Ascaris lacks the piRNA pathway. (A,B) No piRNAs are present in Ascaris, as illustrated by the size distribution of Ascaris germline small RNAs. (A) Testis small RNAs. (B) Ovary small RNAs. Raw reads (before normalization) from the libraries are plotted. Note that no 21U or other U-initiated small RNAs are enriched in either the testis or ovary after miRNAs and siRNAs are removed. This is also observed for other germline and embryo libraries (see Supplemental Data). (C) Ascaris lacks Piwi-clade Argonaute protein. ClustalX was used to generate an alignment of Ascaris and C. elegans Argonaute proteins. The alignment was used to generate a distance matrix and then analyzed by neighbor-joining and bootstrapping methods using PHYLIP software. TreeView (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html) was used to display the resulted tree. Numbers on the branches show the support value for 1000 bootstraps. Argonaute proteins from Ascaris are colored in blue and red for AGO-clade and WAGO-clade proteins, respectively. C. elegans Piwi-clade Argonautes are colored in green. Accession numbers for the Ascaris proteins can be found in Supplemental Table S9. Note that Ascaris also lacks an ortholog of C. elegans RDE-1. (D) RNAs with a 2′-O-methyl are resistant to periodate treatment and β-elimination. Control RNAs (22 nt) were 5′ labeled by kinasing and then either mock treated (−) or treated with periodate followed by β-elimination (+).(E) Ascaris 26G-RNAs are not resistant to periodate/β-elimination and thus are not 3′ modified. Northern blots of Ascaris RNAs treated with periodate/β-elimination and probed with Ascaris 26G-1 and 26G-4 probes. (F) Ascaris 22G-RNAs are not 3′ modified. Northern blot as in E using an Ascaris 22G-RNA probe. (G) Ascaris miRNAs are not 3′ modified. Northern blot as in E using an Ascaris miRNA probe (asu-miR-5342). (H) C. elegans 21U RNAs are 3′ modified and resistant to periodate/β-elimination. Northern blot of C. elegans RNAs treated with periodate/β-elimination and probed with a C. elegans 21U-RNA probe.
