Interaction and cross-talk between non-coding RNAs.

Non-coding RNA (ncRNA) has been shown to regulate diverse cellular processes and functions through controlling gene expression. Long non-coding RNAs (lncRNAs) act as a competing endogenous RNAs (ceRNAs) where microRNAs (miRNAs) and lncRNAs regulate each other through their biding sites. Interactions of miRNAs and lncRNAs have been reported to trigger decay of the targeted lncRNAs and have important roles in target gene regulation. These interactions form complicated and intertwined networks. Certain lncRNAs encode miRNAs and small nucleolar RNAs (snoRNAs), and may regulate expression of these small RNAs as precursors. SnoRNAs have also been reported to be precursors for PIWI-interacting RNAs (piRNAs) and thus may regulate the piRNAs as a precursor. These miRNAs and piRNAs target messenger RNAs (mRNAs) and regulate gene expression. In this review, we will present and discuss these interactions, cross-talk, and co-regulation of ncRNAs and gene regulation due to these interactions

The biogenesis pathway of tRNA-derived piRNAs in Bombyx germ cells.

Transfer RNAs (tRNAs) function in translational machinery and further serves as a source of short non-coding RNAs (ncRNAs). tRNA-derived ncRNAs show differential expression profiles and play roles in many biological processes beyond translation. Molecular mechanisms that shape and regulate their expression profiles are largely unknown. Here, we report the mechanism of biogenesis for tRNA-derived Piwi-interacting RNAs (td-piRNAs) expressed in Bombyx BmN4 cells. In the cells, two cytoplasmic tRNA species, tRNAAspGUC and tRNAHisGUG, served as major sources for td-piRNAs, which were derived from the 5\u27-part of the respective tRNAs. cP-RNA-seq identified the two tRNAs as major substrates for the 5\u27-tRNA halves as well, suggesting a previously uncharacterized link between 5\u27-tRNA halves and td-piRNAs. An increase in levels of the 5\u27-tRNA halves, induced by BmNSun2 knockdown, enhanced the td-piRNA expression levels without quantitative change in mature tRNAs, indicating that 5\u27-tRNA halves, not mature tRNAs, are the direct precursors for td-piRNAs. For the generation of tRNAHisGUG-derived piRNAs, BmThg1l-mediated nucleotide addition to -1 position of tRNAHisGUG was required, revealing an important function of BmThg1l in piRNA biogenesis. Our study advances the understanding of biogenesis mechanisms and the genesis of specific expression profiles for tRNA-derived ncRNAs

Computational methods for analyzing small RNAs and their interaction partners with large-scale techniques

This thesis describes the computational tools and analyzes developed to characterize small regulatory RNAs and their interaction partners using large-scale techniques. Following an introduction into the emerging world of small regulatory RNAs, our methodology for analyzing small RNAs from deep-sequencing data is described (chapter 2). This methodology allows the classification of small RNAs obtained by sequencing and provides several downstream analysis tools such as expression profiling and miRNA sample comparison. It has been integrated with a miRNA target prediction method into a web server which allows one to explore tissue-specific miRNA targeting (chapter 3). In the fourth chapter, an experimental procedure for genome-wide identification of miRNA targets is outlined. With this procedure, we identified the mRNAs, that are targeted by the most abundant miRNAs in HEK293 cells. Importantly, the experimental protocol enabled us to identify the exact location of the miRNA-mRNA interaction site within the mRNA as well as the precise position of the mRNA-protein crosslink. The fifth and sixth chapter describe our studies of murine embryonic stem cells and oocytes that are devoid of Dicer. The murine specific miR-290 cluster has been identified as an important regulator in embryonic stem cells. The loss of pluripotency in Dicer-/- embryonic stem cells has been linked to primary and secondary targets of the miR-290 cluster. In contrast, our analysis of Dicer-/- oocytes revealed that the miRNA pathway plays only a minor part during oocyte maturation, and loss of Dicer affects mainly the endo-siRNA pathway. Finally, we reanalyzed piRNA sequence reads from various species (chapter 6). This analysis revealed an unexpected 19 nt long processing product which is generated during piRNA biogenesis

Computational discovery of animal small RNA genes and targets

Though recently discovered, small RNAs appear to play a wealth of regulatory roles, being involved in degradation of target mRNAs, translation silencing of target genes, chromatin remodeling and transposon silencing. Presented here are the computational tools that I developed to annotate and characterize small RNA genes and to identify their targets. One of these tools is oligomap, a novel software for fast and exhaustive identi�cation of nearly-perfect matches of small RNAs in sequence databases. Oligomap is part of an automated annotation pipeline used in our laboratory to annotate small RNA sequences. The application of these tools to samples of small RNAs obtained from mouse and human germ cells together with subsequent computational analyses lead to the discovery of a new class of small RNAs which are now called piRNAs. The computational analysis revealed that piRNAs have a strong uridine preference at their 5' end, that unlike miRNAs, piRNAs are not excised from fold-back precursors but rather from long primary transcripts, and that the genome organization of their genes is conserved between human and mouse even though piRNAs on the sequence level are poorly conserved. In vertebrates, the most studied class of small regulatory RNAs are the miRNAs which bind to mRNAs and block translation. A computational framework is introduced to identify miRNA targets in mammals, ies, worms and �sh. The method uses extensive cross species conservation information to predict miRNA binding sites that are under evolutionary pressure. A downstream analysis of predicted miRNA targets revealed novel properties of miRNA target sites, one of which is a positional bias of miRNA target sites in long mammalian 3' untranslated regions. Intersection of our predictions with biochemical pathway annotation data suggested novel functions for some of the miRNAs. To gain further insights into the mechanism of miRNA targeting, I studied microarray data obtained in siRNA experiments. SiRNAs have been shown to produce o�- targets that resemble miRNA targets. This analysis suggests the presence of additional determinants of miRNA target site functionality (beyond complementarity between the miRNA 5' end and the target) in the close vicinity (about 150 nucleotides) of the miRNA-complementary site. Finally, as part of a study aiming to reduce siRNA o�-target e�ects by introducing chemical modi�cations in the siRNA, I performed microarray data analysis of siRNA transfection experiments. Presented are the methods used to quantify o�- target activity of siRNAs carrying di�erent types of chemical modi�cations. The analysis revealed that o�-targets caused by the passenger strand of the siRNA can be reduced by 5'-O-methylation

Increasing cell density globally enhances the biogenesis of Piwi-interacting RNAs in Bombyx mori germ cells.

Piwi proteins and their bound Piwi-interacting RNAs (piRNAs) are predominantly expressed in the germline and play crucial roles in germline development by silencing transposons and other targets. Bombyx mori BmN4 cells are culturable germ cells that equip the piRNA pathway. Because of the scarcity of piRNA-expressing culturable cells, BmN4 cells are being utilized for the analyses of piRNA biogenesis. We here report that the piRNA biogenesis in BmN4 cells is regulated by cell density. As cell density increased, the abundance of Piwi proteins and piRNA biogenesis factors was commonly upregulated, resulting in an increased number of perinuclear nuage-like granules where Piwi proteins localize. Along with these phenomena, the abundance of mature piRNAs also globally increased, whereas levels of long piRNA precursor and transposons decreased, suggesting that increasing cell density promotes piRNA biogenesis pathway and that the resultant accumulation of mature piRNAs is functionally significant for transposon silencing. Our study reveals a previously uncharacterized link between cell density and piRNA biogenesis, designates cell density as a critical variable in piRNA studies using BmN4 cell system, and suggests the alteration of cell density as a useful tool to monitor piRNA biogenesis and function

Domestic chickens activate a piRNA defense against avian leukosis virus

PIWI-interacting RNAs (piRNAs) protect the germ line by targeting transposable elements (TEs) through the base-pair complementarity. We do not know how piRNAs co-evolve with TEs in chickens. Here we reported that all active TEs in the chicken germ line are targeted by piRNAs, and as TEs lose their activity, the corresponding piRNAs erode away. We observed de novo piRNA birth as host responds to a recent retroviral invasion. Avian leukosis virus (ALV) has endogenized prior to chicken domestication, remains infectious, and threatens poultry industry. Domestic fowl produce piRNAs targeting ALV from one ALV provirus that was known to render its host ALV resistant. This proviral locus does not produce piRNAs in undomesticated wild chickens. Our findings uncover rapid piRNA evolution reflecting contemporary TE activity, identify a new piRNA acquisition modality by activating a pre-existing genomic locus, and extend piRNA defense roles to include the period when endogenous retroviruses are still infectious. DOI: http://dx.doi.org/10.7554/eLife.24695.00

Unveiling Molecular Mechanisms of piRNA Pathway from Small Signals in Big Data: A Dissertation

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\u27 cleavage products are not degraded but loaded into the third PIWI protein Argonaute3 (Ago3). It is believed that an unidentified nuclease trims the 3\u27 ends of those cleavage products to 23–29 nt, becoming mature piRNAs remained in Ago3. Such piRNAs whose 5\u27 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

Using Experimental and Computational Strategies to Understand the Biogenesis of microRNAs and piRNAs: A Dissertation

Small RNAs are single-stranded, 18–36 nucleotide RNAs that can be categorized as miRNA, siRNA, and piRNA. miRNA are expressed ubiquitously in tissues and at particular developmental stages. They fine-tune gene expression by regulating the stability and translation of mRNAs. piRNAs are mainly expressed in the animal gonads and their major function is repressing transposable elements to ensure the faithful transfer of genetic information from generation to generation. My thesis research focused on the biogenesis of miRNAs and piRNAs using both experimental and computational strategies. The biogenesis of miRNAs involves sequential processing of their precursors by the RNase III enzymes Drosha and Dicer to generate miRNA/miRNA* duplexes, which are subsequently loaded into Argonaute proteins to form the RNA-induced silencing complex (RISC). We discovered that, after assembled into Ago1, more than a quarter of Drosophila miRNAs undergo 3′ end trimming by the 3′-to-5′ exoribonuclease Nibbler. Such trimming occurs after removal of the miRNA* strand from pre-RISC and may be the final step in RISC assembly, ultimately enhancing target messenger RNA repression. Moreover, by developing a specialized Burrow-Wheeler Transform based short reads aligner, we discovered that in the absence of Nibbler a subgroup of miRNAs undergoes increased tailing—non-templated nucleotide addition to their 3′ ends, which are usually associated with miRNA degradation. Therefore, the 3′ trimming by Nibbler might increase miRNA stability by protecting them from degradation. In Drosophila germ line, piRNAs associate with three PIWI-clade Argonaute proteins, Piwi, Aub, and Ago3. piRNAs bound by Aub and Ago3 are generated by reciprocal cleavages of sense and antisense transposon transcripts (a.k.a., the “Ping-Pong” cycle), which amplifies piRNA abundance and degrades transposon transcripts in the cytoplasm. On the other hand, Piwi and its associated piRNA repress the transcription of transposons in the nucleus. We discovered that Aub- and Ago3-mediated transposon RNA cleavage not only generates piRNAs bound to each other, but also produces substrates for the endonuclease Zucchini, which processively cleaves those substrates in a periodicity of ~26 nt and generates piRNAs that predominantly load into Piwi. Without Aub or Ago3, the abundance of Piwi-bound piRNAs drops and transcriptional silencing is compromised. Our discovery revises the current model of piRNA biogenesis

Gene Expression Profiling in Cancer

The contribution of modern-day genetics in designing efficient gene expression profiles for cancer is immense. The progress of technology and science in recent years provides the opportunity for discovery and application of new techniques for treating various diseases that affect humanity. Methods for finding and analyzing the profile of gene expression of infected cells give scientists the ability to develop more targeted and effective treatments, especially for diseases such as cancer. The development of gene expression profiling is one of the most important achievements in cancer genetics in our time. It is essentially the driving force behind personalized and precision medicine. This book highlights recent developments, applications, and breakthroughs in the field of gene expression profiling in cancer

Small Noncoding RNA Expression in Cancer

Despite an inability to encode proteins, small noncoding RNAs (sncRNAs) have critical functions in the regulation of gene expression. They have demonstrated roles in cancer development and progression and are frequently dysregulated. Here we review the biogenesis and mechanism of action, expression patterns, and detection methods of two types of sncRNAs frequently described in cancer: miRNAs and piRNAs. Both miRNAs and piRNAs have been observed to play both oncogenic and tumor-suppressive roles, with miRNAs acting to directly regulate the mRNA of key cancer-associated genes, while piRNAs play crucial roles in maintaining the integrity of the epigenetic landscape. Elucidating these important functions of sncRNAs in normal and cancer biology relies on numerous in silico workflows and tools to profile sncRNA expression. Thus, we also discuss the key detection methods for cancer-relevant sncRNAs, including the discovery of genes that have yet to be described