LTR) retrotransposon encoding a single polypeptide with reverse transcriptase, DNA endonuclease and nucleic acid-binding domains. The current model of R2 retrotransposition involves an ordered series of cleavage and polymerization steps carried out by at least two R2 protein subunits, one bound upstream and one bound downstream of the integration site. The role in the retrotransposition reaction of two conserved DNA-binding motifs, a C2H2 zinc finger (ZF) and a Myb motif, located within the N-terminal domain of the protein are explored in this report. These motifs do not appear to play a role in RT or the ability of the protein to bind the R2 RNA transcript. Methylation and missing nucleoside interference-based DNA footprints using polypeptides to the N-terminal domain suggest the ZF and Myb motifs bind to regions -3 to -1 and +10 to +15 with reference to the insertion site. Mutations in these DNA sites or of the N-terminal protein domain blocked binding and the activity of the downstream subunit. Mutations of the protein domain also affected binding of the upstream subunit but not its function, suggesting the primary path to DNA target recognition by R2 involves both upstream and downstream subunits.

R2 target-primed reverse transcription: ordered cleavage and polymerization steps by protein subunits asymmetrically bound to the target DNA.

R2 elements are non-long terminal repeat retrotransposons that specifically insert into 28S rRNA genes of many animal groups. These elements encode a single protein with reverse transcriptase and endonuclease activities as well as specific DNA and RNA binding properties. In this report, gel shift experiments were conducted to investigate the stoichiometry of the DNA, RNA, and protein components of the integration reaction. The enzymatic functions associated with each of the protein complexes were also determined, and DNase I digests were used to footprint the protein onto the target DNA. Additionally, a short polypeptide containing the N-terminal putative DNA-binding motifs was footprinted on the DNA target site. These combined findings revealed that one protein subunit binds the R2 RNA template and the DNA 10 to 40 bp upstream of the insertion site. This subunit cleaves the first DNA strand and uses that cleavage to prime reverse transcription of the R2 RNA transcript. Another protein subunit(s) uses the N-terminal DNA binding motifs to bind to the 18 bp of target DNA downstream of the insertion site and is responsible for cleavage of the second DNA strand. A complete model for the R2 integration reaction is presented, which with minor modifications is adaptable to other non-LTR retrotransposons.