Pachytene PIWI-interacting RNAs (piRNAs), which comprise >80% of small RNAs in the adult mouse testis, have been proposed to bind and regulate target RNAs like microRNAs, cleave targets like short ...interfering RNAs or lack biological function altogether. Although piRNA pathway protein mutants are male sterile, no biological function has been identified for any mammalian piRNA-producing locus. Here, we report that males lacking piRNAs from a conserved mouse pachytene piRNA locus on chromosome 6 (pi6) produce sperm with defects in capacitation and egg fertilization. Moreover, heterozygous embryos sired by pi6
fathers show reduced viability in utero. Molecular analyses suggest that pi6 piRNAs repress gene expression by cleaving messenger RNAs encoding proteins required for sperm function. pi6 also participates in a network of piRNA-piRNA precursor interactions that initiate piRNA production from a second piRNA locus on chromosome 10, as well as pi6 itself. Our data establish a direct role for pachytene piRNAs in spermiogenesis and embryo viability.
In the male germ cells of placental mammals, 26-30-nt-long PIWI-interacting RNAs (piRNAs) emerge when spermatocytes enter the pachytene phase of meiosis. In mice, pachytene piRNAs derive from ~100 ...discrete autosomal loci that produce canonical RNA polymerase II transcripts. These piRNA clusters bear 5' caps and 3' poly(A) tails, and often contain introns that are removed before nuclear export and processing into piRNAs. What marks pachytene piRNA clusters to produce piRNAs, and what confines their expression to the germline? We report that an unusually long first exon (≥ 10 kb) or a long, unspliced transcript correlates with germline-specific transcription and piRNA production. Our integrative analysis of transcriptome, piRNA, and epigenome datasets across multiple species reveals that a long first exon is an evolutionarily conserved feature of pachytene piRNA clusters. Furthermore, a highly methylated promoter, often containing a low or intermediate level of CG dinucleotides, correlates with germline expression and somatic silencing of pachytene piRNA clusters. Pachytene piRNA precursor transcripts bind THOC1 and THOC2, THO complex subunits known to promote transcriptional elongation and mRNA nuclear export. Together, these features may explain why the major sources of pachytene piRNA clusters specifically generate these unique small RNAs in the male germline of placental mammals.
Male gametogenesis, spermatogenesis, is a stepwise developmental process to generate mature sperm. The most intricate process of spermatogenesis is meiosis during which two successive cell divisions ...ensue with dramatic cellular and molecular changes to produce haploid cells. After entry into meiosis, several forms of regulatory events control the orderly progression of meiosis and the timely entry into post-meiotic sperm differentiation. Among other mechanisms, changes to gene expression are controlled by key transcription factors. In this review, we will discuss the gene regulatory mechanisms underlying meiotic entry, meiotic progression, and post-meiotic differentiation with a particular emphasis on the MYBL1/TCFL5 regulatory architecture and how this architecture involves in various forms of transcription network motifs to regulate gene expression.
Adrenocortical carcinoma (ACC) is an aggressive tumor showing frequent metastatic spread and poor survival. Although recent genome-wide studies of ACC have contributed to our understanding of the ...disease, major challenges remain for both diagnostic and prognostic assessments. The aim of this study was to identify specific microRNAs (miRNAs) associated with malignancy and survival of ACC patients. miRNA expression profiles were determined in a series of ACC, adenoma, and normal cortices using microarray. A subset of miRNAs showed distinct expression patterns in the ACC compared with adrenal cortices and adenomas. Among others, miR-483-3p, miR-483-5p, miR-210, and miR-21 were found overexpressed, while miR-195, miR-497, and miR-1974 were underexpressed in ACC. Inhibition of miR-483-3p or miR-483-5p and overexpression of miR-195 or miR-497 reduced cell proliferation in human NCI-H295R ACC cells. In addition, downregulation of miR-483-3p, but not miR-483-5p, and increased expression of miR-195 or miR-497 led to significant induction of cell death. Protein expression of p53 upregulated modulator of apoptosis (PUMA), a potential target of miR-483-3p, was significantly decreased in ACC, and inversely correlated with miR-483-3p expression. In addition, high expression of miR-503, miR-1202, and miR-1275 were found significantly associated with shorter overall survival among patients with ACC (P values: 0.006, 0.005, and 0.042 respectively). In , we identified additional miRNAs associated with ACC, elucidated the functional role of four miRNAs in the pathogenesis of ACC cells, demonstrated the potential involvement of the pro-apoptotic factor PUMA (a miR-483-3p target) in adrenocortical tumors, and found novel miRNAs associated with survival in ACC.
In animals, PIWI-interacting RNAs (piRNAs) silence transposons, fight viral infections, and regulate gene expression. piRNA biogenesis concludes with 3′ terminal trimming and 2′-O-methylation. Both ...trimming and methylation influence piRNA stability. Our biochemical data show that multiple mechanisms destabilize unmethylated mouse piRNAs, depending on whether the piRNA 5′ or 3′ sequence is complementary to a trigger RNA. Unlike target-directed degradation of microRNAs, complementarity-dependent destabilization of piRNAs in mice and flies is blocked by 3′ terminal 2′-O-methylation and does not require base pairing to both the piRNA seed and the 3′ sequence. In flies, 2′-O-methylation also protects small interfering RNAs (siRNAs) from complementarity-dependent destruction. By contrast, pre-piRNA trimming protects mouse piRNAs from a degradation pathway unaffected by trigger complementarity. In testis lysate and in vivo, internal or 3′ terminal uridine- or guanine-rich tracts accelerate pre-piRNA decay. Loss of both trimming and 2′-O-methylation causes the mouse piRNA pathway to collapse, demonstrating that these modifications collaborate to stabilize piRNAs.
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•2′-O-methylation protects mouse and fly piRNAs from complementarity-dependent decay•Distinct pathways decay mouse piRNAs with triggers complementary to a piRNA 5′ or 3′ end•Mouse pre-piRNAs with U- or G-rich tracts decay faster in testis lysate and in vivo•MIWI-bound pre-piRNAs decay faster than MILI-bound pre-piRNAs
piRNAs are an animal-specific class of small RNAs that repress transposons and regulate genes. piRNA biogenesis concludes with 3′ terminal trimming and 2′-O-methylation. Gainetdinov et al. show that trimming and 2′-O-methylation protect piRNAs from multiple degradation mechanisms triggered by extensively complementary RNAs or sequence motifs in the piRNA.
Little is known about how interactions of diet, intestinal stem cells (ISCs), and immune cells affect early-stage intestinal tumorigenesis. We show that a high-fat diet (HFD) reduces the expression ...of the major histocompatibility complex class II (MHC class II) genes in intestinal epithelial cells, including ISCs. This decline in epithelial MHC class II expression in a HFD correlates with reduced intestinal microbiome diversity. Microbial community transfer experiments suggest that epithelial MHC class II expression is regulated by intestinal flora. Mechanistically, pattern recognition receptor (PRR) and interferon-gamma (IFNγ) signaling regulates epithelial MHC class II expression. MHC class II-negative (MHC-II−) ISCs exhibit greater tumor-initiating capacity than their MHC class II-positive (MHC-II+) counterparts upon loss of the tumor suppressor Apc coupled with a HFD, suggesting a role for epithelial MHC class II-mediated immune surveillance in suppressing tumorigenesis. ISC-specific genetic ablation of MHC class II increases tumor burden cell autonomously. Thus, HFD perturbs a microbiome-stem cell-immune cell interaction that contributes to tumor initiation in the intestine.
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•HFD dampens MHC class II expression in IECs, including ISCs•HFD perturbs intestinal microbes that correlate with MHC class II expression in ISCs•PRR and IFNγ signaling mediates epithelial MHC class II expression•Loss of MHC class II on premalignant ISCs enhances tumor initiation
The mechanisms that link pro-obesity high-fat diets (HFDs) to increased colon cancer risk are not well understood. Beyaz and colleagues demonstrate that a HFD promotes intestinal tumor initiation by suppressing microbiome-stem cell-immune cell crosstalk that is mediated by MHC class II expression on intestinal stem cells.
In male mice, the transcription factor A MYB initiates the transcription of pachytene piRNA genes during meiosis. Here, we report that A MYB activates the transcription factor Tcfl5 produced in ...pachytene spermatocytes. Subsequently, A MYB and TCFL5 reciprocally reinforce their own transcription to establish a positive feedback circuit that triggers pachytene piRNA production. TCFL5 regulates the expression of genes required for piRNA maturation and promotes transcription of evolutionarily young pachytene piRNA genes, whereas A-MYB activates the transcription of older pachytene piRNA genes. Intriguingly, pachytene piRNAs from TCFL5-dependent young loci initiates the production of piRNAs from A-MYB-dependent older loci ensuring the self-propagation of pachytene piRNAs. A MYB and TCFL5 act via a set of incoherent feedforward loops that drive regulation of gene expression by pachytene piRNAs during spermatogenesis. This regulatory architecture is conserved in rhesus macaque, suggesting that it was present in the last common ancestor of placental mammals.
In animals, PIWI-interacting RNAs (piRNAs) of 21-35 nucleotides in length silence transposable elements, regulate gene expression and fight viral infection. piRNAs guide PIWI proteins to cleave ...target RNA, promote heterochromatin assembly and methylate DNA. The architecture of the piRNA pathway allows it both to provide adaptive, sequence-based immunity to rapidly evolving viruses and transposons and to regulate conserved host genes. piRNAs silence transposons in the germ line of most animals, whereas somatic piRNA functions have been lost, gained and lost again across evolution. Moreover, most piRNA pathway proteins are deeply conserved, but different animals employ remarkably divergent strategies to produce piRNA precursor transcripts. Here, we discuss how a common piRNA pathway allows animals to recognize diverse targets, ranging from selfish genetic elements to genes essential for gametogenesis.
Sequencing data has become a standard measure of diverse cellular activities. For example, gene expression is accurately measured by RNA sequencing (RNA-Seq) libraries, protein-DNA interactions are ...captured by chromatin immunoprecipitation sequencing (ChIP-Seq), protein-RNA interactions by crosslinking immunoprecipitation sequencing (CLIP-Seq) or RNA immunoprecipitation (RIP-Seq) sequencing, DNA accessibility by assay for transposase-accessible chromatin (ATAC-Seq), DNase or MNase sequencing libraries. The processing of these sequencing techniques involves library-specific approaches. However, in all cases, once the sequencing libraries are processed, the result is a count table specifying the estimated number of reads originating from each genomic locus. Differential analysis to determine which loci have different cellular activity under different conditions starts with the count table and iterates through a cycle of data assessment, preparation and analysis. Such complex analysis often relies on multiple programs and is therefore a challenge for those without programming skills.
We developed DEBrowser as an R bioconductor project to interactively visualize every step of the differential analysis, without programming. The application provides a rich and interactive web based graphical user interface built on R's shiny infrastructure. DEBrowser allows users to visualize data with various types of graphs that can be explored further by selecting and re-plotting any desired subset of data. Using the visualization approaches provided, users can determine and correct technical variations such as batch effects and sequencing depth that affect differential analysis. We show DEBrowser's ease of use by reproducing the analysis of two previously published data sets.
DEBrowser is a flexible, intuitive, web-based analysis platform that enables an iterative and interactive analysis of count data without any requirement of programming knowledge.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The testis-specific transcription factor, TCFL5, expressed in pachytene spermatocytes regulates the meiotic gene expression program in collaboration with the transcription factor A-MYB.
In male mice, ...the transcription factors STRA8 and MEISON initiate meiosis I. We report that STRA8/MEISON activates the transcription factors A-MYB and TCFL5, which together reprogram gene expression after spermatogonia enter into meiosis. TCFL5 promotes the transcription of genes required for meiosis, mRNA turnover, miR-34/449 production, meiotic exit, and spermiogenesis. This transcriptional architecture is conserved in rhesus macaque, suggesting TCFL5 plays a central role in meiosis and spermiogenesis in placental mammals. Tcfl5em1/em1 mutants are sterile, and spermatogenesis arrests at the mid- or late-pachytene stage of meiosis. Moreover, Tcfl5+/em1 mutants produce fewer motile sperm.
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