Biological significance of piRNA in liver cancer: a review

Abstract

Liver cancer is one of the most common and deadly cancers in the world. In recent years, non-coding RNA has been a hot topic in liver cancer research. piRNAs (PIWI-interacting RNAs) are a new type of small non-coding RNA, which are formed by the PIWI proteins interacting with RNA. The latest research shows that piRNA and PIWI proteins are abnormally expressed in various cancers, including pancreatic, colorectal, breast, etc. piRNA plays an important regulatory role in liver cancer. In this review, we discuss the biological function of piRNAs and new progress in the development of liver cancer, and new targets and ideas for piRNA and PIWI proteins in the diagnosis and treatment of liver cancer.     

PIWI/piRNA“机器”与雄性生殖细胞发育

PIWI(P-element-induced wimpy testis)蛋白在动物生殖系细胞中特异性表达,为动物生殖细胞发育分化所必需。piRNA(PIWI-interacting RNAs)是最近在动物生殖系细胞中发现的一类非编码小分子RNA,这类小RNA特异性地与PIWI家族蛋白相互作用。PIWI/piRNA"机器"通过沉默转座元件和调控编码mRNA等方式在动物生殖细胞发育分化过程中发挥重要作用。本文围绕PIWI/piRNA"机器"的生物学功能及分子机制,对近期取得的相关研究进展进行了系统性总结。     

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.     

Functional involvement of Tudor and dPRMT5 in the piRNA processing pathway in Drosophila germlines

In Drosophila, the PIWI proteins, Aubergine (Aub), AGO3, and Piwi are expressed in germlines and function in silencing transposons by associating with PIWI‐interacting RNAs (piRNAs). Recent studies show that PIWI proteins contain symmetric dimethyl‐arginines (sDMAs) and that dPRMT5/Capsuleen/DART5 is the modifying enzyme. Here, we show that Tudor (Tud), one of Tud domain‐containing proteins, associates with Aub and AGO3, specifically through their sDMA modifications and that these three proteins form heteromeric complexes. piRNA precursor‐like molecules are detected in these complexes. The expression levels of Aub and AGO3, along with their degree of sDMA modification, were not changed by tud mutations. However, the population of transposon‐derived piRNAs associated with Aub and AGO3 was altered by tud mutations, whereas the total amounts of small RNAs on Aub and AGO3 was increased. Loss of dprmt5 did not change the stability of Aub, but impaired its association with Tud and lowered piRNA association with Aub. Thus, in germline cells, piRNAs are quality‐controlled by dPRMT5 that modifies PIWI proteins, in tight association with Tud.     

Multifunctionality of PIWI proteins in control of germline stem cell fate

PIWI proteins interacting with specific type of small RNAs (piRNAs) repress transposable elements in animals. Besides, they have been shown to participate in various cellular processes: in the regulation of heterochromatin formation including telomere structures, in the control of translation and the cell cycle, and in DNA rearrangements. PIWI proteins were first identified by their roles in the self-renewal of germline stem cells. PIWI protein functions are not limited to gonadogenesis, but the role in determining the fate of stem cells is their specific feature conserved throughout the evolution of animals. Molecular mechanisms underlying these processes are far from being understood. This review focuses on the role of PIWI proteins in the control of maintenance and proliferation of germinal stem cells and its relation to the known function of PIWI in transposon repression.     

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.     

piRNA biogenesis during adult spermatogenesis in mice is independent of the ping-pong mechanism

piRNAs, a class of small non-coding RNAs associated with PIWI proteins, have broad functions in germline development, transposon silencing, and epigenetic regulation. In diverse organisms, a subset of piRNAs derived from repeat sequences are produced via the interplay between two PIWI proteins. This mechanism, termed “ping-pong” cycle, operates among the PIWI proteins of the primordial mouse testis; however, its involvement in postnatal testes remains elusive. Here we show that adult testicular piRNAs are produced independent of the ping-pong mechanism. We identified and characterized large populations of piRNAs in the adult and postnatal developing testes associated with MILI and MIWI, the only PIWI proteins detectable in these testes. No interaction between MILI and MIWI or sequence feature for the ping-pong mechanism among their piRNAs was detected in the adult testis. The majority of MILI- and MIWI-associated piRNAs originate from the same DNA strands within the same loci. Both populations of piRNAs are biased for 5′ Uracil but not for Adenine on the 10th nucleotide position, and display no complementarity. Furthermore, in Miwi mutants, MILI-associated piRNAs are not downregulated, but instead upregulated. These results indicate that the adult testicular piRNAs are predominantly, if not exclusively, produced by a primary processing mechanism instead of the ping-pong mechanism. In this primary pathway, biogenesis of MILI- and MIWI-associated piRNAs may compete for the same precursors; the types of piRNAs produced tend to be non-selectively dictated by the available precursors in the cell; and precursors with introns tend to be spliced before processed into piRNAs.     

Retrotransposons and piRNA: The missing link in central nervous system

From times when the whole genome were not available to the present explosion of genome knowledge, the biology of non-coding RNA molecules are an unknown ocean of gems. One among them are PIWI-interacting RNAs (piRNAs) that restrict the mobility of various retrotransposons. PIWI proteins and piRNAs once thought to be germline specific was now explored to be expressed in different somatic cells. Emerging proofs of piRNAs from central nervous system has raised serious questions regarding the role of retrotransposons and its silencing mechanism. In this review, we have focused on the existing knowledge of retrotransposons and piRNAs in the central nervous system and have provided future insights. Meta-analysis of retrotransposons in various mammalian genomes and piRNA targets showcased the abundance of LINE transposon and the possibility of piRNA mediated retrotransposon expression. Thus, understanding the retrotransposons-piRNA pathway will provide a new vision for the study of development, physiology and pathology of the central nervous system.     

Mitochondrial protein BmPAPI modulates the length of mature piRNAs

PIWI proteins and their associated PIWI-interacting RNAs (piRNAs) protect genome integrity by silencing transposons in animal germlines. The molecular mechanisms and components responsible for piRNA biogenesis remain elusive. PIWI proteins contain conserved symmetrical dimethylarginines (sDMAs) that are specifically targeted by TUDOR domain-containing proteins. Here we report that the sDMAs of PIWI proteins play crucial roles in PIWI localization and piRNA biogenesis in Bombyx mori-derived BmN4 cells, which harbor fully functional piRNA biogenesis machinery. Moreover, RNAi screenings for Bombyx genes encoding TUDOR domain-containing proteins identified BmPAPI, a Bombyx homolog of Drosophila PAPI, as a factor modulating the length of mature piRNAs. BmPAPI specifically recognized sDMAs and interacted with PIWI proteins at the surface of the mitochondrial outer membrane. BmPAPI depletion resulted in 3′-terminal extensions of mature piRNAs without affecting the piRNA quantity. These results reveal the BmPAPI-involved piRNA precursor processing mechanism on mitochondrial outer membrane scaffolds.     

PNLDC1, mouse pre‐piRNA Trimmer,is required for meiotic and post‐meiotic male germ cell development

PIWI‐interacting RNAs (piRNAs) are germ cell‐specific small RNAs essential for retrotransposon gene silencing and male germ cell development. In piRNA biogenesis, the endonuclease MitoPLD/Zucchini cleaves long, single‐stranded RNAs to generate 5′ termini of precursor piRNAs (pre‐piRNAs) that are consecutively loaded into PIWI‐family proteins. Subsequently, these pre‐piRNAs are trimmed at their 3′‐end by an exonuclease called Trimmer. Recently, poly(A)‐specific ribonuclease‐like domain‐containing 1 (PNLDC1) was identified as the pre‐piRNA Trimmer in silkworms. However, the function of PNLDC1 in other species remains unknown. Here, we generate Pnldc1 mutant mice and analyze small RNAs in their testes. Our results demonstrate that mouse PNLDC1 functions in the trimming of both embryonic and post‐natal pre‐piRNAs. In addition, piRNA trimming defects in embryonic and post‐natal testes cause impaired DNA methylation and reduced MIWI expression, respectively. Phenotypically, both meiotic and post‐meiotic arrests are evident in the same individual Pnldc1 mutant mouse. The former and latter phenotypes are similar to those of MILI and MIWI mutant mice, respectively. Thus, PNLDC1‐mediated piRNA trimming is indispensable for the function of piRNAs throughout mouse spermatogenesis.     

piRNA clusters as a main source of small RNAs in the animal germline

PIWI subfamily Argonaute proteins and small RNAs bound to them (PIWI interacting RNA, piRNA) control mobilization of transposable elements (TE) in the animal germline. piRNAs are generated by distinct genomic regions termed piRNA clusters. piRNA clusters are often extensive loci enriched in damaged fragments of TEs. New TE integration into piRNA clusters causes production of TE-specific piRNAs and repression of cognate sequences. piRNAs are thought to be generated from long single-stranded precursors encoded by piRNA clusters. Special chromatin structures might be essential to distinguish these genomic loci as a source for piRNAs. In this review, we present recent findings on the structural organization of piRNA clusters and piRNA biogenesis in Drosophila and other organisms, which are important for understanding a key epigenetic mechanism that provides defense against TE expansion.