Search

Supplementary Table 1. Primers used for qPCR reactions and piRNA mimics used for in vitro functional analyses.Supplementary Table 2. Characteristics of GliomaScan subjects included in piRNA SNP imputation and association analysis. Supplementary Table 3. Differentially expressed transcripts 24-hours post piR-598-WT or NC transfection in U87 (FDR-adjusted P < 0.05) by fold-change. Supplementary Figure 1. Flowchart of piRNA SNP selection, imputation, and analysis for association study. Supplementary Figure 2. Quantile-quantile (QQ) plot of observed vs. expected chi-square distribution from association analysis of input genotyping data. Supplementary Figure 3. piR-18913, piR-598, piR-11714, and piR-3266 expression in U87, A172, and NHA cell lines by qPCR. Supplementary Figure 4. Expression array validation results. Five genes were selected for validation of expression array results by qPCR.

Background: PIWI-interacting RNAs (piRNAs), the largest class of noncoding RNAs in mammals, cooperate with PIWI proteins to safeguard the genome from insertional mutations during germline development. Although a growing number of studies have linked the PIWI–piRNA pathway to carcinogenesis, the role of piRNAs in glioma has not been explored.Methods: Utilizing directly measured and imputed genotypes from the GliomaScan genome-wide association study (1,840 cases and 2,401 controls), genetic variants in 1,428 piRNAs were analyzed for association with glioma risk. In vitro assays were performed to interrogate the functional impact of a top identified piRNA and its variant allele.Results: Variants in five piRNAs were considered to be associations of interest and four of these showed narrow clusters of enhanced association signals surrounding the index variant. Functional analyses of one of these piRNAs, piR-598, revealed that transfection of the wild-type piRNA impacted expression of genes involved in cell death/survival and reduced glioma cell viability and colony formation. However, upon delivery of piR-598 containing the variant allele at rs147061479 [OR, 1.80; 95% confidence interval (CI), 1.33–2.46; P = 1.69 × 10−4], cell proliferation was sharply increased.Conclusions: The genetic association analysis identifies several piRNAs associated with glioma risk, and follow-up functional analyses suggest that variant rs147061479 in piR-598 increases glioma risk by abolishing the tumor-suppressive function of piR-598, instead conferring growth-promoting properties.Impact: This transdisciplinary study demonstrates a role of piRNAs in gliomagenesis by evidence from both post-GWAS and in vitro functional analyses and supports expanded investigation into the link between the PIWI–piRNA pathway and cancer. Cancer Epidemiol Biomarkers Prev; 25(7); 1073–80. ©2016 AACR.

In our 20

Transcription and splicing of pre-messenger RNA are closely coordinated, but how this functional coupling is disrupted in human disease remains unexplored. Here, we investigated the impact of non-synonymous mutations in SF3B1 and U2AF1, two commonly mutated splicing factors in cancer, on transcription. We find that the mutations impair RNA Polymerase II (RNAPII) transcription elongation along gene bodies leading to transcription-replication conflicts, replication stress and altered chromatin organization. This elongation defect is linked to disrupted pre-spliceosome assembly due to impaired association of HTATSF1 with mutant SF3B1. Through an unbiased screen, we identified epigenetic factors in the Sin3/HDAC complex, which, when modulated, normalize transcription defects and their downstream effects. Our findings shed light on the mechanisms by which oncogenic mutant spliceosomes impact chromatin organization through their effects on RNAPII transcription elongation and present a rationale for targeting the Sin3/HDAC complex as a potential therapeutic strategy.GRAPHICAL ABSTRACTHIGHLIGHTS-Oncogenic mutations of SF3B1 and U2AF1 cause a gene-body RNAPII elongation defect-RNAPII transcription elongation defect leads to transcription replication conflicts, DNA damage response, and changes to chromatin organization and H3K4me3 marks-The transcription elongation defect is linked to disruption of the early spliceosome formation through impaired interaction of HTATSF1 with mutant SF3B1.-Changes to chromatin organization reveal potential therapeutic strategies by targeting the Sin3/HDAC pathway

Targeted cancer therapy aims to achieve specific elimination of cancerous but not normal cells. Recently, PIWI proteins, a subfamily of the PAZ-PIWI Domain (PPD) protein family, have emerged as promising candidates for targeted cancer therapy. PPD proteins are essential for small non-coding RNA pathways. The Argonaute subfamily partners with microRNA and small interfering RNA, whereas the PIWI subfamily partners with PIWI-interacting RNA (piRNA). Both PIWI proteins and piRNA are mostly expressed in the germline and best known for their function in transposon silencing, with no detectable function in mammalian somatic tissues. However, PIWI proteins become aberrantly expressed in multiple types of somatic cancers, thus gaining interest in targeted therapy. Despite this, little is known about the regulatory mechanism of PIWI proteins in cancer. Here we report that one of the four PIWI proteins in humans, PIWIL1, is highly expressed in gastric cancer tissues and cell lines. Knocking out PIWIL1 expression (PIWIL1-KO) drastically reduces gastric cancer cell proliferation, migration, metastasis, and tumorigenesis. RNA deep sequencing of gastric cancer cell line SNU-1 reveals that PIWIL1-KO significantly changes the transcriptome, causing the up-regulation of most of its associated transcripts. Surprisingly, fewbona fidepiRNAs exist in gastric cancer cells. Furthermore, abolishing the piRNA-binding activity of PIWIL1 does not affect its oncogenic function. Thus, PIWIL1 function in gastric cancer cells is independent of piRNA. This piRNA-independent regulation involves interaction with the UPF1-mediated nonsense-mediated mRNA decay (NMD) mechanism. Altogether, our findings reveal a novel and piRNA-independent function of PIWIL1 in promoting gastric cancer.SIGNIFICANCEPrecision medicine aims to cure cancer without affecting normal tissues. PIWI proteins provide a promising opportunity for precision medicine because they are normally expressed only in the testis for male fertility but gain expression in diverse types of cancers. Thus, inhibitingPIWIexpression may stop cancer development (and spermatogenesis) without affecting normal body function. To establish causality between PIWI and cancer, we show here that the expression of PIWIL1, a human PIWI protein, promotes gastric cancer. Surprisingly, this oncogenic function does not require piRNA, the expected partner of PIWI proteins, but involves the nonsense-mediated mRNA decay mechanism. These findings reveal a new function and action mechanism of PIWI proteins in oncogenesis, guiding the identification of PIWI inhibitors to cure cancer.

Gene regulation in embryonic stem cells (ESCs) has been extensively studied at the epigenetic-transcriptional level, but not at the posttranscriptional level. Pumilio (Pum) proteins are among the few known translational regulators required for stem-cell maintenance in invertebrates and plants. Here we report the essential function of two murine Pum proteins, Pum1 and Pum2, in ESCs and early embryogenesis. Pum1/2 double-mutant ESCs display severely reduced self-renewal and differentiation, and Pum1/2 double-mutant mice are developmentally delayed at the morula stage and lethal by embryonic day 8.5. Remarkably, Pum1-deficient ESCs show increased expression of pluripotency genes but not differentiation genes, whereas Pum2-deficient ESCs show decreased pluripotency markers and accelerated differentiation. Thus, despite their high homology and overlapping target messenger RNAs (mRNAs), Pum1 promotes differentiation while Pum2 promotes self-renewal in ESCs. Pum1 and Pum2 achieve these two complementary aspects of pluripotency by forming a negative interregulatory feedback loop that directly regulates at least 1,486 mRNAs. Pum1 and Pum2 regulate target mRNAs not only by repressing translation, but also by promoting translation and enhancing or reducing mRNA stability of different target mRNAs. Together, these findings reveal distinct roles of individual mammalian Pum proteins in ESCs and their essential functions in ESC pluripotency and embryogenesis.

Splicing factor mutations are common among cancers, recently emerging as drivers of myeloid malignancies. U2AF1 carries hotspot mutations in its RNA-binding motifs; however, how they affect splicing and promote cancer remain unclear. The U2AF1/U2AF2 heterodimer is critical for 3' splice site (3'SS) definition. To specifically unmask changes in U2AF1 function in vivo, we developed a crosslinking and immunoprecipitation procedure that detects contacts between U2AF1 and the 3'SS AG at single-nucleotide resolution. Our data reveal that the U2AF1 S34F and Q157R mutants establish new 3'SS contacts at -3 and +1 nucleotides, respectively. These effects compromise U2AF2-RNA interactions, resulting predominantly in intron retention and exon exclusion. Integrating RNA binding, splicing, and turnover data, we predicted that U2AF1 mutations directly affect stress granule components, which was corroborated by single-cell RNA-seq. Remarkably, U2AF1-mutant cell lines and patient-derived MDS/AML blasts displayed a heightened stress granule response, pointing to a novel role for biomolecular condensates in adaptive oncogenic strategies.

Hydrocephalus, characterized by cerebral ventricular dilatation, is routinely attributed to primary defects in cerebrospinal fluid (CSF) homeostasis. This fosters CSF shunting as the leading reason for brain surgery in children despite considerable disease heterogeneity. In this study, by integrating human brain transcriptomics with whole-exome sequencing of 483 patients with congenital hydrocephalus (CH), we found convergence of CH risk genes in embryonic neuroepithelial stem cells. Of all CH risk genes, TRIM71/lin-41 harbors the most de novo mutations and is most specifically expressed in neuroepithelial cells. Mice harboring neuroepithelial cell-specific Trim71 deletion or CH-specific Trim71 mutation exhibit prenatal hydrocephalus. CH mutations disrupt TRIM71 binding to its RNA targets, causing premature neuroepithelial cell differentiation and reduced neurogenesis. Cortical hypoplasia leads to a hypercompliant cortex and secondary ventricular enlargement without primary defects in CSF circulation. These data highlight the importance of precisely regulated neuroepithelial cell fate for normal brain-CSF biomechanics and support a clinically relevant neuroprogenitor-based paradigm of CH.

Transposable elements (TEs) can damage genomes, thus organisms use a variety of mechanisms to repress TE expression. The PIWI–piRNA pathway is a small RNA pathway that represses TE expression in the germline of animals. Here we explore the function of the pathway in the somatic stem cells of Hydra, a long-lived freshwater cnidarian. Hydra have three stem cell populations, all of which express PIWI proteins; endodermal and ectodermal epithelial stem cells (ESCs) are somatic, whereas the interstitial stem cells have germline competence. To study somatic function of the pathway, we isolated piRNAs from Hydra that lack the interstitial lineage and found that these somatic piRNAs map predominantly to TE transcripts and display the conserved sequence signatures typical of germline piRNAs. Three lines of evidence suggest that the PIWI–piRNA pathway represses TEs in Hydra ESCs. First, epithelial knockdown of the Hydra piwi gene hywi resulted in up-regulation of TE expression. Second, degradome sequencing revealed evidence of PIWI-mediated cleavage of TE RNAs in epithelial cells using the ping-pong mechanism. Finally, we demonstrated a direct association between Hywi protein and TE transcripts in epithelial cells using RNA immunoprecipitation. Altogether, our data reveal that the PIWI–piRNA pathway represses TE expression in the somatic cell lineages of Hydra, which we propose contributes to the extreme longevity of the organism. Furthermore, our results, in combination with others, suggest that somatic TE repression is an ancestral function of the PIWI–piRNA pathway.

PIWI proteins are key regulators of germline and somatic stem cells throughout different evolutionary lineages. However, how PIWI proteins themselves are regulated remains largely unknown. To identify candidate proteins that interact with PIWI proteins and regulate their stability, here we established a yeast two-hybrid (Y2H) assay in the planarian species Schmidtea mediterranea. We show that DNAJA1, a heat shock protein 40 family member, interacts with the PIWI protein SMEDWI-2, as validated by the Y2H screen and co-immunoprecipitation assays. We found that DNAJA1 is enriched in planarian adult stem cells, the nervous system, and intestinal tissues. DNAJA1-knockdown abolished planarian regeneration and homeostasis, compromised stem cell maintenance and PIWI-interacting RNA (piRNA) biogenesis, and deregulated SMEDWI-1/2 target genes. Mechanistically, we observed that DNAJA1 is required for the stability of SMEDWI-1 and SMEDWI-2 proteins. Furthermore, we noted that human DNAJA1 binds to Piwi-like RNA-mediated gene silencing 1 (PIWIL1) and is required for PIWIL1 stability in human gastric cancer cells. In summary, our results reveal not only an evolutionarily conserved functional link between PIWI and DNAJA1 that is essential for PIWI protein stability and piRNA biogenesis, but also an important role of DNAJA1 in the control of proteins involved in stem cell regulation.

In many animals, germline development is initiated by proteins and RNAs that are expressed maternally. PIWI proteins and their associated small noncoding PIWI-interacting RNAs (piRNAs), which guide PIWI to target RNAs by base-pairing, are among the maternal components deposited into the germline of the early embryo in Drosophila. Piwi has been extensively studied in the adult ovary and testis, where it is required for transposon suppression, germline stem cell self-renewal, and fertility. Consequently, loss of Piwi in the adult ovary using piwi-null alleles or knockdown from early oogenesis results in complete sterility, limiting investigation into possible embryonic functions of maternal Piwi. In this study, we show that the maternal Piwi protein persists in the embryonic germline through gonad coalescence, suggesting that maternal Piwi can regulate germline development beyond early embryogenesis. Using a maternal knockdown strategy, we find that maternal Piwi is required for the fertility and normal gonad morphology of female, but not male, progeny. Following maternal piwi knockdown, transposons were mildly derepressed in the early embryo but were fully repressed in the ovaries of adult progeny. Furthermore, the maternal piRNA pool was diminished, reducing the capacity of the PIWI/piRNA complex to target zygotic genes during embryogenesis. Examination of embryonic germ cell proliferation and ovarian gene expression showed that the germline of female progeny was partially masculinized by maternal piwi knockdown. Our study reveals a novel role for maternal Piwi in the germline development of female progeny and suggests that the PIWI/piRNA pathway is involved in germline sex determination in Drosophila.

Somatic mutations in splicing factors are of significant interest in myeloid malignancies and other cancers. U2AF1, together with U2AF2, is essential for 3’ splice site recognition. U2AF1 mutations result in aberrant splicing, but the molecular mechanism and the full spectrum of consequences on RNA biology have not been fully elucidated to date. We performed multi-omics profiling of in vivo RNA binding, splicing and turnover for U2AF1 S34F and Q157R mutants. We dissected specific binding signals of U2AF1 and U2AF2 and showed that U2AF1 mutations individually alter U2AF1-RNA binding, resulting in defective U2AF2 recruitment. We demonstrated a complex relationship between differential binding and splicing, expanding upon the currently accepted loss-of-binding model. Finally, we observed that U2AF1 mutations increase the formation of stress granules in both cell lines and primary acute myeloid leukemia samples. Our results uncover U2AF1 mutation-dependent pathogenic RNA mechanisms and provide the basis for developing targeted therapeutic strategies.

High-throughput sequencing reveals the complex landscape of small noncoding RNAs (sRNAs). However, it is limited by requiring 5′-monophosphate and 3′-hydroxyl in RNAs for adapter ligation and hindered by methylated nucleosides that interfere with reverse transcription. Here we develop Cap-Clip acid pyrophosphatase (Cap-Clip), T4 polynucleotide kinase (PNK) and AlkB/AlkB(D135S)-facilitated small ncRNA sequencing (CPA-seq) to detect and quantify sRNAs with terminus multiplicities and nucleoside methylations. CPA-seq identified a large number of previously undetected sRNAs. Comparison of sRNAs with or without AlkB/AlkB(D135S) treatment reveals nucleoside methylations on sRNAs. Using CPA-seq, we profiled the sRNA transcriptomes (sRNomes) of nine mouse tissues and reported the extensive tissue-specific differences of sRNAs. We also observed the transition of sRNomes during hepatic reprogramming. Knockdown of mesenchymal stem cell-enriched U1-5′ snsRNA promoted hepatic reprogramming. CPA-seq is a powerful tool with high sensitivity and specificity for profiling sRNAs with methylated nucleosides and diverse termini.

PUMILIO (PUM) proteins belong to the highly conserved PUF family post-transcriptional regulators involved in diverse biological processes. However, their function in carcinogenesis remains under explored. Here, we found that the expression of Pum1 and Pum2 are increased in clinical colorectal cancer (CRC). Intestine-specific knockout of Pum1 and Pum2 significantly inhibited the progression of colitis associated cancer in the AOM/DSS model. Knockout or knockdown of Pum1 and/or Pum2 resulted in a significant decrease in the tumorigenicity. In addition, delayed G1/S transition was observed. We identified p21/Cdkn1a as direct target of PUM1, and abrogation of the PUM1 binding site in p21 resulted in decreased tumor cell growth as well as delayed G1/S transition. Furthermore, intravenous injection of nanoparticle-encapsulated anti-Pum1 and Pum2 siRNAs reduced colorectal tumor growth in murine orthotopic colon cancer models. These findings reveal a tumor growth promoting role of PUM proteins in CRC and its potential as therapeutic targets.

PUMILIO (PUM) proteins belong to the highly conserved PUF family post-transcriptional regulators involved in diverse biological processes. However, their function in carcinogenesis remains under-explored. Here, we report that Pum1 and Pum2 display increased expression in human colorectal cancer (CRC). Intestine-specific knockout of Pum1 and Pum2 in mice significantly inhibits the progression of colitis-associated cancer in the AOM/DSS model. Knockout or knockdown of Pum1 and/or Pum2 in human CRC cells result in a significant decrease in the tumorigenicity and delayed G1/S transition. We identify p21/Cdkn1a as a direct target of PUM1. Abrogation of the PUM1 binding site in the p21 mRNA also results in decreased cancer cell growth and delayed G1/S transition. Furthermore, intravenous injection of nanoparticle-encapsulated anti-Pum1 and Pum2 siRNAs reduces colorectal tumor growth in murine orthotopic colon cancer models. These findings reveal the requirement of PUM proteins for CRC progression and their potential as therapeutic targets.

Small noncoding RNA pathways have been implicated in diverse mechanisms of gene regulation. In Drosophila ovaries, Piwi binds to Piwi-interacting RNAs (piRNAs) of mostly 24–28 nucleotides (nt) and plays an important role in germline stem cell maintenance, transposon repression, and epigenetic regulation. To understand the mechanism underlying these functions, we report the application of the DamID-seq method to identify genome-wide binding sites of Piwi in Drosophila ovaries. Piwi localizes to at least 4535 euchromatic regions that are enriched with piRNA target sites. Surprisingly, the density of Piwi binding to euchromatin is much higher than in heterochromatin. Disrupting the piRNA binding of Piwi results in an overall change of the genomic binding profile, which indicates the role of piRNAs in directing Piwi to specific genomic sites. Most Piwi binding sites were either within or in the vicinity of protein-coding genes, particularly enriched near the transcriptional start and termination sites. The methylation signal near the transcriptional termination sites is significantly reduced when Piwi was mutated to become defective in piRNA binding. These observations indicate that Piwi might directly regulate the expression of many protein-coding genes, especially through regulating the 3' ends of targeted transcripts.

SETDB1 is a histone-lysine N-methyltransferase critical for germline development. However, its function in early meiotic prophase I remains unknown. Here we report that Setdb1-null spermatocytes display aberrant centromere clustering during leptotene, bouquet formation during zygotene, and subsequent failure in pairing and synapsis of homologous chromosomes as well as compromised meiotic silencing of unsynapsed chromatin, which leads to meiotic arrest before pachytene and apoptosis of spermatocytes. H3K9m3 is enriched in (peri-)centromeric regions and is present in many sites throughout the genome, with a subset changed in the Setdb1 mutant. These observations indicate that SETDB1-mediated H3K9 trimethylation is essential for the bivalent formation in early meiosis. Transcriptome analysis reveals the function of SETDB1 in repressing transposons and transposon-proximal genes and in regulating meiotic and somatic lineage genes. These findings highlight a mechanism in which SETDB1-mediated H3K9 trimethylation during early meiosis ensures the formation of homologous bivalents and survival of spermatocytes.

MIWI, a murine member of PIWI proteins mostly expressed during male meiosis, is crucial for piRNA biogenesis, post-transcriptional regulation, and spermiogenesis. However, its meiotic function remains unknown. Here, we report that MIWI deficiency alters meiotic kinetochore assembly, significantly increases chromosome misalignment at the meiosis metaphase I plate, and causes chromosome mis-segregation. Consequently, Miwi-deficient mice show elevated aneuploidy in metaphase II and spermatid death. Furthermore, in Miwi-null and Miwi slicer-deficient mutants, major and minor satellite RNAs from centromeric and pericentromeric satellite repeats accumulate in excess. Over-expression of satellite repeats in wild-type spermatocytes also causes elevated chromosome misalignment, whereas reduction of both strands of major or minor satellite RNAs results in lower frequencies of chromosome misalignment. We show that MIWI, guided by piRNA, cleaves major satellite RNAs, generating RNA fragments that may form substrates for subsequent Dicer cleavage. Furthermore, Dicer cleaves all satellite RNAs in conjunction with MIWI. These findings reveal a novel mechanism in which MIWI- and Dicer-mediated cleavage of the satellite RNAs prevents the over-expression of satellite RNAs, thus ensuring proper kinetochore assembly and faithful chromosome segregation during meiosis.

Transposable elements (TEs) can damage genomes, thus organisms employ a variety of mechanisms to repress TE expression. However, these mechanisms often fail over time leading to de-repression of TEs in aging tissues. The PIWI-piRNA pathway is a small RNA pathway that represses TE expression in the germline of animals. Here we explore the function of the pathway in the epithelial stem cells ofHydra, a long-lived freshwater cnidarian.Hydrahave three stem cell populations; endodermal and ectodermal epithelial stem cells are strictly somatic, whereas the interstitial stem cells retain germline competence. In our previous study, we found that the PIWI proteins are expressed in all threeHydrastem cell types. In this study, we focus on the ectodermal and endodermal epithelial stem cells to understand the somatic function of the pathway. We isolated piRNAs fromHydrathat lack the interstitial lineage and found that these somatic piRNAs map predominantly to TE transcripts and display the conserved sequence signatures typical of germline piRNAs. Three lines of evidence suggest that the PIWI-piRNA pathway represses TEs inHydraepithelial stem cells. First, epithelial knockdown of theHydraPIWI proteinhywiresulted in upregulation of TE expression. Second, degradome sequencing revealed evidence of PIWI-mediated cleavage of TE RNAs in epithelial cells using the ping-pong mechanism. Finally, we demonstrated a direct association between Hywi protein and TE transcripts in epithelial cells using RNA immunoprecipitation. Interestingly, we found that RNAi knockdown ofhywileads to an upregulation of genes involved in innate immunity, which may be in response to TE upregulation; this is consistent with recent studies on TE expression in mammalian cells. Altogether, this study suggests a function for the PIWI-piRNA pathway in maintaining the long-lived somatic cell lineages ofHydraand may point to a broader role for this pathway in protecting somatic tissue from TE-induced damage.

Heterochromatin protein1a (HP1a) is a highly conserved epigenetic factor that associates with heterochromatin in gene silencing. Though it can activate transcription, the function of HP1a in development has been under-investigated..... Heterochromatin protein 1a (HP1a) is a highly conserved and versatile epigenetic factor that can both silence and activate transcription. However, the function of HP1a in development has been underinvestigated. Here, we report the role of maternal HP1a in producing maternal transcripts that drive early Drosophila embryogenesis. Maternal HP1a upregulates genes involved in translation, mRNA splicing, and cell division, but downregulates genes involved in neurogenesis, organogenesis, and germline development, which all occur later in development. Our study reveals the earliest contribution of HP1a during oogenesis in regulating the production of maternal transcripts that drive early Drosophila embryogenesis.

Triple-negative breast cancer (TNBC), characterized by the lack of expression of the estrogen receptor, the progesterone receptor, and the human epidermal growth factor receptor 2, is an aggressive form of cancer that conveys unpredictable and poor prognosis due to limited treatment options and lack of effective targeted therapies. Wnt/β-catenin signaling is hyperactivated in TNBC, which promotes the progression of TNBC. However, the molecular mechanism of Wnt/β-catenin activation in TNBC remains unknown. Here, we report the drastic overexpression of miR-221/222 in all of four TNBC cell lines and TNBC primary tumor samples from patients. Furthermore, we demonstrate by both ex vivo and xenograft experiments that inhibiting miR-221/222 expression in a TNBC cell line (MDA-MB-231) suppresses its proliferation, viability, epithelial-to-mesenchymal transition, and migration; whereas expressing miR-221/222 in a non-TNBC line (MCF7) promotes all of the above cancer properties. miR-221/222 achieve so by directly repressing multiple negative regulators of the Wnt/β-catenin signaling pathway, including WIF1, SFRP2, DKK2, and AXIN2, to activate the pathway. Notably, the level of miR-221/222 expression is inversely correlated whereas that of WIF1, DKK2, SFRP2, and AXIN2 expression is positively correlated with the patient survival. Last, we show that anti-miR-221/222 significantly increases apoptotic cells with tamoxifen/Wnt3a treatment but not with cyclophosphamide/Wnt3a treatment. These results demonstrate that miR-221/222 activate the Wnt/β-catenin signaling to promote the aggressiveness and TNBC properties of breast cancers, and thus reveal a new prospect for TNBC treatment.

Piwi-interacting RNAs (piRNAs) are a class of small noncoding RNAs that bind Piwi proteins to silence transposons and to regulate gene expression. In Drosophila germ cells, the Aubergine (Aub)-Argonaute 3 (Ago3)–dependent ping-pong cycle generates most germline piRNAs. Loading of antisense piRNAs amplified by this cycle enables Piwi to enter the nucleus and silence transposons. Nuclear localization is crucial for Piwi function in transposon silencing, but how this process is regulated remains unknown. It is also not known whether any of the components of the nuclear pore complex (NPC) directly function in the piRNA pathway. Here, we show that nucleoporin 358 (Nup358) and Piwi interact with each other and that a germline knockdown (GLKD) of Nup358 with short hairpin RNA prevents Piwi entry into the nucleus. The Nup358 GLKD also activated transposons, increased genomic instability, and derailed piRNA biogenesis because of a combination of decreased piRNA precursor transcription and a collapse of the ping-pong cycle. Our results point to a critical role for Nup358 in the piRNA pathway, laying the foundation for future studies to fully elucidate the mechanisms by which Nup358 contributes to piRNA biogenesis and transposon silencing.

Somatic mutations in splicing factor genes are drivers of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). The splicing factors U2AF1 and U2AF2 form the U2AF heterodimer that is critical in the 3' splice site (3'SS) recognition and in the recruitment of U2 small nuclear ribonucleoproteins for the activation of the spliceosome complex. U2AF1 carries hotspot mutations in its two RNA binding motifs; yet the molecular mechanisms affecting the splicing process and promoting clonal advantage remain unclear, albeit necessary to develop effective targeted therapies. We applied a multi-omics approach comparing the activities of two U2AF1 mutants (S34F and Q157R) in MDS/AML cell lines and primary samples. Using a novel approach of fractionated enhanced crosslinking immunoprecipitation coupled with deep RNA-sequencing (freCLIP-seq), we mapped transcriptome-wide binding at nucleotide resolution and we identified conformational changes in mutant vs wild-type U2AF1 binding. Specifically, we observed an emergent peak in position -3 of the 3'SS for the S34F mutant and in position +1 for the Q157R mutant, matching the critical positions observed by differential splicing analysis on RNA-seq data. Altered U2AF1-RNA binding compromised U2AF2-RNA interactions, resulting predominantly in exon exclusion and intron retention. Combined binding-splicing analysis showed that while the Q157R mutant mainly exhibits loss of binding, the S34F mutant follows a gain-of-binding pattern, where splicing progression appears impaired by increased mutant binding. Functional analysis of genes affected by both binding and splicing alterations revealed that U2AF1 mutants alter RNA granule biology, affecting in particular stress granule-enriched transcripts and proteins. Stress granules are membrane-less cytoplasmic assemblies of RNAs and RNA binding proteins that improve cellular adaptation in response to stress conditions. Increased stress granule formation has been linked to tumorigenesis as a strategy exploited by cancer cells to regulate gene expression and signal transduction, enhancing their fitness under stress. To probe how aberrant binding and splicing of stress granule components affected stress granule biology, we assessed stress granule formation in U2AF1 mutant vs wild-type cells at steady state and after stress induction with sodium arsenite treatment. Immunofluorescent staining followed by confocal imaging demonstrated that U2AF1 mutations enhance stress granule formation upon arsenite stress in both cell lines and primary samples. RNA turnover analysis by TimeLapse-seq confirmed that U2AF1 S34F and Q157R mutations promote stability/synthesis of transcripts that are enriched in stress granules and determine degradation/shutdown of transcripts that are depleted in stress granules, providing a molecular explanation for the increase in stress granules observed by imaging. Finally, we were able to corroborate our observations by single-cell RNA-seq in patient-derived U2AF1-mutant MDS blasts, establishing the causal link between U2AF1 mutations and upregulation of stress granule components. Collectively, this multi-omics analysis identified biological processes directly influenced by mutant U2AF1 binding and splicing, laying the foundation for a new paradigm where splicing factor mutations enhance stress granule formation by acting on the availability of their RNA and protein components. The enhanced formation of stress granules potentially fosters the stress adaptation of U2AF1-mutant cells, contributing to their clonal advantage in MDS/AML. Stress granule perturbations may therefore represent a novel therapeutic vulnerability in U2AF1-mutant MDS/AML patients and possibly in patients carrying other splicing factor mutations. Disclosures Hunck: Boehringer Ingelheim: Other: Fellowship.

Spliceosomal gene mutations function as drivers of hematologic malignancies and other cancers with an occurrence of more than 50% in myelodysplastic syndromes and secondary acute myeloid leukemia. Hotspot mutations S34F and Q157R in the two zinc finger domains of the splicing factor U2AF1, forming with U2AF2 the U2AF complex that recognizes 3' splice site (3'SS) of U2 introns, alter exon usage in a sequence-specific manner. However, how pathological U2AF1 mutations disrupt ordered splicing, from binding to recruitment of cooperating RNA binding proteins and ultimately splicing kinetics, is still not known at the molecular level. To obtain unique insights into in vivo RNA binding mechanisms, we performed fractionated enhanced crosslinking immunoprecipitation coupled with deep RNA sequencing (freCLIP-seq) on human erythroleukemia (HEL) cells expressing wild-type (WT) or mutant (S34F, Q157R) U2AF1. Transcriptome-wide analysis of binding at single nucleotide resolution in light and heavy fractions, corresponding respectively to U2AF1 only and U2AF complex, allowed to: i) deconvolute U2AF1 signal peaking over the AG dinucleotide at the intronic end of the 3'SS region, and U2AF2 signal sitting on the adjacent polypyrimidine tract (PPT); ii) identify conformational changes in mutant U2AF1 binding with a novel peak in position -3 of the 3'SS region for S34F and in position +1 for Q157R. Alternative splicing analysis on newly collected RNA-seq data showed that less included exons present higher probability of U in position -3 for S34F and A in position +1 for Q157R, pinpointing a match with nucleotide positions affected by aberrant binding in freCLIP-seq. In both U2AF1 mutants, aberrant binding and splicing mechanisms affected genes involved in mRNA processing and transport (P-value We then performed a combined analysis of differential binding and aberrant splicing in U2AF1 mutants vs WT considering 4 categories: ">inclusion/>binding", "binding", ">inclusion/binding" with 123 events out of 309 (Figure 1A). Moreover, differential binding was not dependent on specific nucleotides in position -3: events characterized by increased S34F binding (Figure 1B, top), as well as events characterized by decreased S34F binding (Figure 1B, bottom), showed -3U in less included exons or -3C in more included exons. The binding analysis across the 4 categories showed that increased S34F binding was associated with reduced U2AF2 binding (Figure 1C, top) particularly in less included exons, while decreased S34F binding was associated with increased U2AF2 binding (Figure 1C, bottom) especially in more included exons. Finally, analysis of branch point and splice junction features revealed that PPT strength influences the splicing outcome with "binding" category characterized by a weak PPT that impairs U2AF2 binding in the presence of skewed U2AF1 S34F binding (Figure 1D). Additionally, transcriptome-wide RNA kinetics analysis by TimeLapse-seq demonstrated that U2AF1 S34F and Q157R, compared to WT, globally decrease synthesis of aberrantly spliced and bound 3'SS regions. Of note, this shutdown effect was particularly evident in the downstream exons pointing towards a role of U2AF1 mutations in a widespread alteration of RNA synthesis and splicing dynamics. Collectively, these results disclose novel molecular mechanisms of pathogenic U2AF1 mutations in the context of myeloid malignancies and provide the basis for the development of effective U2AF1 directed therapeutic strategies. Disclosures Hunck: Boehringer Ingelheim Fonds: Other: MD Fellowship.

Specific patterns of DNA mutations in normal skin show history of UV exposure and association with cancer risk., In ultraviolet (UV) radiation–exposed skin, mutations fuel clonal cell growth. The relationship between UV exposure and the accumulation of clonal mutations (CMs) and the correlation between CMs and skin cancer risk are largely unexplored. We characterized 450 individual-matched sun-exposed (SE) and non-SE (NE) normal human skin samples. The number and relative contribution of CMs were significantly different between SE and NE areas. Furthermore, we identified hotspots in TP53, NOTCH1, and GRM3 where mutations were significantly associated with UV exposure. In the normal skin from patients with cutaneous squamous cell carcinoma, we found that the cancer burden was associated with the UV-induced mutations, with the difference mostly conferred by the low-frequency CMs. These findings provide previously unknown information on UV’s carcinogenic effect and pave the road for future development of quantitative assessment of subclinical UV damage and skin cancer risk.

Gene regulation in embryonic stem cells (ESCs) has been extensively studied at the epigenetic-transcriptional levels, but not at the post-transcriptional levels. Pumilio (Pum) proteins are among the few known translational regulators required for stem cell maintenance in invertebrates and plants. Here we report the essential function of two murine Pum proteins, Pum1 and Pum2, in ESCs and early embryogenesis. Pum1/2 double mutants are developmentally delayed at the morula stage and lethal by embryonic day 8.5 (e8.5). Correspondingly, Pum1/2 double mutant ESCs display severely reduced self-renewal and differentiation, revealing the combined function of Pum1 and Pum2 in ESC pluripotency. Remarkably, Pum1-deficient ESCs show increased expression of pluripotency genes but not differentiation genes, indicating that Pum1 mainly promote differentiation; whereas Pum2-deficient ESCs show decreased expression of pluripotency genes and accelerated differentiation, indicating that Pum2 promotes self-renewal. Thus, Pum1 and Pum2 each uniquely contributes to one of the two complementary aspects of pluripotency. Furthermore, we show that Pum1 and Pum2 achieve ESC functions by forming a negative auto- and inter-regulatory feedback loop that directly regulates at least 1,486 mRNAs. Pum1 and Pum2 regulate target mRNAs not only by repressing translation as expected but also by promoting translation and enhancing or reducing mRNA stability of different target mRNAs. Together, these findings reveal the distinct roles of individual mammalian Pum proteins in ESCs and their collectively essential functions in ESC pluripotency and embryogenesis. Moreover, they demonstrate three novel modes of regulation of Pum proteins towards target mRNAs.SIGNIFICANCE STATEMENTThis report demonstrates the essential functions of mammalian Pumilio (Pum) proteins for embryonic stem cells (ESCs) pluripotency and embryogenesis. Moreover, it reveals the contrasting but complementary function of individual Pum proteins in regulating distinct aspects of ESC pluripotency, despite their largely overlapping expression and extremely high homology. Furthermore, it unravels a complex regulatory network in which Pum1 and Pum2 form a negative auto- and inter-regulatory feedback loop that regulates 1,486 mRNAs not only by translational repression as expected but also by promoting translation and enhancing or reducing stability of different target mRNAs, which reveals novel modes of post-transcriptional regulation mediated by Pum.

Transposable elements (TEs) can damage genomes, thus organisms use a variety of mechanisms to repress TE expression. The PIWI–piRNA pathway is a small RNA pathway that represses TE expression in the germline of animals. Here we explore the function of the pathway in the somatic stem cells of Hydra, a long-lived freshwater cnidarian. Hydra have three stem cell populations, all of which express PIWI proteins; endodermal and ectodermal epithelial stem cells (ESCs) are somatic, whereas the interstitial stem cells have germline competence. To study somatic function of the pathway, we isolated piRNAs from Hydra that lack the interstitial lineage and found that these somatic piRNAs map predominantly to TE transcripts and display the conserved sequence signatures typical of germline piRNAs. Three lines of evidence suggest that the PIWI–piRNA pathway represses TEs in Hydra ESCs. First, epithelial knockdown of the Hydra piwi gene hywi resulted in up-regulation of TE expression. Second, degradome sequencing revealed evidence of PIWI-mediated cleavage of TE RNAs in epithelial cells using the ping-pong mechanism. Finally, we demonstrated a direct association between Hywi protein and TE transcripts in epithelial cells using RNA immunoprecipitation. Altogether, our data reveal that the PIWI–piRNA pathway represses TE expression in the somatic cell lineages of Hydra, which we propose contributes to the extreme longevity of the organism. Furthermore, our results, in combination with others, suggest that somatic TE repression is an ancestral function of the PIWI–piRNA pathway.

EML4-ALK and NPM-ALK fusion proteins possess constitutively activated ALK (anaplastic lymphoma kinase) activity, which in turn leads to the development of non-small cell lung cancer and anaplastic large-cell lymphomas (ALCLs). FDA-approved ALK inhibitor drugs cause significant cancer regression. However, drug resistance eventually occurs and it becomes a big obstacle in clinic. Novel proteolysis targeting chimera (PROTAC) technology platform provides a potential therapeutic strategy for drug resistance. Herein, we designed and synthesized a series of ALK PROTACs based on Brigatinib and VHL-1 conjunction, and screened SIAIS117 as the best degrader which not only blocked the growth of SR and H2228 cancer cell lines, but also degraded ALK protein. In addition, SIAIS117 also showed much better growth inhibition effect than Brigatinib on 293T cell line that exogenously expressed G1202R-resistant ALK proteins. Furthermore, it also degraded G1202R mutant ALK protein in vitro. At last, it has the potentially anti-proliferation ability of small cell lung cancer. Thus, we have successfully generated the degrader SIAIS117 that can potentially overcome resistance in cancer targeted therapy.

Background RNA binding proteins play a pivotal role during the oocyte-to-embryo transition and maternal phase of embryogenesis in invertebrates, but their function in these processes in mammalian systems remain largely understudied. Results Here we report that a member of the Pumilio/FBF family of RNA binding proteins in mice, Pumilio 1 (Pum1), is a maternal effect gene. The absence of maternal PUM1 in the oocyte does not affect meiotic maturation but leads to abnormal preimplantation development. Furthermore, genome-wide transcriptome analysis of oocytes and embryos revealed that there is a concomitant perturbation of the mRNA milieu. Of note, putative PUM1 mRNA targets were equally perturbed as non-direct targets, which indicates that PUM1 regulates the stability of maternal mRNAs both directly and indirectly. We show Cdk1 mRNA, a known PUM1 target essential for meiosis and preimplantation development, is not degraded appropriately during meiosis, leading to an increase in CDK1 protein in mature oocytes, which indicates that PUM1 post-transcriptionally regulates Cdk1 mRNA; this could partially explain the observed abnormal preimplantation development. Furthermore, our results show that maternal and zygotic PUM1 are required for postnatal survival. Conclusions These findings indicate that PUM1 is essential in the process of cytoplasmic maturation and developmental competence of the oocyte. These results reveal an important function of maternal PUM1 as a post-transcriptional regulator during mammalian embryogenesis. Electronic supplementary material The online version of this article (10.1186/s13578-018-0251-1) contains supplementary material, which is available to authorized users.