RNA interference genes in platypus. Phylogenetic trees of EIF2C (A) and PIWI (B) families in multiple vertebrate species. The platypus PIWIL4 (oanPIWIL4) sequence was incomplete, and a partial coding sequence was used to place this gene on the tree (broken line) (Supplemental Table 2). Platypus genes are highlighted in red. Scale: 0.1 = 0.1 base substitutions expected. (oan) Ornithorhynchus anatinus; (cfa) Canis familiaris; (hsa) Homo sapiens; (mmu) Mus musculus; (rno) Rattus norvegicus; (mdo) Monodelphis domesctica; (gga) Gallus gallus; (xtr) Xenopus tropicalis; (dre) Danio rerio.

Novel microRNAs in monotremes. (A) Pie graph of monotreme species distribution of 180 novel miRNAs. Eighty-one miRNAs were sequenced in both platypus and echidna small (gray); 95 were only in platypus (black); and four were cloned only in echidna (white), although their sequences were present in the platypus genome. (B) Novel miRNA tissue expression profiles. The expression score represented by blue squares is a ratio of the number of normalized reads in that tissue versus all tissues in that organism. Expression data for 10 of the most highly expressed novel miRNAs is shown. (C) Testis miRNA classification. Ninety-two novel miRNAs were highly expressed in testis and were derived from one of seven related hairpin precursors. The number of miRNAs and miRNA seed sequences derived from each precursor category is shown. (D) Five out of nine contigs containing testis-expressed novel miRNAs map to X1q. Fluorescence in situ hybridization with BAC and Fosmid clones containing miRNA clusters on male platypus metaphase chromosomes. (i) BAC 175h10 from contig 8388 (green) and BAC 273L19 from contig 7359 (red) on the long arm of X1. (ii) Colocalization of BAC 175h10 from contig 8388 (green) and BAC 272K3 from contig 22,847 (red) on X1q. (iii) Localization of Fosmid 0879F15 from contig 7160 (green) on X1q. (Insert) Localization of Fosmid 1002L21 (contig 11,344) on X1q. (iv) BAC clones 686C10 (green) and 802P20 (red) from contig 1754 map to a metacentric autosome. (E) Novel testis-expressed chromosome X1 miRNA cluster. Only small RNAs that map to the genome three times or less are shown. The cloning frequency divided by the number of genome matches for each miRNA is represented by a bar. Platypus miRNAs are plotted in green, echidna in red; platypus and echidna bars are independent and not additive. Gaps in the genome assembly are shown by //. A LINE element insertion that disrupted an echidna small RNA precursor is represented in yellow. (F) Orientation and sequence conservation of 53 miRNAs derived from the miR-1421 group of precursors. Multiple sequence alignment of 53 miR-1421 precursors with the sequence of the mature miRNA highlighted in blue and the miRNA star in yellow. The percent of nucleotides in the alignment that match the consensus for each position is graphed below. Perfectly conserved nucleotides are marked with an asterisk (*).

Conservation of microRNA expression patterns in monotremes. Comparison of platypus/echidna and platypus/chicken miRNA tissue expression profiles. miRNA orthology was determined by perfect match (platypus and echidna) or at least 16-nt identity in the mature miRNA (mouse, human, chicken, and fish). The expression score represented by blue squares is a ratio of the number of normalized reads in that tissue versus all tissues in that organism, and the difference score represented by red squares is the difference between the confidence intervals of the expression ratios for each platypus/echidna or platypus/chicken pair of miRNAs. A subset of miRNAs shared between monotremes, mouse/human, and chicken; monotreme and chicken; monotreme and fish; and monotreme and mouse/human are shown. miRNAs with the most distinct tissue-specific expression patterns are shown here clustered by tissue expression; the complete data sets can be found in Supplemental Figs. 1–4.

piRNAs in platypus. (A) Total RNA labeling from mouse, platypus, and echidna testis reveals a distinct ∼30-nt piRNA species in monotremes. RNA was dephosphorylated, end labeled with ³²P, and run on a denaturing 15% polyacrylamide gel. Sizes of markers (left) are in nucleotides. (B) Comparison of platypus and mouse piRNA libraries. A platypus piRNA library was generated by cloning small RNAs in a 25- to 33-nt size window from total platypus testis RNA. Sequences from this library and from an equivalent mouse piRNA library (Girard et al. 2006) were classed as repeat (red), unannotated (blue), or other (beige). The repeat piRNAs were broken down into classes of repeat (bars). (C) 10A profiles for platypus piRNAs. An A at piRNA position 10 is a signature of an active piRNA amplification system. “Clustered” piRNAs are defined as those that map to the genome once and fall into one of 50 piRNA clusters (Supplemental Table 4), while “non-clustered” piRNAs are those that map to the genome once but do not fall into a cluster. 10A percentages for all piRNAs are in black, those that are derived from LINE2 elements in blue, and those from Mon1 SINE elements in white. (D) Characteristics of “small” piRNAs. “Percent 1U” includes all small RNAs that match the genome; “percent in clusters” includes only small RNAs that map only once to the genome.