Somatic piRNA and PIWI-mediated post-transcriptional gene regulation in stem cells and disease

Abstract

PIWI-interacting RNAs (piRNAs) are small non-coding RNAs that bind to the PIWI subclass of the Argonaute protein family and are essential for maintaining germline integrity. Initially discovered in Drosophila, PIWI proteins safeguard piRNAs, forming ribonucleoprotein (RNP) complexes, crucial for regulating gene expression and genome stability, by suppressing transposable elements (TEs). Recent insights revealed that piRNAs and PIWI proteins, known for their roles in germline maintenance, significantly influence mRNA stability, translation and retrotransposon silencing in both stem cells and bodily tissues. In the current review, we explore the multifaceted roles of piRNAs and PIWI proteins in numerous biological contexts, emphasizing their involvement in stem cell maintenance, differentiation, and the development of human diseases. Additionally, we discussed the up-and-coming animal models, beyond the classical fruit fly and earthworm systems, for studying piRNA-PIWIs in self-renewal and cell differentiation. Further, our review offers new insights and discusses the emerging roles of piRNA-dependent and independent functions of PIWI proteins in the soma, especially the mRNA regulation at the post-transcriptional level, governing stem cell characteristics, tumor development, and cardiovascular and neurodegenerative diseases.

Keywords: PIWI; disease; gene regulation; mRNA; piRNA; retrotransposons; stem cells.

FIGURE 1

PIWI proteins and their functional domains in the piRNA-PIWI complex, and the overview of the piRNA pathway in Drosophila. (A) Phylogenetic tree of the Argonaute (AGO) protein family with each class of AGOs specific to the type of small non-coding RNAs. Hs: Homo sapiens, Mm: Mus musculus, Sm: Schmidtea mediterranea, Dr: Danio rerio, Hv: Hydra vulgaris, Dm: Drosophila melanogaster, Dj: Dugesia japonica, Ce: Caenorhabditis elegans. (B) (left) The MID domain of PIWI proteins binds to the 5′phosphate group of piRNA (shown in blue), and the PAZ domain binds to the 2′-O-methylated 3′end of piRNA (right). The target RNA is captured by piRNA through base-pair complementarity, and the PIWI domain, which possesses ribonuclease activity, cleaves the target RNA (shown in red). (C) The piRNA biogenesis in Drosophila: First, the piRNA cluster on the chromosome is transcribed by the RNA Pol II to produce piRNA precursors with the assistance of proteins, namely Rhino (Rhi), Cutoff (Cuff), Deadlock (Del), Trf2, and Moonshiner (Moon). The piRNA precursors are transported out of the nucleus by proteins such as Nxf3 and Bootlegger (Boot), and their secondary structure is removed by Armitage (Armi), and cut by Zucchini to form piRNA intermediates. Following, some piRNA intermediates, after being trimmed by Nibbler (Nbr) and methylated at the 3′end by Hen1, bind to Piwi (PIWIL1 homolog) to form a Piwi-piRNA complex (i). Other piRNA intermediates bind to Aub (PIWIL2 homolog) and then enter the ping-pong cycle dominated by Aub and Ago3 (PIWI4 homolog) (ii). (D) The ping-pong cycle, widely conserved in zebrafish, mice, humans, and other animals, silences target RNAs (such as TE transcripts) and amplifies piRNAs. The initial piRNA precursor, transcribed by RNA Pol II is exported from the nucleus to the cell cytoplasm, where it is processed into a mature form. After binding to the Aub protein, the mature piRNA cleaves the target TE transcript. The fragments of the cleaved transcript, bound by the Ago3 protein, act as a template and facilitate the processing of the piRNA precursor. The processed piRNA precursor, bound by the Aub protein and cleaved with the help of the protein Trimmer, enters the cycle to continue the silencing of complementary transcripts and the generation of piRNAs.

FIGURE 2

The functions of PIWI proteins in planarian adult pluripotent stem cells (aPSCs) and mouse adult neural progenitor cells (aNPCs). (A) In planarian aPSCs (neoblasts), TEs are mainly silenced in the nucleus. The SMEDWI-2 complex binds to TEs that are being transcribed, induces H3K9 methylation, and prevents further transcription. For other TE transcripts that successfully enter the cytoplasm, the SMEDWI-1 complex silences them through the ping-pong cycle. In addition, poorly translated transcripts are recognized and silenced by the SMEDWI-1 complex, facilitated by its interaction with the small ribosomal subunit. (B) Knockdown of DNAJA1 in neoblasts reduced the abundance of SMEDWI-2 protein but had little effect on the expression levels of SMEDWI-2 transcripts. (C) The effects of SMEDWI-1 and SMEDWI-3 on mRNA in neoblasts: (i) SMEDWI-1 and SMEDWI-3 assist germinal histone H4 (gH4) transcripts in localizing to chromatoid bodies. (ii) SMEDWI-1 and SMEDWI-3, guided by the piRNAs, cleave mRNAs that code for all the histone proteins, Traf-6, and Npk1-like proteins. The cleaved mRNAs become templates for the newly generated piRNA-SMEDWI-1 complexes. (iii) The piRNA-SMEDWI-3 complexes bind other mRNAs, such as Y2R mRNA. However, no changes were observed in the mRNA levels, and this association’s outcome is yet to be determined (denoted as ?). (D) The piRNA-MILI complex is involved in the preservation of mouse aNPC pluripotency. (left) Artificial knockout of MILI causes aNPCs to differentiate into unhealthy astrocytes, also referred to, as reactive glia. (right) The piRNA-MILI complex inhibits protein synthesis in aNPCs by silencing tRNA, 5S rRNA, SINEB1 and mRNAs encoding ribosomal proteins, thereby slowing down their differentiation.

FIGURE 3

piRNA-PIWI-mediated mRNA regulation in Drosophila adult stem cells. (A) Aub activating mRNA translation in the early Drosophila embryo: The piRNA-Aub complex binds to complementary regions within mRNAs bound by the poly(A)-binding protein Wispy. When the mRNA needs to be activated, the Aub complex interacts with Wispy, and recruits eIF3d subunit, which is part of eIF3 complex, along with the 40S ribosomal subunit, thereby initiating the process of translation. (B) Role of JAK/STAT pathway and PIWI proteins in Drosophila ISCs: The JAK/STAT pathway activates ISC proliferation and induces the expression of Drosophila Piwi protein, which silences transposons and ensures gene stability. Knocking down Piwi led to the accumulation of multiple TE transcripts, including Ty3 retrotransposon, mdg1 and hetA.

FIGURE 4

piRNA-specific and independent function of PIWIs in stem cell differentiation. (A) PIWIL1 overexpression in glioblastoma stem cells promotes self-renewal and tumorigenic potential. Knockdown of PIWIL1 upregulates tumor suppressors, namely BTG2 and FBXW7, inhibiting GSC self-renewal and proliferation, while promoting differentiation and senescence. (B) piRNAs in the differentiation of BMSCs: piR-36741 promotes the differentiation of BMSCs into osteoblasts, while piR-63049 inhibits this process. (C) piRNAs in exosomes/microvesicles have anti-viral roles. Mouse NSCs excrete Ex/Mvs carrying piRNAs out of the cells via exocytosis. These Ex/Mvs specific piRNAs bind and degrade target viral RNAs of HIV and SARS-Cov-2. MILI was identified as cargo in Ex/Mvs, and however, its role in the piRNA-guided antiviral functions is yet to be demonstrated (denoted as ?).

FIGURE 5

piRNA and PIWI functions in emerging models of adult stem cells in animals. (A) In planarian neoblasts, the SMEDWI-1-piRNA complex binds to the small ribosome subunit to cleave rRNAs, snRNAs, or pseudogene mRNAs. The cleaved RNA fragments will form new SMEDWI-1-piRNA complexes and continue to participate in target RNA cleavage. (B) In actively dividing neoblasts, the expression of PIWI-1 and TSPAN-1 has been detected to increase simultaneously. However, the connection between them has not been fully established in terms of their expression changes and the downstream effects on neoblast differentiation into a variety of progenitor cells. (C) High piwi-1 expression was detected during the differentiation of neoblasts expressing the histone variant H3.3, into epidermal, muscle and neural cells. The likely interplay between piwi-1, H3.3 and TFs involved in the process, is yet to be explored (denoted as ?). (D) In Cnidaria, Tfap2 and Piwi-1 jointly induce i-cells to differentiate into germ cells, with little information known in terms of how Piwi-1 controls Tfap2 expression.

FIGURE 6

piRNA-PIWI-mediated regulation of mating-induced germline hyperactivity and somatic collapse in the aging of C. elegans. (i) Mating initiates hyperactivity in the germline. (ii) This germline hyperactivity leads to the downregulation of piRNAs. (iii) The downregulation of piRNAs results in the de-silencing and activation of Hedgehog-like ligands, such as wrt-1 and wrt-10. (iv) The activation of these Hedgehog-like ligands enhances Hedgehog signalling, which impacts somatic cells through specific receptors, PTR-6 and PTR-16. (v) The increased Hedgehog signalling in somatic cells triggers a cascade of events leading to somatic collapse, characterized by a decline in somatic cell function and health, ultimately accelerating aging and reducing lifespan.

FIGURE 7

piRNA and PIWI functions in cancer. (A) (i) PIWIL1 downregulates tumor suppressor mRNAs (VCL, LAMC3, FLNA, MYO18B, SRCIN1, TPM2) by recruiting the NMD machinery (SMG1, UPF2, UPF1), promoting gastric cancer progression. (ii) PIWIL1’s role in upregulating oncogene mRNAs (CCND3, ORC6, PCNA, CDC25A, MCM10) remains unexplored. (B) (i) PIWIL1/HIWI upregulation, along with piR-017724, is associated with the progression of hepatocellular carcinoma (HCC). (ii) Downregulation of PIWIL1/HIWI along with piR-017724 prevents the development of HCC. (C) PIWIL1/HIWI in pancreatic cancer: (i) The upregulation of piR-017061, which binds with PIWIL1, inhibits the mRNA coding for EFNA5. (ii) Conversely, the downregulation of piR-017061 resulted in the increased expression of EFNA5, an oncogene implicated in the progression of pancreatic cancer. (D) PIWIL2 in lung cancer: PIWIL2 induces the expression of CDK2 and Cyclin A, which inhibit apoptosis and prevent G2/M cycle arrest, thus progressing NSCLC. Inhibition of PIWIL2 leads to apoptosis and G2/M cycle arrest.

FIGURE 8

Dysregulated expression and functions of piRNA and PIWIs in other human diseases. (A) Dysregulated piRNAs and PIWI proteins in cardiovascular, neurodegenerative and respiratory tract diseases. (B) piRNA HNEAP in cardiomyocyte necroptosis: (i) HNEAP recruits DNMT1 to Atf7 mRNA, inhibiting its m⁵C methylation, which results in increased Atf7 mRNA transcription and protein expression. Whether HNEAP forms a complex with PIWIL2 and PIWIL4 is yet to be determined (denoted as ?). (ii) Elevated Atf7 expression inhibits the transcription of CHMP2A, an anti-necroptotic factor, leading to increased cardiomyocyte necroptosis. (iii) Knockdown of Atf7 reduces cardiomyocyte necroptosis induced by pathological stimuli, such as hypoxia/reoxygenation (H/R) exposure., indicating Atf7’s role as a pro-necroptotic transcription factor.