Profiling the Mismatch Tolerance of Argonaute 2 through Deep Sequencing of Sliced Polymorphic Viral RNAs

Low allelic and clonal variability among endogenous RNA interference (RNAi) targets has focused mismatch tolerance studies to RNAi-active guide strands. However, the inherent genomic instability of RNA viruses such as hepatitis C virus (HCV) gives rise to quasi-species mutants within discrete clones: this facilitates mismatch tolerance studies from a target perspective. We recently quantified the slicing imprecision of Argonaute 2 using small interfering RNA (siRNA) analogues of the DNA-directed RNAi drug TT-034 and next generation sequencing of 5’ RNA Ligase-Mediated Rapid Amplification of cDNA Ends (RACE-SEQ). Here, we present an open source, customizable, and computationally light RACE-SEQ bioinformatic pipeline, describing adaptations that semi-quantitatively report the impact of RNAi hybridisation site mismatches from the target perspective. The analysis shows Argonaute 2 has a substitution-specific, 3-5 log activity window between fully complementary targets and targets with mismatches across positions 10-11. It further focuses the endonucleotic Slicer imprecision around positions 13-17, demonstrating its dependence on guide strand central region complementarity, and potentiation by even a single mismatch. We further propose pharmacogenomics value in testing endogenous targets using recombinant replicon systems and RACE-SEQ to report the pharmacodynamics of sequence-specific oligonucleotide therapeutics against all possible polymorphisms in a population, in a minimally-biased, patient-free manner

HENMT1 and piRNA stability are required for adult male germ cell transposon repression and to define the spermatogenic program in the mouse

piRNAs are critical for transposable element (TE) repression and germ cell survival during the early phases of spermatogenesis, however, their role in adult germ cells and the relative importance of piRNA methylation is poorly defined in mammals. Using a mouse model of HEN methyltransferase 1 (HENMT1) loss-of-function, RNA-Seq and a range of RNA assays we show that HENMT1 is required for the 2' O-methylation of mammalian piRNAs. HENMT1 loss leads to piRNA instability, reduced piRNA bulk and length, and ultimately male sterility characterized by a germ cell arrest at the elongating germ cell phase of spermatogenesis. HENMT1 loss-of-function, and the concomitant loss of piRNAs, resulted in TE de-repression in adult meiotic and haploid germ cells, and the precocious, and selective, expression of many haploid-transcripts in meiotic cells. Precocious expression was associated with a more active chromatin state in meiotic cells, elevated levels of DNA damage and a catastrophic deregulation of the haploid germ cell gene expression. Collectively these results define a critical role for HENMT1 and piRNAs in the maintenance of TE repression in adult germ cells and setting the spermatogenic program.Shu Ly Lim, Zhi Peng Qu, R. Daniel Kortschak, David M. Lawrence, Joel Geoghegan, Anna-Lena Hempfling, Martin Bergmann, Christopher C. Goodnow, Christopher J. Ormandy, Lee Wong, Jeff Mann, Hamish S. Scott, Duangporn Jamsai, David L. Adelson, Moira K. O, Brya

HENMT1 is required for mouse spermatogenesis.

<p><b>(A-D)</b> Periodic acid-Schiff (PAS) stained testis sections from <i>Henmt1</i>^<i>WT/WT</i> and <i>Henmt1</i>^{<i>PIN/PIN</i>} mice. Arrows indicate the presence of pinhead-shaped sperm heads. Arrow heads indicate symplasts composed of coalesced germ cells. <b>(E)</b> Testis weight in <i>Henmt1</i>^<i>WT/WT</i> and <i>Henmt1</i>^{<i>PIN/PIN</i>} mice (n = 6 / genotype +/- SEM, * p<0.05). <b>(F)</b> Daily sperm production in <i>Henmt1</i>^<i>WT/WT</i> and <i>Henmt1</i>^{<i>PIN/PIN</i>} mice expressed as percentage of <i>Henmt1</i>^<i>WT/WT</i> (n = 6 / genotype +/- SEM, ** p<0.01) <b>(G-H)</b> PAS stained sections of epididymides from <i>Henmt1</i>^<i>WT/WT</i> and <i>Henmt1</i>^{<i>PIN/PIN</i>} mice. <b>(I)</b> Epididymal sperm content in <i>Henmt1</i>^<i>WT/WT</i> and <i>Henmt1</i>^{<i>PIN/PIN</i>} mice expressed as percentage of <i>Henmt1</i>^<i>WT/WT</i> (n = 3 / genotype +/- SD, ****p<0.0001). <b>(J)</b> Hematoxylin and eosin stained sperm from <i>Henmt1</i>^<i>WT/WT</i> and <i>Henmt1</i>^{<i>PIN/PIN</i>} mice showing the pin-shaped heads and the absence of a mitochondrial sheath (open arrow) in <i>Henmt1</i>^{<i>PIN/PIN</i>} sperm. <b>(K)</b> The percentage of sperm showing any forms of motility in <i>Henmt1</i>^<i>WT/WT</i> and <i>Henmt1</i>^{<i>PIN/PIN</i>} mice (n = 3 / genotype +/- SD, ****p<0.0001). <b>(L)</b> The percentage of sperm showing progressive motility in <i>Henmt1</i>^<i>WT/WT</i> and <i>Henmt1</i>^{<i>PIN/PIN</i>} mice (n = 3 / genotype +/- SD, ****p<0.001). <b>(M-N)</b> Electron microscopy of stage VIII pachytene spermatocytes showing the presence of inter-mitochondrial cement (arrow) and the chromatoid body (red square) in <i>Henmt1</i>^<i>WT/WT</i> and <i>Henmt1</i>^{<i>PIN/PIN</i>} mice. All data was collected from 10 week-old mice. A two-tailed unpaired student T-test was performed for statistical analyses.</p

HENMT1 is required for the repression of <i>Line-1</i> and <i>IAP</i> retrotransposons in spermatocytes and round spermatids.

<p><b>(A-D)</b> qPCR analyses of (<b>A</b>) <i>L1_A</i> (<b>B</b>) <i>L1_TF14</i> (<b>C</b>) <i>IAP_LTR</i> (<b>D</b>) <i>IAP_GAG</i> in <i>Henmt1</i>^<i>WT/WT</i> and <i>Henmt1</i>^{<i>PIN/PIN</i>} spermatocytes and round spermatids (round) purified from 30 day old mice. A two-tailed unpaired student T-test was performed for statistical analysis, n = 5/genotype, +/- SEM, * p<0.05; ** p<0.01; ***p<0.001; ****p<0.0001. <b>(E-F)</b> RNA <i>in situ</i> hybridisation for <i>Line-1</i> expression in <i>Henmt1</i>^<i>WT/WT</i> and <i>Henmt1</i>^{<i>PIN/PIN</i>} 10 weeks-old testes. Black arrows indicate pachytene spermatocytes. Red arrows indicate round spermatids. <b>(G)</b> Staining for LINE-1 in in <i>Henmt1</i>^<i>WT/WT</i> and <i>Henmt1</i>^{<i>PIN/PIN</i>} in 10 weeks-old testes. The boxed areas are magnified in the panel to the immediate right of each genotype. The scale bar = 50 μm. (<b>H</b>) Immunofluorescence staining for γH2AX (red) as a marker of DNA double stranded break respectively in <i>Henmt1</i>^<i>WT/WT</i> and <i>Henmt1</i>^{<i>PIN/PIN</i>} in 10 weeks-old testes. The white arrow indicates elevated γH2AX staining in spermatids compared to wild type cells. The boxed areas are magnified in the panel to the immediate right of each genotype. The scale bar = 50 μm.</p

Altered gene expression in <i>Henmt1</i>^{<i>PIN/PIN</i>} spermatocytes and round spermatids.

<p><b>(A)</b> A heatmap showing log2 fold change in differentially expressed genes in spermatocytes and their corresponding log2 fold change in round spermatids (round). Color coding represent log2 fold changes of genes. <i>Prm1</i>, <i>Tnp1</i>, <i>Tnp2</i>, <i>Prm2</i> indicate the position of transcripts subjected to further analysis. <b>(B)</b> A volcano plot showing expression changes of all detected genes in spermatocytes and round spermatids. Dots in cyan represent significantly differential expressed genes (FDR < 0.05) based on edgeR. Fold changes and p-values were calculated with edgeR. <b>(C-G)</b> qPCR of spermiogenic genes including (C) <i>Tnp1</i> (D) <i>Tnp2</i> (E) <i>Prm1</i> (F) <i>Prm2</i> (G) <i>Gapdhs</i> in 28 day-old <i>Henmt1</i>^<i>WT/WT</i> and <i>Henmt1</i>^{<i>PIN/PIN</i>} spermatocytes and round spermatids (n = 5 / genotype +/- SEM, * p<0.05, **p<0.01). White bar represents <i>Henmt1</i>^<i>WT/WT</i> and black bar is <i>Henmt1</i>^{<i>PIN/PIN</i>}. S’cytes = spermatocytes, S’tids = round spermatids. A two-tailed unpaired student T-test was performed for statistical analyses.</p

Enrichment of active histone mark at the promoter regions of spermiogenic genes in <i>Henmt1</i>^{<i>PIN/PIN</i>} spermatocytes.

<p>ChIP and qPCR analyses were performed on <i>Henmt1</i>^<i>WT/WT</i> and <i>Henmt1</i>^{<i>PIN/PIN</i>} spermatocytes (n = 5/genotypes, 3 biological replicates, * p<0.05, **p<0.01, *** p<0.001, **** p<0.0001). qPCR for the promoter regions of (<b>A</b>) <i>Tnp1</i>, (<b>B</b>) <i>Tnp2</i>, (<b>C</b>) <i>Prm1</i>, (<b>D</b>) <i>Prm2</i>, (<b>E</b>) <i>Gapdhs</i>, and (<b>F</b>) <i>Ppia</i> (as a house keeping control). Histone enrichment was normalised to histone H3. The data is presented in the ratio of <i>Henmt1</i>^{<i>PIN/PIN</i>} enrichment/ <i>Henmt1</i>^<i>WT/WT</i> enrichment. A two-tailed unpaired student T-test was performed for statistical analyses.</p

The <i>Henmt1</i>^{<i>PIN/PIN</i>} mutation resulted in exon 3 skipping, the production of unstable <i>Henmt1</i> mRNAs and an absence of HENMT1 protein in the testis.

<p><b>(A)</b> A schematic representation of the three <i>Henmt1</i> transcripts and the effect of the <i>Henmt1</i>^{<i>PIN/PIN</i>} on exon 3 splicing. Boxes represent exons, open bars represent introns. <b>(B)</b> RT-PCR analysis using primers located in exons 2 and 4 confirmed the skipping of exon 3 in the <i>Henmt1</i>^<i>PIN</i> allele. <b>(C)</b> qPCR analysis of <i>Henmt1</i> transcript 1 and 2 levels in <i>Henmt1</i>^{<i>PIN/PIN</i>} and <i>Henmt1</i>^<i>WT/WT</i> spermatocytes and round spermatids (round). 8 weeks old, n = 3 /genotype (* p<0.05, mean ± SD). <b>(D)</b> The relative expression of <i>Henmt1</i> transcripts 1 and 2, and transcript 3 in <i>Henmt1</i>^<i>WT/WT</i> spermatocytes and round spermatids. 8 weeks old. n = 3 /genotype (p*<0.05, ** p<0.01, mean ± SD). <b>(E)</b> HENMT1 localization (red) in <i>Henmt1</i>^<i>WT/WT</i> (upper two panels) and 8 week old <i>Henmt1</i>^{<i>PIN/PIN</i>} testes (lower two panels). DNA is labelled with DAPI (blue). <b>(F)</b> A representative western blot for HENMT1 in 8 week old <i>Henmt1</i>^<i>WT/WT</i> and <i>Henmt1</i>^{<i>PIN/PIN</i>} testes. Beta actin was used as a loading control. Where relevant a two-tailed unpaired student T-test was performed for statistical analyses. Scale bars = 50μm.</p

HENMT1 loss results in decreased piRNA bulk and the absence of 3’ 2’-O-methylation and stability.

<p><b>(A-B)</b> A representative image illustrating a ~51% reduction in the abundance of piRNAs in <i>Henmt1</i>^{<i>PIN/PIN</i>} and <i>Henmt1</i>^<i>WT/WT</i> 30 day-old testes as indicated on an acrylamide gel (total piRNAs, A) and a small RNA northern blotting for pachytene piRNA1 (B). 5.8S and 5S rRNAs were used as loading controls. Statistics were carried out on n-4 biological replicates per genotype. <b>(C)</b> The effects of β-elimination on piRNA length. 5.8S and 5S rRNAs were used as loading controls. <b>(D-E)</b> piRNA length distributions, expressed as a percentage of total, in <i>Henmt1</i>^<i>WT/WT</i> and <i>Henmt1</i>^{<i>PIN/PIN</i>}. <b>(F-G)</b> The percentage of piRNAs with (F) adenylation (last base of read was A when the reference base was not A) and (G) uridylation (last base of read was T, reference base was not T) at their 3’ end in spermatocytes and round spermatids (n = 2 / <i>Henmt1</i>^<i>WT/WT</i> spermatocytes, n = 3 <i>Henmt1</i>^<i>WT/WT</i> round, <i>Henmt1</i>^{<i>PIN/PIN</i>} spermatocytes and round +/- SD, * p<0.05; ** p<0.01; ***p<0.001; ****p<0.0001). Round = round spermatids. A two-tailed unpaired student T-test was performed for statistical analysis.</p

HENMT1 and piRNA Stability Are Required for Adult Male Germ Cell Transposon Repression and to Define the Spermatogenic Program in the Mouse.

piRNAs are critical for transposable element (TE) repression and germ cell survival during the early phases of spermatogenesis, however, their role in adult germ cells and the relative importance of piRNA methylation is poorly defined in mammals. Using a mouse model of HEN methyltransferase 1 (HENMT1) loss-of-function, RNA-Seq and a range of RNA assays we show that HENMT1 is required for the 2' O-methylation of mammalian piRNAs. HENMT1 loss leads to piRNA instability, reduced piRNA bulk and length, and ultimately male sterility characterized by a germ cell arrest at the elongating germ cell phase of spermatogenesis. HENMT1 loss-of-function, and the concomitant loss of piRNAs, resulted in TE de-repression in adult meiotic and haploid germ cells, and the precocious, and selective, expression of many haploid-transcripts in meiotic cells. Precocious expression was associated with a more active chromatin state in meiotic cells, elevated levels of DNA damage and a catastrophic deregulation of the haploid germ cell gene expression. Collectively these results define a critical role for HENMT1 and piRNAs in the maintenance of TE repression in adult germ cells and setting the spermatogenic program