Deep small RNA sequencing from the nematodeAscarisreveals conservation, functional diversification, and novel developmental profiles

Abstract

Eukaryotic cells express several classes of small RNAs that regulate gene expression and ensure genome maintenance. Endogenous siRNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs) mainly control gene and transposon expression in the germline, while microRNAs (miRNAs) generally function in post-transcriptional gene silencing in both somatic and germline cells. To provide an evolutionary and developmental perspective on small RNA pathways in nematodes, we identified and characterized known and novel small RNA classes through gametogenesis and embryo development in the parasitic nematode Ascaris suum and compared them with known small RNAs of Caenorhabditis elegans. piRNAs, Piwi-clade Argonautes, and other proteins associated with the piRNA pathway have been lost in Ascaris. miRNAs are synthesized immediately after fertilization in utero, before pronuclear fusion, and before the first cleavage of the zygote. This is the earliest expression of small RNAs ever described at a developmental stage long thought to be transcriptionally quiescent. A comparison of the two classes of Ascaris endo-siRNAs, 22G-RNAs and 26G-RNAs, to those in C. elegans, suggests great diversification and plasticity in the use of small RNA pathways during spermatogenesis in different nematodes. Our data reveal conserved characteristics of nematode small RNAs as well as features unique to Ascaris that illustrate significant flexibility in the use of small RNAs pathways, some of which are likely an adaptation to Ascaris' life cycle and parasitism.

Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway

“…The functional meaning of this small RNA size discrepancy needs further investigation. Given that each Argonaute protein has very specific requirements for small RNA association, it is possible that the larger small RNAs generated against Ago2-2 do not load productively into Ago2-2 and thus cannot mediate gene silencing (70). In Drosophila, it has been demonstrated that modifications of the 3Ј terminus of siRNAs and miRNAs are coupled to efficient assembly into Ago2-RNA-induced silencing complex (71).…”

Identification, expression, and molecular evolution of microRNAs in the “living fossil” Triops cancriformis (tadpole shrimp)

“…Each sample was ground with a mortar and pestle, and the total RNAs were extracted with TRIzol Reagent (Invitrogen), according to manufacturer's instructions. To normalize the reads among the developmental stages, two small RNA spikes (spike1: p-AACUGUGUCUUUUCUGAAUAGA; and spike2: p-UAUUUAGAAUGGCGCUGAUCUG) corresponding to mammal-specific small RNAs (Wang et al 2011) were added at different concentrations (spike 1, 20 fmol; spike 2, 0.5 fmol) to 20 μg of total RNA (or 30 μg for egg RNA only). The small RNA fraction (∼12-45 nt), including miRNAs, was isolated and purified from these total RNA samples by gel electrophoresis.…”

“…To compare the relative levels of small RNA expression in the six different developmental stages, read normalization was performed using two spike RNAs, as described previously (Wang et al 2011). The read number obtained from the second instar larvae was defined as the standard by setting it to 20 million, and the read numbers for the other stages were normalized to it.…”

“…In addition to analyses of miRNA sequences, it is also important to focus on the components of the RNAi machinery. For example, Ascaris suum reportedly lacks the piRNAs, Piwi-clade Argonautes, and other proteins associated with the piRNA pathway, indicating that the piRNA pathway was lost in A. suum (Wang et al 2011). These findings indicate that the investigation of miRNAs and components of RNAi machinery in nonmodel species is important for elucidating their evolutionary pathways.…”

Guest list or black list: heritable small RNAs as immunogenic memories

“…While piRNAs are involved in many inherited immunity effects, they are obviously not central to all such mechanisms. piRNAs are absent in several organisms that display heritable genome immunity, such as plants, fungi, and even in some worms, for example in the parasitic nematode, Ascaris suum, a relative of C.elegans [45,46].…”