A Single Mechanism of Biogenesis, Initiated and Directed by PIWI Proteins, Explains piRNA Production in Most Animals [preprint]

In animals, piRNAs guide PIWI-proteins to silence transposons and regulate gene expression. The mechanisms for making piRNAs have been proposed to differ among cell types, tissues, and animals. Our data instead suggest a single model that explains piRNA production in most animals. piRNAs initiate piRNA production by guiding PIWI proteins to slice precursor transcripts. Next, PIWI proteins direct the stepwise fragmentation of the sliced precursor transcripts, yielding tail-to-head strings of phased pre-piRNAs. Our analyses detect evidence for this piRNA biogenesis strategy across an evolutionarily broad range of animals including humans. Thus, PIWI proteins initiate and sustain piRNA biogenesis by the same mechanism in species whose last common ancestor predates the branching of most animal lineages. The unified model places PIWI-clade Argonautes at the center of piRNA biology and suggests that the ancestral animal--the Urmetazoan--used PIWI proteins both to generate piRNA guides and to execute piRNA function

Understanding the Production and Stability of Mouse PIWI-Interacting RNAs

PIWI-interacting RNAs (piRNAs) are small non-coding RNAs unique to animals that guard the germline genome integrity by regulating transposons, viruses, and genes. In mice, piRNAs are highly expressed in testis and guide one of the three PIWI proteins to regulate their targets. The purpose of the 3′ end 2′-O-methyl modification in piRNAs is unknown. It has been speculated that the modification increases stability and facilitates the function of piRNAs, but the direct evidence is lacking. My dissertation addresses two unanswered questions about mouse piRNAs: (1) how are piRNAs produced and how conserved is the piRNA pathway in all animals, and (2) why are mouse piRNAs 2′-O-methylated at their 3′ ends? How piRNAs are generated is still poorly characterized in several model organisms. Studies of these model organisms imply the mechanisms that produce piRNAs differ among animals, tissues and cell types. Here, we demonstrate that a single unified mechanism can explain piRNA production in most animals, from human to the non-bilateral animal hydra. Our analysis elucidated that, in male mouse and female fly germlines, PIWI proteins guided by the initiator piRNA slice long piRNA precursor transcripts, and this PIWI-guided slicing action starts the piRNA biogenesis. PIWI proteins also position the endonuclease to further fragment long piRNA precursor transcripts into a string of tail-to-head, phased trailing piRNAs in a stepwise manner. Our discovery shows the central role of PIWI proteins in the piRNA pathway: both initiating and sustaining the production of piRNAs. For the second question, we discovered that pre-piRNA trimming and piRNA 2′-O-methylation protect piRNAs from separate decay mechanisms. We showed that in the absence of 2′-O-methylation, mouse piRNAs with extensive complementarity to long RNAs are destabilized and destroyed by a mechanism similar to target-directed microRNA degradation (TDMD). On the other hand, untrimmed pre-piRNAs are destroyed by a different mechanism, independent of their extensive complementarity to long RNAs. In the absence of both 2′-O-methylation and trimming, the piRNA pathway collapses which supports the idea of piRNA trimming and methylation collaborating to stabilize piRNAs. Our work suggests that 2′-O-methylation and trimming are important for maintaining the steady-state abundance of piRNAs which is necessary for their function in either transposon silencing or gene regulation

Maelstrom Represses Canonical Polymerase II Transcription within Bi-directional piRNA Clusters in Drosophila melanogaster

In Drosophila, 23-30 nt long PIWI-interacting RNAs (piRNAs) direct the protein Piwi to silence germline transposon transcription. Most germline piRNAs derive from dual-strand piRNA clusters, heterochromatic transposon graveyards that are transcribed from both genomic strands. These piRNA sources are marked by the heterochromatin protein 1 homolog Rhino (Rhi), which facilitates their promoter-independent transcription, suppresses splicing, and inhibits transcriptional termination. Here, we report that the protein Maelstrom (Mael) represses canonical, promoter-dependent transcription in dual-strand clusters, allowing Rhi to initiate piRNA precursor transcription. Mael also represses promoter-dependent transcription at sites outside clusters. At some loci, Mael repression requires the piRNA pathway, while at others, piRNAs play no role. We propose that by repressing canonical transcription of individual transposon mRNAs, Mael helps Rhi drive non-canonical transcription of piRNA precursors without generating mRNAs encoding transposon proteins

Maelstrom Represses Canonical Polymerase II Transcription within Bi-directional piRNA Clusters in Drosophila melanogaster

In Drosophila, 23-30 nt long PIWI-interacting RNAs (piRNAs) direct the protein Piwi to silence germline transposon transcription. Most germline piRNAs derive from dual-strand piRNA clusters, heterochromatic transposon graveyards that are transcribed from both genomic strands. These piRNA sources are marked by the heterochromatin protein 1 homolog Rhino (Rhi), which facilitates their promoter-independent transcription, suppresses splicing, and inhibits transcriptional termination. Here, we report that the protein Maelstrom (Mael) represses canonical, promoter-dependent transcription in dual-strand clusters, allowing Rhi to initiate piRNA precursor transcription. Mael also represses promoter-dependent transcription at sites outside clusters. At some loci, Mael repression requires the piRNA pathway, while at others, piRNAs play no role. We propose that by repressing canonical transcription of individual transposon mRNAs, Mael helps Rhi drive non-canonical transcription of piRNA precursors without generating mRNAs encoding transposon proteins

Dicer cooperates with p53 to suppress DNA damage and skin carcinogenesis in mice.

Dicer is required for the maturation of microRNA, and loss of Dicer and miRNA processing has been found to alter numerous biological events during embryogenesis, including the development of mammalian skin and hair. We have previously examined the role of miRNA biogenesis in mouse embryonic fibroblasts and found that deletion of Dicer induces cell senescence regulated, in part, by the p53 tumor suppressor. Although Dicer and miRNA molecules are thought to have either oncogenic or tumor suppressing roles in various types of cancer, a role for Dicer and miRNAs in skin carcinogenesis has not been established. Here we show that perinatal ablation of Dicer in the skin of mice leads to loss of fur in adult mice, increased epidermal cell proliferation and apoptosis, and the accumulation of widespread DNA damage in epidermal cells. Co-ablation of Dicer and p53 did not alter the timing or extent of fur loss, but greatly reduced survival of Dicer-skin ablated mice, as these mice developed multiple and highly aggressive skin carcinomas. Our results describe a new mouse model for spontaneous basal and squamous cell tumorigenesis. Furthermore, our findings reveal that loss of Dicer in the epidermis induces extensive DNA damage, activation of the DNA damage response and p53-dependent apoptosis, and that Dicer and p53 cooperate to suppress mammalian skin carcinogenesis

Ablation of <i>Dicer</i> by the K5-Cre transgene induces fur loss and epidermal dysmorphology in older mice.

<p><b>A</b>) Indistinguishable appearance of <i>Dicer</i>-conditional littermates with (Dicer^Δ/Δ) or without (Dicer^c/c) the K5-Cre transgene at four weeks of age. <b>B</b>) Normal morphology of skin and hair follicles in four-week old control mice (Dicer^c/c mice, left two panels) and age-matched Dicer^Δ/Δ mice (Dicer^c/c, K5Cre+, right two panels). Scale bars equal 100 microns. <b>C</b>) Appearance of litter of K5-Cre negative (Dicer^c/c) and K5-Cre+ (Dicer^Δ/Δ) mice at 10 weeks of age. <b>D</b>) Appearance of 8-month old Dicer heterozygous mouse (top) and Dicer-ablated mouse (bottom) Haplo-insufficieny for Dicer does not alter the fur of mice. <b>E</b>) PCR analysis of tissue-derived genomic DNA samples from representative K5Cre+, Dicer^c/c (Dicer^Δ/Δ) mouse indicates presence of <i>Dicer</i>-ablated allele (283 bp product) in the tail and skin of the mouse, but not in the liver or spleen. PCR product of Dicer conditional allele and Dicer wildtype allele = 476 bp and 396 bp, respectively. Three control genomic DNA samples (Dicer^c/c; K5Cre+, Dicer^c/c; and wildtype) were used as controls. <b>F</b>) Kaplan-Meier survival curves for control (wildytype or Dicer^c/c) cohorts and Dicer^Δ/Δ mouse cohorts. <b>G</b>) Hematoxylin and eosin staining of skin tissue sections of 7-month old mice. Deletion of <i>Dicer</i> in the epidermis (Dicer^Δ/Δ mice-bottom panels) results in defective follicle morphology. Wildtype (wt) and K5-Cre+ skin samples are used as controls (top panels). Scale bars equal 100 microns.</p

Dicer and p53 cooperate to suppress skin carcinogenesis.

<p><b>A</b>) Kaplan-Meier tumor curves for various mouse cohorts. K5-Cre+ mice that bear <i>Dicer</i> and <i>p53</i> wildtype alleles (n = 18) do not present with spontaneous tumors (black curve). Tumorigenesis in K5-Cre+ cohorts that contain one ablated allele of <i>p53</i> (blue curves), with (n = 13) and without (n = 12) <i>Dicer</i> co-ablation, or in K5-Cre+ cohorts that bear two ablated alleles of <i>p53</i> (orange curves), with (n = 25) or without (n = 12) <i>Dicer</i> co-ablation reveals that loss of Dicer (Dicer^Δ/Δ) accelerates cancer formation in p53-haploinsufficient mice (p<0.0001) and p53-ablated mice (p<0.0001). <b>B</b>) Representative Dicer^Δ/Δ, p53^Δ/Δ mouse (on right) presenting with multiple incidence of tumor formation. Dicer^Δ/Δ mouse (on left) is an age-matched, 12-month old mouse with wildtype <i>p53</i> alleles. <b>C</b>) PCR analysis of tumor-derived and tissue-derived genomic DNA samples from representative Dicer^Δ/Δ, p53^Δ/Δ mice reveals the presence of the Dicer-ablated allele and p53 ablated allele in the skin and tumor of the mouse, but not in control mice (right panels). <b>D</b>) Hematoxylin and eosin staining of skin tumors arising in <i>Dicer</i>-ablated mice deleted for one (p53^Δ/wt) or both copies (p53^Δ/Δ) of p53. Top left panel is a BCC. Mice co-deleted for both <i>Dicer</i> and <i>p53</i> form moderately or poorly differentiated SCC (top right panel) and poorly differentiated and invasive carcinomas (bottom two panels). Scale bars equal 100 microns.</p

Co-ablation of p53 decreases apoptosis and the survival of Dicer^Δ/Δ mice.

<p><b>A</b>) Hematoxylin and eosin staining of skin tissue sections of 5-month old mice. Control (K5-Cre+ transgene) was similar in appearance to wildtype control samples, whereas ablation of <i>Dicer</i> showed reduced and misshapen follicle morphology, with increased epithelial thickening, mitotic figures (arrow), and incidence of apoptosis (arrowheads). Ablation of p53 did not alter follicle dysmorphology seen in Dicer^Δ/Δ mice (upper right panel), though epithelial cellularity was more pronounced in some regions (see star on bottom right side panel) and fewer apoptotic events were noted. Scale bars equal 100 microns. B) Measurements of epidermal thickness in microns for control skin (white bar), Dicer-ablated skin (gray bar), and Dicer, p53 co-ablated skin (black bar). Bars represent average thickness, with standard deviation shown. Asterisks indicate p values<0.01 between control and Dicer-ablated skin or control and Dicer, p53 co-ablated skin. <b>C</b>) Average number of apoptotic cells observed in 40X field. No apoptotic figures were seen in control tissues (white bar) whereas apoptosis was increases in Dicer-ablated skin (gray bar) and in Dicer, p53 co-ablated skin (black bar) relative to controls. Apoptosis was decreased in Dicer-ablated skin co-deleted for functional p53. Asterisks indicate p values<0.01. <b>D</b>) Average number of mitotic figures seen in 40X field. Mitosis was increased in Dicer-ablated skin (gray bar) and in Dicer, p53 co-ablated skin (black bar) relative to controls (white bar). Asterisks indicate p values<0.01 between control and Dicer-ablated skin or control and Dicer, p53 co-ablated skin. <b>E</b>) Immuno-staining for the proliferation antigen Ki-67 confirms increased cell proliferation in the epidermis of <i>Dicer</i>-ablated mice in the presence or absence of functional <i>p53</i>. Scale bars equal 100 microns. <b>F</b>) PCR-based genotyping of tissues harvested from Dicer conditional, p53 conditional mice in the absence (Dicer^c/c, p53^c/c) or presence (Dicer^Δ/Δ p53^Δ/Δ) of the K5-Cre transgene. Deletion of functional Dicer and p53 alleles is detected specifically in the skin and tumor tissues of K5-Cre+ mice. <b>G</b>) Kaplan-Meier survival curves for Dicer-ablated mice bearing one (purple; n = 17), two (red; n = 14), or no (black; n = 45) functional alleles of p53. Ablation of both <i>Dicer</i> and <i>p53</i> in the skin greatly reduced viability of these mice relative to mice ablated for <i>Dicer</i> in skin (p<0.01) or mice ablated for <i>Dicer</i> and one allele of <i>p53</i> in skin (p<0.01).</p

Co-ablation of p53 does not alter fur loss in Dicer^Δ/Δ mice.

<p><b>A</b>) Appearance of mice at 5-months of age, with genotypes of the mice given beneath. As the two middle mice lack the K5-Cre transgene, not all conditional (c) alleles have undergone deletion. <b>B</b>) Fur loss was scored in <i>Dicer</i>-ablated mice bearing one (purple), two (red), or no (black) functional alleles of p53. Regardless of genotype, all Dicer^Δ/Δ mice began to lose fur between ages 5 and 6 weeks of age, and almost all fur is lost by week 12. <b>C</b>) Hematoxylin and eosin staining of skin tissue sections of two 8-week old K5-Cre transgenic control mice. <b>D</b>) Hematoxylin and eosin staining of skin tissue sections of two 8-week old Dicer skin-ablated mice. <b>E</b>) Hematoxylin and eosin staining of skin tissue sections of two 8-week old mice co-ablated in skin for Dicer and p53. Scale bars for 2C, 2D, and 2E equal 100 microns.</p

Tumor spectrum of mice deleted for <i>p53</i> and/or <i>Dicer</i> in skin.

<p>A majority of tumors forming in the mice were fixed and classified by morphology after sectioning and staining with hematoxylin and eosin. The number of mice in cohort and the number of mice presenting with one or more tumors are given in second and third columns. Tumors undergoing histopathologic analysis and classified are given in fourth column, as are the numbers (and percentages) of mice presenting with multiple, simultaneous tumor formations (last column). SCC = squamous cell carcinoma, BCC = basal cell carcinoma, Carc = carcinoma, wd = well differentiated, md = moderately differentiated, pd = poorly differentiated, vpd = very poorly differentiated-undifferentiated.</p