Cílem práce je seznámení se s problematikou uchovávání informace v DNA, provést rešerši na téma transpozony, bioinformatické nástroje a algoritmy, které jsou používány k jejich detekci v nasekvenovaných genomech a vytvořit tak stručný úvod do obsáhle problematiky, včetně jejího zasazení do kontextu současně probíhajícího výzkumu v dané oblasti. Na základě přehledu stávajících algoritmů a nástrojů pro detekci transpozonů je navržen a implementován nástroj pro hledání tzv. LTR transpozonů.

Upon fertilization, the early embryo sustains most of the cellular processes using the maternally deposited reserves in the egg itself until the zygotic gene expression takes charge. Among the plethora of essential components provided by the mother are small non-coding RNAs called PIWI-interacting RNAs (piRNAs), which provide immunity to the zygote against transposon challenge. In this thesis, I have presented three different functions of piRNAs in Drosophila melanogaster- in maintenance of genomic integrity, telomere protection and their role as an adaptive immune system against genomic parasites. In Chapter 2, I have described the phenotypic effects of the loss of piRNA function in early embryos. The mutations affecting the piRNA pathway are known to cause embryonic lethality. To describe this lethality in detail, I have shown that all the characterized piRNA mutants show compromised zygotic genomic integrity during early embryogenesis. In addition, two piRNA pathway components, Aubergine (Aub) and Armitage (Armi) are also required for telomere resolution during early embryogenesis. Aub and Armi recruit telomeric protection complex proteins, HOAP and HP1, to the telomeric ends and thus avoid activation of the Non-homologous end joining (NHEJ) DNA repair pathway at the telomeres. There are about 120 transposon families in Drosophila melanogaster and piRNA pathway mutations cause activation of many of the resident transposons in the genome. In Chapter 3, I have described the effects of infection by a single transposon, P-element, in naïve strains by introduction through the zygote. Activation of the P-element leads to desilencing of unrelated transposons, causing accumulation of germline DNA damage which is linked to severely reduced fertility in the hybrid females. However, there is partial restoration of fertility as the hybrid progeny age, which correlates with P-element piRNA production and thus P-element silencing. Additionally, a number of transposons mobilize into piRNA generating heterochromatic clusters in the genome, and these insertions are stably inherited in the progeny. Collectively our data shows that piRNA production can be triggered in the adults in an absence of maternal contribution and that piRNAs serve as an adaptive immune system which helps resolve an internal genetic conflict between the host and the parasite. In an effort to understand the phenotypic effects of piRNA dysfunction in Drosophila, we have uncovered new exciting roles for piRNAs in development and presented evidence how transposons can act as architects in restructuring the host genome.

Small interfering RNAs (siRNAs), microRNAs (miRNAs), and piRNAs (piRNA) are conserved classes of small single-stranded ~21-30 nucleotide (nt) RNA guides that repress eukaryotic gene expression using distinct RNA Induced Silencing Complexes (RISCs). At its core, RISC is composed of a single-stranded small RNA guide bound to a member of the Argonaute protein family, which together bind and repress complementary target RNA. miRNAs target protein coding mRNAs—a function essential for normal development and broadly involved in pathways of human disease; small interfering RNAs (siRNA) defend against viruses, but can also be engineered to direct experimental or therapeutic gene silencing; piwi associated RNAs (piRNAs) protect germline genomes from expansion of parasitic nucleic acids such as transposons. Using the fruit fly, Drosophila melanogaster, as a model organism we seek to understand how small silencing RNAs are made and how they function. In Drosophila, miRNAs and siRNAs are proposed to have parallel, but separate biogenesis and effector machinery. miRNA duplexes are excised from imperfectly paired hairpin precursors by Dicer1 and loaded into Ago1; siRNA duplexes are hewn from perfectly paired long dsRNA by Dicer2 and loaded into Ago2. Contrary to this model we found one miRNA, miR-277, is made by Dicer1, but partitions between Ago1 and Ago2 RISCs. These two RISCs are functionally distinct—Ago2 could silence a perfectly paired target, but not a centrally bulged target; Ago1 could silence a bulged target, but not a perfect target. This was surprising since both Ago1 and Ago2 have endonucleolytic cleavage activity necessary for perfect target cleavage in vitro. Our detailed kinetic studies suggested why—Ago2 is a robust multiple turnover enzyme, but Ago1 is not. Along with a complementary in vitro study our data supports a duplex sorting mechanism in which Diced duplexes are released, and rebind to Ago1 or Ago2 loading machinery, regardless of which Dicer produced them. This allows structural information embedded in small RNA duplexes to direct small RNA loading into Ago1 and/or Ago2, resulting in distinct regulatory outputs. Small RNA sorting also has chemical consequences for the small RNA guide. Although siRNAs were presumed to have the signature 2′, 3′ hydroxyl ends left by Dicer, we found that small RNAs loaded into Ago2 or Piwi proteins, but not Ago1, are modified at their 3´ ends by the RNA 2´-O-methyltransferase DmHen1. In plants Hen1 modifies the 3´ ends all small RNAs duplexs, protecting and stabilizing them. Implying a similar function in flies, piRNAs are smaller, less abundant, and their function is perturbed in hen1 mutants. But unlike plants, small RNAs are modified as single-strands in RISC rather than as duplexes. This nicely explains why the dsRNA binding domain in plant Hen1 was discarded in animals, and why both dsRNA derived siRNAs and ssRNA derived piRNAs are modified. The recent discovery that both piRNAs and siRNAs target transposons links terminal modification and transposon silencing, suggesting that it is specialized for this purpose.

Transposon silencing is required to maintain genome stability. The non-coding piRNAs effectively suppress of transposon activity during germline development. In the Drosophila female germline, long precursors of piRNAs are transcribed from discrete heterochromatic clusters and then processed into primary piRNAs in the perinuclear nuage. However, the detailed mechanism of piRNA biogenesis, specifically how the nuclear and cytoplasmic processes are connected, is not well understood. The nuclear DEAD box protein UAP56 has been previously implicated in protein-coding gene transcript splicing and export. I have identified a novel function of UAP56 in piRNA biogenesis. In Drosophila egg chambers, UAP56 co-localizes with the cluster-associated HP1 variant Rhino. Nuage is a germline-specific perinuclear structure rich in piRNA biogenesis proteins, including Vasa, a DEAD box with an established role in piRNA production. Vasa-containing nuage granules localize directly across the nuclear envelope from cluster foci containing UAP56 and Rhino, and cluster transcripts immunoprecipitate with both Vasa and UAP56. Significantly, a charge-substitution mutation that alters a conserved surface residue in UAP56 disrupts co-localization with Rhino, germline piRNA production, transposon silencing, and perinuclear localization of Vasa. I therefore propose that UAP56 and Vasa function in a piRNA-processing compartment that spans the nuclear envelope.

PIWI-interacting RNAs (piRNA) are a group of 23–35 nucleotide (nt) short RNAs that protect animal gonads from transposon activities. In Drosophila germ line, piRNAs can be categorized into two different categories— primary and secondary piRNAs— based on their origins. Primary piRNAs, generated from transcripts of specific genomic regions called piRNA clusters, which are enriched in transposon fragments that are unlikely to retain transposition activity. The transcription and maturation of primary piRNAs from those cluster transcripts are poorly understood. After being produced, a group of primary piRNAs associates Piwi proteins and directs them to repress transposons at the transcriptional level in the nucleus. Other than their direct role in repressing transposons, primary piRNAs can also initiate the production of secondary piRNA. piRNAs with such function are loaded in a second PIWI protein named Aubergine (Aub). Similar to Piwi, Aub is guided by piRNAs to identify its targets through base-pairing. Differently, Aub functions in the cytoplasm by cleaving transposon mRNAs. The 5' cleavage products are not degraded but loaded into the third PIWI protein Argonaute3 (Ago3). It is believed that an unidentified nuclease trims the 3' ends of those cleavage products to 23–29 nt, becoming mature piRNAs remained in Ago3. Such piRNAs whose 5' ends are generated by another PIWI protein are named secondary piRNAs. Intriguingly, secondary piRNAs loaded into Ago3 also cleave transposon mRNA or piRNA cluster transcripts and produce more secondary piRNAs loaded into Aub. This reciprocal feed-forward loop, named the “Ping-Pong cycle”, amplified piRNA abundance. By dissecting and analyzing data from large-scale deep sequencing of piRNAs and transposon transcripts, my dissertation research elucidates the biogenesis of germline piRNAs in Drosophila. How primary piRNAs are processed into mature piRNAs remains enigmatic. I discover that primary piRNA signal on the genome display a fixed periodicity of ~26 nt. Such phasing depends on Zucchini, Armitage and some other primary piRNA pathway components. Further analysis suggests that secondary piRNAs bound to Ago3 can initiate phased primary piRNA production from cleaved transposon RNAs. The first ~26 nt becomes a secondary piRNA that bind Aub while the subsequent piRNAs bind Piwi, allowing piRNAs to spread beyond the site of RNA cleavage. This discovery adds sequence diversity to the piRNA pool, allowing adaptation to changes in transposon sequence. We further find that most Piwi-associated piRNAs are generated from the cleavage products of Ago3, instead of being processed from piRNA cluster transcripts as the previous model suggests. The cardinal function of Ago3 is to produce antisense piRNAs that direct transcriptional silencing by Piwi, rather to make piRNAs that guide post-transcriptional silencing by Aub. Although Ago3 slicing is required to efficiently trigger phased piRNA production, an alternative, slicing-independent pathway suffices to generate Piwi-bound piRNAs that repress transcription of a subset of transposon families. The alternative pathway may help flies silence newly acquired transposons for which they lack extensively complementary piRNAs. The Ping-Pong model depicts that first ten nucleotides of Aub-bound piRNAs are complementary to the first ten nt of Ago3-bound piRNAs. Supporting this view, piRNAs bound to Aub typically begin with Uridine (1U), while piRNAs bound to Ago3 often have adenine at position 10 (10A). Furthermore, the majority of Ping-Pong piRNAs form this 1U:10A pair. The Ping-Pong model proposes that the 10A is a consequence of 1U. By statistically quantifying those target piRNAs not paired to g1U, we discover that 10A is not directly caused by 1U. Instead, fly Aub as well as its homologs, Siwi in silkmoth and MILI in mice, have an intrinsic preference for adenine at the t1 position of their target RNAs. On the other hand, this t1A (and g10A after loading) piRNA directly give rise to 1U piRNA in the next Ping-Pong cycle, maximizing the affinity between piRNAs and PIWI proteins.