Sequence characterization of piRNA clusters.

<p>(A) TE composition of predicted piRNA clusters compared to the genomic sequence of the pig. Percentages represent the share of a TE group in the genome. A ratio above 1 indicates an enrichment of a TE group in piRNA clusters, while a ratio below 1 indicates the depletion of a TE group in piRNA clusters. Different colors express the sequence divergence of a TE group to its consensus. (B) Sequence shares of TEs, protein-coding genes, pseudogenes, and uncharacterized transcribed sequences within piRNA clusters compared to the whole genome of the pig. (C) Number of piRNA clusters containing sequences of protein-coding genes, pseudogenes or both within the same piRNA cluster.</p

Transposon-derived piRNAs.

<p>(A) Shares of TE sequences in mapped piRNA reads and in the porcine genome. (B) Representation of TE families within the sequences of piRNA reads in sense and antisense direction compared to the genomic TE family distribution. (C) 5’ overlap of TE-derived piRNAs. (D) Mapping of piRNA reads to the sequence of a PRE1f2 element, a member of the tRNA-derived SINE subfamily of porcine repetitive elements. Regions from positions 24–79 nt and 128–179 nt are shown as exemplary target sites of ping-pong processing.</p

piRNAs from Pig Testis Provide Evidence for a Conserved Role of the Piwi Pathway in Post-Transcriptional Gene Regulation in Mammals

<div><p>Piwi-interacting (pi-) RNAs guide germline-expressed Piwi proteins in order to suppress the activity of transposable elements (TEs). But notably, the majority of pachytene piRNAs in mammalian testes is not related to TEs. This raises the question of whether the Piwi/piRNA pathway exerts functions beyond TE silencing. Although gene-derived piRNAs were described many times, a possible gene-regulatory function was doubted due to the absence of antisense piRNAs. Here we sequenced and analyzed piRNAs expressed in the adult testis of the pig, as this taxon possesses the full set of mammalian Piwi paralogs while their spermatozoa are marked by an extreme fitness due to selective breeding. We provide an exhaustive characterization of porcine piRNAs and genomic piRNA clusters. Moreover, we reveal that both sense and antisense piRNAs derive from protein-coding genes, while exhibiting features that clearly show that they originate from the Piwi/piRNA-mediated post-transcriptional silencing pathway, commonly referred to as ping-pong cycle. We further show that the majority of identified piRNA clusters in the porcine genome spans exonic sequences of protein-coding genes or pseudogenes, which reveals a mechanism by which primary antisense piRNAs directed against mRNA can be generated. Our data provide evidence that spliced mRNAs, derived from such loci, are not only targeted by piRNAs but are also subject to ping-pong cycle processing. Finally, we demonstrate that homologous genes are targeted and processed by piRNAs in pig, mouse and human. Altogether, this strongly suggests a conserved role for the mammalian Piwi/piRNA pathway in post-transcriptional regulation of protein-coding genes, which did not receive much attention so far.</p></div

piRNA clusters containing protein-coding genes or pseudogenes.

<p>Mapping of piRNA reads on plus and minus strands of piRNA cluster sequences combined with RefSeq (NCBI) annotation of transcribed sequences and RepeatMasker annotation of TEs. NCBI GeneIDs for transcribed sequences are stated in brackets. Directions of transcription for RefSeq sequences are indicated by white arrows.</p

Model of post-transcriptional regulation of protein-coding genes by the Piwi/piRNA pathway.

<p>(A) piRNA cluster containing a protein-coding gene. (B) Hypothetical model of post-transcriptional gene regulation mediated by piRNA clusters, based on data of porcine piRNAs. piRNA clusters containing sequences of genes or pseudogenes in reverse orientation relative to the cluster directionality can presumably produce primary piRNAs complementary to spliced mRNA, which can direct the decay of such transcripts and produce secondary piRNAs within the ping-pong amplification loop.</p

Basic characterization of putative piRNAs in porcine testes.

<p>(A) Length distribution of small RNAs. The mapped sRNA reads show an approximately Gaussian length distribution, ranging mostly from 24 to 33 nt with a peak at 30 nt. The majority of each size fraction maps to predicted piRNA clusters. (B) 5’ overlap of sRNAs. Sense and antisense sRNA reads produce a high rate of 10 nt 5’ overlaps. (C) Positional nucleotide frequencies starting from 5’ end. (D) Positional nucleotide frequencies starting from 3’ end.</p

Gene-derived piRNAs.

<p>(A) Portions of piRNA reads mapping to introns, exons and pseudogenes in the porcine genome. (B) 5’ overlap of testis piRNAs from pig, mouse and human, mapping to corresponding annotated cDNA. In all three species a high rate of 10 nt 5’ overlaps is detectable. (C) Length distribution of sense and antisense exon-derived piRNAs. (D) Mapping of piRNA reads to the mRNA sequence of the protein-coding gene NUTM2G. Exemplary sites with 10 nt 5’ overlap between sense and antisense piRNA reads are indicated by arrows.</p

tRNA-derived small RNAs.

<p>(A) Fractions of sRNAs that were annotated as known ncRNA in a sodium periodate treated and untreated sRNA library. (B) Length distribution of tRNA-derived sRNA reads. (C) Positions on tRNAs matched by 5’ ends of sRNA reads. (D) Shares of sRNA reads mapping to distinct tRNAs. (E) Alignment of tRNA sequences and their most abundant matching sRNAs (numbers refer to read counts).</p

Evolutionary Conservation of Mammalian Sperm Proteins Associates with Overall, not Tyrosine, Phosphorylation in Human Spermatozoa

We investigated possible associations between sequence evolution of mammalian sperm proteins and their phosphorylation status in humans. As a reference, spermatozoa from three normozoospermic men were analyzed combining two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry. We identified 99 sperm proteins (thereof 42 newly described) and determined the phosphorylation status for most of them. Sequence evolution was studied across six mammalian species using nonsynonymous/synonymous rate ratios (dN/dS) and amino acid distances. Site-specific purifying selection was assessed employing average ratios of evolutionary rates at phosphorylated versus nonphosphorylated amino acids (α). According to our data, mammalian sperm proteins do not show statistically significant sequence conservation difference, no matter if the human ortholog is a phosphoprotein with or without tyrosine (Y) phosphorylation. In contrast, overall phosphorylation of human sperm proteins, i.e., phosphorylation at serine (S), threonine (T), and/or Y residues, associates with above-average conservation of sequences. Complementary investigations suggest that numerous protein–protein interactants constrain sequence evolution of sperm phosphoproteins. Although our findings reject a special relevance of Y phosphorylation for sperm functioning, they still indicate that overall phosphorylation substantially contributes to proper functioning of sperm proteins. Hence, phosphorylated sperm proteins might be considered as prime candidates for diagnosis and treatment of reduced male fertility

Evolutionary Conservation of Mammalian Sperm Proteins Associates with Overall, not Tyrosine, Phosphorylation in Human Spermatozoa

We investigated possible associations between sequence evolution of mammalian sperm proteins and their phosphorylation status in humans. As a reference, spermatozoa from three normozoospermic men were analyzed combining two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry. We identified 99 sperm proteins (thereof 42 newly described) and determined the phosphorylation status for most of them. Sequence evolution was studied across six mammalian species using nonsynonymous/synonymous rate ratios (dN/dS) and amino acid distances. Site-specific purifying selection was assessed employing average ratios of evolutionary rates at phosphorylated versus nonphosphorylated amino acids (α). According to our data, mammalian sperm proteins do not show statistically significant sequence conservation difference, no matter if the human ortholog is a phosphoprotein with or without tyrosine (Y) phosphorylation. In contrast, overall phosphorylation of human sperm proteins, i.e., phosphorylation at serine (S), threonine (T), and/or Y residues, associates with above-average conservation of sequences. Complementary investigations suggest that numerous protein–protein interactants constrain sequence evolution of sperm phosphoproteins. Although our findings reject a special relevance of Y phosphorylation for sperm functioning, they still indicate that overall phosphorylation substantially contributes to proper functioning of sperm proteins. Hence, phosphorylated sperm proteins might be considered as prime candidates for diagnosis and treatment of reduced male fertility