Ascaris endo-siRNAs to mRNAs. (A) Size distribution of small RNA raw reads corresponding to mRNAs from 17 5′ all-phosphate libraries. (Upper panel) Antisense small RNAs; (lower panel) sense small RNAs. (B) Comparison, classification, and levels of normalized small RNAs corresponding to mRNAs. Area of the circles is proportional to the amount of different types of small RNAs (see text) present in the 5′-mono- and 5′-all-phosphate libraries. (C) Twenty-two-nucleotide small RNAs are polyphosphate RNAs, whereas 26G-RNAs are monophosphate RNAs. miRNAs were used to normalize the reads between 5′ monophosphate and 5′ all-phosphate libraries. In these and all subsequent box-and-whisker plots, the top and bottom ends of each box represent the 75th and 25th percentile, respectively; the middle line represents the median value; the extended lines illustrate the range (highest and lowest value); and dots represent values beyond the extremes of the whisker. (D) siRNA distribution on mRNA transcripts. Different types of small RNA reads from 5′ all-phosphate libraries are plotted for 8279 full-length cDNAs. Each mRNA is equally divided into 20 bins, and small RNAs mapped to each bin were counted. Plotted is the sum of the counts (y-axis) against the relative position (x-axis) on the cDNA from 5′ to 3′, with the 5′ end of the mRNA designated as 1 and the site of polyadenylation at the 3′ end designated as 100. (E) Twenty-two-nucleotide monophosphate RNAs are likely derived from 22-nt polyphosphate RNAs. Plots illustrate a comparison of small RNA reads between 5′ all-phosphate libraries (x-axis) and 5′ monophosphate libraries (y-axis). The data sets are the top 200 siRNAs in read frequency.

Ascaris reproductive systems and early development. (A) Ascaris male and female reproductive systems. The male seminal vesicle contains spermatids that are activated on fertilization of a female. Sperm are stored in the female seminal receptacle. As mature oocytes move through the seminal receptacle, they are fertilized, and for each fertilized egg, an impermeable layer and chitinous shell is immediately formed around the egg in region one of the uterus (zygote-1). The zygote undergoes maturation as it passes through the uterus (within 12–24 h). Note that the figure is not drawn to scale. Extended male and female reproductive systems are ∼140 cm and ∼240 cm in length, respectively. (B) Early Ascaris embryo development. Eggs derived from the region designated as zygote-4 are equivalent to 0-h embryos. Incubation of 0-h embryos at 30°C in high humidity leads to relatively synchronous development (∼85%) as illustrated. The maternal to zygotic transition is thought to occur between 46 and 96 h of development (four to 16 cells). Ascaris larvae L1 and L2 remain within the chitinous shell. The 0-h embryo is ∼100 × 45 μm and does not change in size during development as illustrated. All regions/stages in A and B were analyzed in the study.

Characteristics of Ascaris small RNAs. (A) 5′ end-labeled Ascaris small RNAs. Low-molecular-weight (LMW) enriched RNAs were treated with calf alkaline phosphatase and then 5′ end labeled with ³²P using T4 polynucleotide kinase. RNAs in A–D were resolved on 12.5% denaturing PAGE and autoradiographed. (B) 3′ end-labeled Ascaris small RNAs. LMW enriched RNAs were 3′ end-labeled using ³²pCp and T4 RNA ligase. (C) Ascaris small RNAs ∼22 nt in length can be cap-labeled. LMW enriched RNAs were capped using ³²P-α-GTP and vaccinia guanylyltransferase. Samples were treated with tobacco acid pyrophosphatase (TAP) to remove the cap to confirm cap-labeling. Note the significant labeling of RNAs ∼22 nt in length. (D) The majority of germline and early embryo 22-nt RNAs have 5′ polyphosphates. pCp-labeled small RNAs shown in B were treated with GTP and vaccinia guanylyltransferase. The lower and upper arrows mark the average position of the labeled small RNAs prior to and after capping. Note that the majority of the ³²pCp-labeled RNAs are shifted to a longer length. (E,F) Size distribution of analyzed Ascaris small RNAs. (E) All 28 5′ monophosphate libraries characterized combined. (F) All 19 5′ all-phosphate (5′ tri-, di-, and monophosphate) libraries characterized combined. In these and all subsequent size distribution figures, small RNAs starting with A, C, G, and U in different sizes are plotted against their read frequencies (raw reads, before normalization). (G,H) Comparison, classification, and levels of Ascaris small RNAs characterized. (G) 5′ monophosphate RNAs. (H) 5′ all-phosphate libraries derived from different developmental stages. Area of the circles is proportional to the amount of small RNAs present in each stage or tissue after normalization (see text).

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.

Dynamic miRNA expression profiles in Ascaris development. Heatmap and Northern Blot expression profiles of Ascaris miRNAs. For each miRNA, colors represent log₂ values of fold changes to the average read frequencies of the miRNA during development. Heatmap expression profiles represent normalized mature miRNA read numbers.

Ascaris small RNAs expression and their 5′ phosphate termini. (A) Northern blots illustrating intrauterine zygotic expression of miRNAs (for whole blots, see Supplemental Fig. S6A). (B) Northern blots illustrating dynamic expression of 22G-RNAs. (C) Northern blots illustrating expression of testis-specific 26G-RNAs. (D) Ascaris miRNAs have a 5′ monophosphate. Northern blot of Ascaris LMW RNA untreated or treated with terminator, GTP and guanylyltransferase (capping), or ligation of an RNA oligonucleotide (T4 RNA ligation) and probed with asu-miR-5347 probe. Note that the miRNA is degraded by terminator, is uncapped, and can be ligated to an RNA oligonucleotide, indicating the 5′ end is a monophosphate. (E) Ascaris 22G-RNAs have 5′ polyphosphates. Northern blot as in D probed with a 22G-RNA probe. Note that the 22G-RNA can be capped but is not susceptible to terminator or T4 RNA ligation, suggesting the 5′ end of the RNA is a di- or triphosphate. (F) Ascaris 26G-RNAs have a 5′ monophosphate. Northern blot as in D probed with a 26G-RNA probe. Note that the 26G-RNA is testis specific and is degraded by terminator, is uncapped, and can be ligated to an RNA oligonucleotide, indicating the 5′ end is a monophosphate. The band at ∼32 nt in the RNA+T4 RNA ligation is an artifact in the ovary.

Ascaris 26G-RNAs and 22G-RNAs targets. (A) Examples of distribution of 26G- and 22G-RNAs to mRNAs throughout development. Note that all small RNAs are antisense. (Red) 22G-RNAs; (blue) 26G-RNAs. Vertical dashed lines indicate the start and stop of the ORF. Numbers on left indicate the normalized read frequency of the gray line. Note that for 26G-RNA targets, only the profiles from testis 26G-RNAs are shown; for their complete profiles, see Supplemental Table S7 and Supplemental Figures S9 and S10. (B) Dynamic expression profiles of 22G-RNAs to 159 exemplary targets (see text). Normalized As.22G RNA reads mapping to each target mRNA were used for the heatmap. For each target, colors represent log₂ values of fold changes to the average read frequencies of the As.22G RNAs during development. (C) Distribution of 26G- and 22G-RNAs on mRNAs predominantly targeted by one of the two types of small RNAs. Only mRNAs identified as near full length are used and include 138 26G-targeted mRNAs and 97 22G-targeted mRNAs (see Supplemental Table S7; Supplemental Figs. S9, S10). Plotted is the sum of the counts (y-axis) against the relative position (x-axis) on the cDNA from 5′ to 3′, with the 5′ end of the mRNA designated as 1 and the site of polyadenylation at the 3′ end designated as 100. (D) Venn diagram illustrating the relationship between the mRNAs targeted by 26G- or 22G-RNAs. (E) Ratio of defined testis 26G-RNA/22G-RNAs mRNA targets. (Upper panel) 26G-RNA targets, the 26G/22G ratio; (lower panel) 22G-RNA targets. (F) Expression profile of 26G-RNAs and 22G-RNAs in spermatogenesis is different for Ascaris and C. elegans (see Discussion). Note that for C. elegans, the total level of 26G-RNAs decreases during spermatogenesis with a concomitant and dramatic increase in 22G-RNAs targeting the same mRNAs. In contrast, the ratio of Ascaris 26G/22G RNAs to mRNAs remains the same during spermatogenesis and the 26G-RNAs are always the dominant small RNA species. Note that the figure does not represent absolute levels of the RNAs.