Cloning and expression profiling of small RNAs expressed in the mouse ovary

Small noncoding RNAs have been suggested to play important roles in the regulation of gene expression across all species from plants to humans. To identify small RNAs expressed by the ovary, we generated mouse ovarian small RNA complementary DNA (srcDNA) libraries and sequenced 800 srcDNA clones. We identified 236 small RNAs including 122 microRNAs (miRNAs), 79 piwi-interacting RNAs (piRNAs), and 35 small nucleolar RNAs (snoRNAs). Among these small RNAs, 15 miRNAs, 74 piRNAs, and 21 snoRNAs are novel. Approximately 70% of the ovarian piRNAs are encoded by multicopy genes located within the repetitive regions, resembling previously identified repeat-associated small interference RNAs (rasiRNAs), whereas the remaining approximately 30% of piRNA genes are located in nonrepetitive regions of the genome with characteristics similar to the majority of piRNAs originally cloned from the testis. Since these two types of piRNAs display different structural features, we categorized them into two classes: repeat-associated piRNAs (rapiRNAs, equivalent of the rasiRNAs) and non-repeat-associated piRNAs (napiRNAs). Expression profiling analyses revealed that ovarian miRNAs were either ubiquitously expressed in multiple tissues or preferentially expressed in a few tissues including the ovary. Ovaries appear to express more rapiRNAs than napiRNAs, and sequence analyses support that both may be generated through the "ping-pong" mechanism. Unique expression and structural features of these ovarian small noncoding RNAs suggest that they may play important roles in the control of folliculogenesis and female fertility.     

Cloning, characterization and widespread expression analysis of testicular piRNA-like chicken RNAs

Piwi-interacting RNAs (piRNAs) are small RNAs abundant in the germline that have been implicated in germline development and maintenance of genomic integrity across several animal species including human, mouse, rat, zebrafish and drosophila. Tens of thousands of piRNAs have been discovered, yet abundant piRNAs have still not been detected in various eukaryotic organisms. This is a report on the characterization, cloning and expression profiling of piRNA-like chicken RNAs. Here, we identified 19 piRNAs, each 23–39 nucleotides long, from chicken testis using a small RNA cDNA library and T-A cloning methods. Three different pilRNAs were selected according to size, homology and secondary structure for temporal and spatial expression by Q-PCR technology in different tissues at five growth and four development stages of Chinese indigenous Rugao chickens (RG) and introduced recessive white feather chickens (RW). We found that, consistent to other organisms, pilRNA-encoding sequences within the chicken genome were asymmetrically distributed on the chromosomes while displaying a preference for intergenic regions across the genome. Interestingly, unlike miRNAs with unique stem-loop structures (mature miRNAs form stem section and the rest form loop section), distinct secondary structures of pilRNAs were predicted. In addition, chicken pilRNAs were not only abundant in the germline but also existed in somatic tissues, where, expression levels were influenced mainly by different pilRNAs, breed and gender. Taken together, our results suggest that two distinct secondary structures exist between pilRNAs and miRNAs, which may clarify the splicing and processing mechanisms of the two small RNAs are possible different. Moreover, our results suggest that pilRNAs may not only be confined to development and maintenance of the germline but may also play important roles in somatic tissues. Additionally, different pilRNAs may be involved in the unique regulatory machinery of complex biological processes.     

Characterization of Sus scrofa Small Non-Coding RNAs Present in Both Female and Male Gonads

Small non-coding RNAs (sncRNAs) are indispensable for proper germ cell development, emphasizing the need for greater elucidation of the mechanisms of germline development and regulation of this process by sncRNAs. We used deep sequencing to characterize three families of small non-coding RNAs (piRNAs, miRNAs, and tRFs) present in Sus scrofa gonads and focused on the small RNA fraction present in both male and female gonads. Although similar numbers of reads were obtained from both types of gonads, the number of unique RNA sequences in the ovaries was several times lower. Of the sequences detected in the testes, 2.6% of piRNAs, 9% of miRNAs, and 10% of tRFs were also present in the ovaries. Notably, the majority of the shared piRNAs mapped to ribosomal RNAs and were derived from clustered loci. In addition, the most abundant miRNAs present in the ovaries and testes are conserved and are involved in many biological processes such as the regulation of homeobox genes, the control of cell proliferation, and carcinogenesis. Unexpectedly, we detected a novel sncRNA type, the tRFs, which are 30–36-nt RNA fragments derived from tRNA molecules, in gonads. Analysis of S. scrofa piRNAs show that testes specific piRNAs are biased for 5′ uracil but both testes and ovaries specific piRNAs are not biased for adenine at the 10^th nucleotide position. These observations indicate that adult porcine piRNAs are predominantly produced by a primary processing pathway or other mechanisms and secondary piRNAs generated by ping-pong mechanism are absent.     

PIWI-interacting small RNAs: the vanguard of genome defence

PIWI-interacting RNAs (piRNAs) are a distinct class of small non-coding RNAs that form the piRNA-induced silencing complex (piRISC) in the germ line of many animal species. The piRISC protects the integrity of the genome from invasion by 'genomic parasites'--transposable elements--by silencing them. Owing to their limited expression in gonads and their sequence diversity, piRNAs have been the most mysterious class of small non-coding RNAs regulating RNA silencing. Now, much progress is being made into our understanding of their biogenesis and molecular functions, including the specific subcellular compartmentalization of the piRNA pathway in granular cytoplasmic bodies.     

3' end formation of PIWI-interacting RNAs in vitro

PIWI-interacting RNAs (piRNAs) are 23-30 nucleotides small RNAs that act with PIWI proteins to silence transposon activity in animal gonads. In contrast to microRNAs and small interfering RNAs, the biogenesis of piRNAs, including how 3' ends are formed, remains largely unknown. Here, by using lysate from BmN4, a silkworm ovary-derived cell line, we have developed a cell-free system that recapitulates key steps of piRNA biogenesis: loading of long single-stranded precursor RNAs into PIWI proteins with 5'-nucleotide bias, followed by Mg(2+)-dependent 3' to 5' exonucleolytic trimming and 2'-O-methylation at 3' ends. Importantly, 3' end methylation is tightly coupled with trimming and yet is not a prerequisite for determining the mature piRNA length. Our system provides a biochemical framework for dissecting piRNA biogenesis.     

Small RNA profiling and characterization of piRNA clusters in the adult testes of the common marmoset,a model primate

Small RNAs mediate gene silencing by binding Argonaute/Piwi proteins to regulate target RNAs. Here, we describe small RNA profiling of the adult testes of Callithrix jacchus, the common marmoset. The most abundant class of small RNAs in the adult testis was piRNAs, although 353 novel miRNAs but few endo-siRNAs were also identified. MARWI, a marmoset homolog of mouse MIWI and a very abundant PIWI in adult testes, associates with piRNAs that show characteristics of mouse pachytene piRNAs. As in other mammals, most marmoset piRNAs are derived from conserved clustered regions in the genome, which are annotated as intergenic regions. However, unlike in mice, marmoset piRNA clusters are also found on the X chromosome, suggesting escape from meiotic sex chromosome inactivation by the X-linked clusters. Some of the piRNA clusters identified contain antisense-orientated pseudogenes, suggesting the possibility that pseudogene-derived piRNAs may regulate parental functional protein-coding genes. More piRNAs map to transposable element (TE) subfamilies when they have copies in piRNA clusters. In addition, the strand bias observed for piRNAs mapped to each TE subfamily correlates with the polarity of copies inserted in clusters. These findings suggest that pachytene piRNA clusters determine the abundance and strand-bias of TE-derived piRNAs, may regulate protein-coding genes via pseudogene-derived piRNAs, and may even play roles in meiosis in the adult marmoset testis.     

Piwi-Interacting RNAs (piRNAs) Are Dysregulated in Renal Cell Carcinoma and Associated with Tumor Metastasis and Cancer-Specific Survival

Piwi-interacting RNAs (piRNAs) are a distinct group of small noncoding RNAs (sncRNAs) that silence transposable genetic elements to protect genome integrity. Because of their limited expression in gonads and sequence diversity, piRNAs remain the most mysterious class of small RNAs. Studies have shown piRNAs are present in somatic cells and dysregulated in gastric, breast and liver cancers. By deep sequencing 24 frozen benign kidney and clear cell renal cell carcinoma (ccRCC) specimens and using the publically available piRNA database, we found 26,991 piRNAs present in human kidney tissue. Among 920 piRNAs that had at least two copies in one specimen, 19 were differentially expressed in benign kidney and ccRCC tissues, and 46 were associated with metastasis. Among the metastasis-related piRNAs, we found three piRNAs (piR-32051, piR-39894 and piR-43607) to be derived from the same piRNA cluster at chromosome 17. We confirmed the three selected piRNAs not to be miRNAs or miRNA-like sncRNAs. We further validated the aberrant expression of the three piRNAs in a 68-case formalin-fixed and paraffin-embedded (FFPE) ccRCC tissue cohort and showed the up-regulation of the three piRNAs to be highly associated with ccRCC metastasis, late clinical stage and poor cancer-specific survival.     

Mice deficient for a small cluster of Piwi-interacting RNAs implicate Piwi-interacting RNAs in transposon control

The mammalian testis expresses a class of small noncoding RNAs that interact with mammalian PIWI proteins. In mice, the PIWI-interacting RNAs (piRNAs) partner with mammalian PIWI proteins, PIWIL1 and PIWIL2, also known as MIWI and MILI, to maintain transposon silencing in the germline genome. Here, we demonstrate that inactivation of Nct1/2, two noncoding RNAs encoding piRNAs, leads to derepression of LINE-1 (L1) but does not affect mouse viability, spermatogenesis, testicular gene expression, or fertility. These findings indicate that piRNAs from a cluster on chromosome 2 are necessary to maintain transposon silencing.     

Identification and Characterization of piRNA-Like Small RNAs in the Gonad of Sea Urchin (Strongylocentrotus nudus)

Piwi-interacting RNAs (piRNAs) and their partner PIWI proteins play an essential role in fertility, germline stem cell development, as well as the basic control and evolution of animal genomes. However, research was rare with regard to piRNA population in sea urchin, a model animal intensively used for development and genetics studies. Utilizing Solexa sequencing, we present an identification of 13,051 piRNA-like RNAs expressed in male gonad of Strongylocentrotus nudus. Out of 202 tested RNAs, 94 sequences were confirmed to express in female gonad using microarray assay, suggesting that both male and female gonads are piRNA-like RNA-enriched organs. These RNAs with "U" at the 5' end or "A" at position of 10, in size from 26 to 30 nucleotides, were predominantly 28?nt in length and tend to be clustered in small regions in genome, achieving the longest piRNA-like RNA-enriched region about 5.5?kb in scaffold78427. Alignment results showed 11 RNAs were homologous to the known piRNAs. Furthermore, BLASTn searching against sea urchin repeat element database showed these piRNA-like RNAs matched to 101 types of DNA transposons and retrotransposons, of which SPRP1, Harbinger-N2, piggyBac-N10, SINE2-1, and piggyBac-N11 were the most frequent hit elements, suggesting a transposon silencing function of these piRNA-like RNAs.     

The mouse homolog of HEN1 is a potential methylase for Piwi-interacting RNAs

Piwi-interacting RNAs (piRNAs) are a novel class of small regulatory RNAs that are expressed specifically and abundantly in germ cells. Mammalian piRNAs are 26-31 nucleotides in length and bind to Piwi proteins, but their function and biogenesis remain elusive. We previously showed that mammalian piRNAs are 2'-O-methylated at their 3' termini. The biosynthetic mechanism and function of this modification is unknown. Here, we report that the mouse homolog (mHEN1) of HEN1, a plant microRNA (miRNA) 2'-O-methyltransferase, is expressed specifically in testis and methylates 3' termini of piRNAs in vitro. These findings provide insight into the biogenesis of piRNAs.     

Novel rapidly evolving hominid RNAs bind nuclear factor 90 and display tissue-restricted distribution

Nuclear factor 90 (NF90) is a double-stranded RNA-binding protein implicated in multiple cellular functions, but with few identified RNA partners. Using in vivo cross-linking followed by immunoprecipitation, we discovered a family of small NF90-associated RNAs (snaR). These highly structured non-coding RNAs of ~117 nucleotides are expressed in immortalized human cell lines of diverse lineages. In human tissues, they are abundant in testis, with minor distribution in brain, placenta and some other organs. Two snaR subsets were isolated from human 293 cells, and additional species were found by bioinformatic analysis. Their genes often occur in multiple copies arranged in two inverted regions of tandem repeats on chromosome 19. snaR-A is transcribed by RNA polymerase III from an intragenic promoter, turns over rapidly, and shares sequence identity with Alu RNA and two potential piRNAs. It interacts with NF90's double-stranded RNA-binding motifs. snaR orthologs are present in chimpanzee but not other mammals, and include genes located in the promoter of two chorionic gonadotropin hormone genes. snaRs appear to have undergone accelerated evolution and differential expansion in the great apes.     

Evolution of animal Piwi-interacting RNAs and prokaryotic CRISPRs

Piwi-interacting RNAs (piRNAs) and CRISPR RNAs (crRNAs) are two recently discovered classes of small noncoding RNA that are found in animals and prokaryotes, respectively. Both of these novel RNA species function as components of adaptive immune systems that protect their hosts from foreign nucleic acids-piRNAs repress transposable elements in animal germlines, whereas crRNAs protect their bacterial hosts from phage and plasmids. The piRNA and CRISPR systems are nonhomologous but rather have independently evolved into logically similar defense mechanisms based on the specificity of targeting via nucleic acid base complementarity. Here we review what is known about the piRNA and CRISPR systems with a focus on comparing their evolutionary properties. In particular, we highlight the importance of several factors on the pattern of piRNA and CRISPR evolution, including the population genetic environment, the role of alternate defense systems and the mechanisms of acquisition of new piRNAs and CRISPRs.     

Small non-coding RNAs in animal development

The modulation of gene expression by small non-coding RNAs is a recently discovered level of gene regulation in animals and plants. In particular, microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs) have been implicated in various aspects of animal development, such as neuronal, muscle and germline development. During the past year, an improved understanding of the biological functions of small non-coding RNAs has been fostered by the analysis of genetic deletions of individual miRNAs in mammals. These studies show that miRNAs are key regulators of animal development and are potential human disease loci.     

Bombyx small RNAs: genomic defense system against transposons in the silkworm, Bombyx mori

Selfish genetic elements called transposons can insert themselves at new locations in host genomes to modify gene structure and alter gene expression. Expansion of transposons can occur when novel transposition events are transmitted to subsequent generations after germline hopping. Therefore, organisms seem likely to have evolved defense mechanisms to silence transposons in the germline. Recently, small RNAs interacting with Piwi proteins (piwi-interacting RNAs: piRNAs) have been demonstrated to be involved in genomic defense mechanism against transposons. Here, we show that piRNA-like small RNAs are present abundantly in the Bombyx ovary. We cloned 38,493 kinds of Bombyx small RNA from the ovary and performed functional characterization. Bombyx small RNAs showed a unimodal length distribution with a peak at 28nt and a strong bias for U at the 5' end. We found that 12,869 kinds of Bombyx small RNAs were associated with transposons or repetitive sequences. We classified them as repeat-associated small interfering RNAs (rasiRNAs), a subclass of piRNAs. Notably, antisense rasiRNAs have a strong bias toward U at 5' ends; in contrast, sense rasiRNAs have a strong bias toward A at nucleotide position 10, indicating that the piRNA amplification loop proposed in Drosophila is evolutionarily conserved in Bombyx. These results suggest that Bombyx small RNAs regulate transposon activity.