Nematode Small RNA Pathways in the Absence of piRNAs

TL;DR: In this article, small RNA pathways in the parasitic nematode Ascaris were analyzed, and the authors provided key insights into the conservation and divergence of small RNA pathway in C. elegans.

Abstract: Small RNA pathways play diverse regulatory roles in the nematode C. elegans. However, our understanding of small RNA pathways, their conservation, and their roles in other nematodes is limited. Here, we analyzed small RNA pathways in the parasitic nematode Ascaris. Ascaris has ten Argonautes with five worm-specific Argonautes (WAGOs) that are associated with secondary 59-triphosphate small RNAs (22-24G-RNAs). These Ascaris WAGOs and their small RNAs target repetitive sequences (WAGO-1, WAGO-2, WAGO-3, and NRDE-3) or mature mRNAs (CSR-1, NRDE-3, and WAGO-3) and are similar to the C. elegans mutator, nuclear, and CSR-1 small RNA pathways. Ascaris CSR-1 likely functions to "license" gene expression in the absence of an Ascaris piRNA pathway. Ascaris ALG-4 and its associated 26G-RNAs target and appear to repress specific mRNAs during meiosis in the testes. Notably, Ascaris WAGOs (WAGO-3 and NRDE-3) small RNAs change their targets between repetitive sequences and mRNAs during spermatogenesis or in early embryos illustrating target plasticity of these WAGOs. We provide a unique and comprehensive view of mRNA and small RNA expression throughout nematode spermatogenesis that illustrates the dynamics and flexibility of small RNA pathways. Overall, our study provides key insights into the conservation and divergence of nematode small RNA pathways.

Summary

Introduction

• Small RNAs contribute to many regulatory processes.
• Overall, C. elegans secondary 22G-RNAs are amplified responses to targets identified by 21U- and 26G-RNAs and other primary siRNAs.
• While extensive analyses of C. elegans Argonautes, small RNAs, and pathways have been carried out, relatively little is known regarding the conservation, divergence, or function of these pathways in other nematodes [8, 40, 41].
• Thus, they have been described as defining self vs. non-self [57].
• Here, the authors generated antibodies to Ascaris Argonaute proteins and carried out Argonaute IP and small RNA sequencing to characterize small RNA pathways in several developmental stages.

Results

• The authors previously identified 10 Ascaris Argonautes [51].
• The 5 Ascaris WAGOs were named AsCSR-1, AsWAGO-1, AsWAGO-2, AsWAGO-3, and AsNRDE-3 based on expression pattern, their sequence and phylogenetic similarity to C. elegans, and the small RNAs associated with these Argonautes (see below)(Figure 1).
• These mRNA targeting Argonautes bind distinct sizes of small RNAs, with AsNRDE-3, AsWAGO-3, AsCSR-1 and AsALG-4 associated small RNAs 22G-, 23G-, 24G-, and 26G-RNAs, respectively (Figure S6).
• Most of these repetitive sequences are not expressed and thus appear silenced (Figure 5A and Tables S3).
• Prior to the formation of spermatids, AsCSR-1 in concert with AsALG-4 and AsNRDE-3 small RNAs may also repress and facilitate the turnover of all mRNAs (Figure 6D).

Discussion

• The authors understanding of nematode small RNA pathways comes from detailed studies in C. elegans [8-12, 70, 88].
• Little is known regarding the conservation and functional role of small RNA pathways (other than miRNAs) in these divergent nematodes [51, 89, 90].
• Overall, several small RNA pathways and their functions appear conserved between Ascaris and C. elegans while others have diverged in function and targets or been lost.
• The most abundant small RNAs in Ascaris are 22G-RNA secondary siRNAs.
• A striking feature of Ascaris small RNA pathways is the unique expression of AsALG-4 and associated 26G-RNAs during the later stages of spermatogenesis (M6-M7).

Summary

• Nematoda is a diverse phylum including free-living and parasitic species.
• Ascaris lacks piRNAs but maintains a CSR-1 pathway with small RNAs that target most expressed mRNAs.
• Ascaris ALG-4 associated 26G-RNAs target male meiosisspecific genes commensurate with their mRNA degradation and do not appear to act as primary siRNAs for targeting and generating secondary 22G-RNAs during spermatogenesis.
• Several Ascaris Argonautes, including AsNRDE-3 and AsWAGO-3 targets are stage dependent altering their targets between mRNAs and repeats during spermatogenesis and development.
• The authors data significantly expand their understanding of the conservation, divergence, and flexibility of nematode Argonautes and small RNA pathways.

Antibodies

• The authors generated polyclonal antibodies to Ascaris fusion proteins for AsALG-1, AsALG-4, AsCSR-1, AsWAGO-1, AsWAGO-2, AsWAGO-3, and AsNRDE-3 and also to peptides for AsWAGO-2 and AsWAGO3.
• Ascaris embryo immunohistochemistry was carried as described [61] using a modified freeze-crack method to permeabilize and fix embryos.
• The embryos 22 were then re-suspended in blocking solution (0.5% BSA in PBS pH7.4) for 30 min at RT, followed by overnight incubation in primary antibodies at 4°C, and then a 2 hr incubation in secondary antibodies at room temperature.
• Regions were characterized and defined based on nuclear morphology and presence or absence of mitotic/meiotic structures as follows: 1. Mitotic: round-shape interphase nuclei with evenly distributed chromatin and defined nucleolus and the presence of mitotic metaphases and anaphases.
• After staining of nuclei with DAPI, slides were mounted in anti-fade medium and kept in the dark at room temperature for 24 h before imaging.

Image Acquisition

• Ascaris germline immunohistochemistry and DAPI-stained preparations were imaged on an Applied Precision DeltaVision microscope, using a 60X immersion objective and FITC/DAPI excitation filter set.
• Images were deconvolved with Applied Precision’s Softworx software and analyzed using Fiji software.
• The beads were resuspended in 250 ul of Proteinase K buffer containing 200 µg/ml Proteinase K and incubated for 1 hr at 37°C.
• Following RppH treatment, samples were repurified with Trizol LS (adopted for small RNAs extraction) and stored at -80°C.
• Long RNA (>200 nt) libraries were made using CORALL Total RNA-Seq Library Prep Kit .

Small RNA data analysis

• Bioinformatic processing of small RNA sequencing data was carried out as previously described [51].
• The authors normalized the coverage for each library to 30 million reads, a number close to the 24 average number of raw input reads for the libraries.
• The authors then used the normalized reads (rpkm) mapped to these loci and a 10-fold enrichment in at least one library to define these genome regions as enriched for AsWAGO-1 and AsWAGO-2 small RNAs.
• The majority (~77%) of the sequences defined as AsNRDE-3 targets overlap with AsWAGO-1/2 targets.
• Heatmaps were generated using Treeview 3 [127].

Data access.

• The small RNA and RNA sequencing data is deposited to NCBI GEO database (accession number pending).
• The data is also available in UCSC Genome Browser track data hubs [139] that can be access with this link: http://genome.ucsc.edu/s/jianbinwang/Ascaris_small_RNAs.

Figure Legends

• A. Argonaute protein phylogenetic tree showing the relationship of Ascaris and C. elegans Argonautes.
• A. Size distribution, frequency, and targets of small RNAs associated with specific Argonautes.
• Ascaris male gonad regions and nuclear morphology.
• Targeted loci were sorted based on the same order in the four heatmaps.
• A. Genome browser view of a region of chromosome 1 illustrating AsCSR1, AsNRDE-3 and AsALG-4 associated small RNAs and their mRNA target expression during spermatogenesis.

Full text

Nematode Small RNA Pathways in the Absence of piRNAs
Maxim Zagoskin
1,2,3
*, Jianbin Wang
1,2,3,4
*
,#
, Ashley T. Neff
1
, Giovana M. B. Veronezi
1
, and
Richard E. Davis
1,2#
1
Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora,
CO
2
RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora, CO
3
Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, TN
4
UT-ORNL Graduate School of Genome Science and Technology, University of Tennessee, Knoxville,
TN
*These authors contributed equally
#
Corresponding Authors, richard.davis@cuanschutz.edu, jianbin.wang@utk.edu
.CC-BY-NC-ND 4.0 International licenseavailable under a
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2
Abstract
Small RNA pathways play diverse regulatory roles in the nematode C. elegans. However, our
understanding of small RNA pathways, their conservation, and their roles in other nematodes is limited.
Here, we analyzed small RNA pathways in the parasitic nematode Ascaris. Ascaris has ten Argonautes
with five worm-specific Argonautes (WAGOs) that are associated with secondary 5’-triphosphate small
RNAs (22-24G-RNAs). These Ascaris WAGOs and their small RNAs target repetitive sequences (WAGO-
1, WAGO-2, WAGO-3, and NRDE-3) or mature mRNAs (CSR-1, NRDE-3, and WAGO-3) and are similar
to the C. elegans mutator, nuclear, and CSR-1 small RNA pathways. Ascaris CSR-1 likely functions to
“license” gene expression in the absence of an Ascaris piRNA pathway. Ascaris ALG-4 and its associated
26G-RNAs target and appear to repress specific mRNAs during meiosis in the testes. Notably, Ascaris
WAGOs (WAGO-3 and NRDE-3) small RNAs change their targets between repetitive sequences and
mRNAs during spermatogenesis or in early embryos illustrating target plasticity of these WAGOs. We
provide a unique and comprehensive view of mRNA and small RNA expression throughout nematode
spermatogenesis that illustrates the dynamics and flexibility of small RNA pathways. Overall, our study
provides key insights into the conservation and divergence of nematode small RNA pathways.
Keywords: Nematode, small RNA pathways, Argonautes, WAGOs, CSR-1, NRDE-3, ALG-4, piRNAs,
22G-RNA, 26G-RNA, RdRP, spermatogenesis, germline, and Ascaris
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Introduction
Small RNAs contribute to many regulatory processes. They are associated with diverse functions including
repressing foreign invaders and mobile elements, transcriptional regulation, mRNA translation and
degradation, DNA repair, chromatin regulation and epigenetic inheritance, and ciliate genome
rearrangements [1-5]. One of the first discoveries of small RNAs and their role in gene regulation was in
the free-living nematode Caenorhabditis elegans [6, 7]. Subsequent studies in C. elegans have revealed
a diverse and complex set of small RNAs, pathways, and associated Argonautes [8-11].
C. elegans small RNAs include miRNAs, 21U-RNAs (piRNAs) and small-interfering RNAs (siRNAs). C.
elegans miRNAs are involved in post-transcriptional gene regulation through regulation of mRNA
translation and degradation [12], but have also been associated with transcriptional activation [13]. C.
elegans can generate siRNAs against foreign elements, but also has a large repertoire of endogenous
siRNAs [8-11]. These C. elegans small RNAs are named based on their length and predominant first
nucleotide of the RNA, e.g., 21U-, 22G- and 26G-RNAs. 21U-RNAs have a 5’-monophosphate and 3’ 2’-
O-methylation [14-16]. They are primarily thought to identify foreign (non-self) RNA elements. The
identification of these foreign elements through RNA base-pairing leads to the subsequent synthesis of
secondary siRNAs that repress their targets. 22G-RNAs have a 5’-triphosphate [16-20]. These are
secondary siRNAs as they are generated in response to other small RNAs (21U-RNA, 26G-RNA, or other
siRNAs) [20, 21]. Thus, primary siRNAs base-pair with transcript targets, marking or identifying them for
the synthesis of the more abundant antisense, secondary 22G-RNAs by RNA-dependent RNA
polymerases (RdRPs) [18-23]. 22G-RNAs silence mobile elements, pseudogenes, non-annotated loci, and
select endogenous, germline genes [18]. They also serve to “activate or license”, “tune”, or repress gene
expression [24-27]. 26G-RNAs have a 5’ monophosphate [16, 19, 28, 29]. There are two classes of 26G-
RNAs in C. elegans, one is testis-specific and associated with the Argonautes ALG-3/4 [30-32], the other
is expressed in early embryos and associated with the Argonaute ERGO-1 [28, 29, 32, 33]. Like 21U-
RNAs, 26G-RNAs trigger or act to prime the synthesis of secondary siRNAs (22G-RNAs) through base-
pairing with targets [8-11]. Overall, C. elegans secondary 22G-RNAs are amplified responses to targets
identified by 21U- and 26G-RNAs and other primary siRNAs.
Small RNAs are bound by effector Argonaute proteins. C. elegans Argonautes have undergone significant
expansion and diversification. Twenty-seven Argonaute genes were originally described [23]; 19 are
expressed (Julie Claycomb, personal communication). These include members of the AGO, PIWI, and
WAGO (Worm-specific Argonautes) Argonaute clades [34]. The C. elegans AGO clade Argonautes (5
AGOs) interact with miRNAs and 26G-RNAs, the PIWI clade (1 AGO) with 21U-RNAs (the worm piRNA
ortholog), and the WAGO clade (13 AGOs) with 22G-RNAs. Several of the WAGO clade Argonautes
function in the nucleus regulating transcription and heterochromatin formation [3, 11, 35, 36]. The largest
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expansion of C. elegans Argonautes is in the WAGO clade [23]. Many of these WAGOs are thought to
have redundant functions in C. elegans.
Nematodes are an extremely diverse and abundant phylum adapted to a wide variety of lifestyles [37-39].
While extensive analyses of C. elegans Argonautes, small RNAs, and pathways have been carried out,
relatively little is known regarding the conservation, divergence, or function of these pathways in other
nematodes [8, 40, 41]. Nematodes have been divided into three major classes and five clades with C.
elegans and its relatives as members of Clade V [42-45]. The nematode Ascaris is a parasite of humans
(and pigs) infecting upwards of 800,000 people [46-48]. Ascaris is a Clade III nematode estimated to have
diverged from C. elegans approximately 365-400 million years ago [49, 50]. Previous studies in Ascaris
indicated that piRNAs and PIWI Argonautes are absent in Ascaris [51]. PIWI Argonautes and piRNAs play
a key role in repressing mobile elements in the germline. C. elegans piRNAs have been proposed to serve
in identifying and defining foreign genetic elements acting upstream of secondary siRNA pathways [52-
56]. Thus, they have been described as defining self vs. non-self [57]. The WAGO-associated 22G
secondary RNAs function in silencing and can maintain silencing of the foreign elements over generations
in C. elegans [8, 11]. To counteract silencing, it has been proposed that 22G-RNAs associated with the C.
elegans CSR-1 function to ‘license”, identify self, or protect germline genes from repression and allow for
their expression [24, 26, 58]. Given the key role of piRNAs in many organisms, and their role in C. elegans,
the absence of piRNAs and PIWI in Ascaris raises the question of how Ascaris small RNA pathways have
adapted to the absence of piRNAs and PIWI (e.g., self vs non-self). Does Ascaris still need small RNA
pathways to “license” gene expression without the presence of piRNAs? How are self vs non-self elements
determined?
Here, we generated antibodies to Ascaris Argonaute proteins and carried out Argonaute IP and small RNA
sequencing to characterize small RNA pathways in several developmental stages. Ascaris has Argonautes
that are highly specific for binding miRNAs (AsALG-1) and 26G-RNAs (AsALG-4). AsWAGO Argonautes
bind 5’-triphosphate small RNAs (22-24G-RNAs). These small RNAs target repetitive sequences including
mobile elements, but they also target mRNAs and likely “license”, “tune”, and/or repress gene expression.
Two Ascaris WAGOs change their genomic targets (mRNAs vs repetitive sequences) in different
developmental stages. We exploited the long ~1 meter Ascaris male germline to obtain discrete regions of
the testis and analyzed Ascaris Argonautes and their small RNAs throughout spermatogenesis. These
analyses provide a unique and comprehensive timeline for the expression of Argonautes, their bound small
RNAs and targets, and changes in expression of their corresponding genomic or mRNA targets throughout
nematode spermatogenesis. Our study suggests that in the absence of piRNAs and with extensive
evolutionary divergence from C. elegans, several Ascaris small RNA pathways and Argonautes appear to
bind similar small RNAs, have similar targets, and in many cases appear to serve similar roles in both
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was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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5
nematodes. Therefore, the potential “licensing” of gene expression in another nematode by Ascaris CSR-
1 does not appear to be a consequence or counter response to the presence of piRNAs. Overall, our
studies illustrate the conservation, loss of Argonautes, and divergence of small RNA pathways that
illustrate the flexibility and adaptability of Argonautes and small RNA pathways in nematodes.
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was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 23, 2021. ; https://doi.org/10.1101/2021.07.23.453445doi: bioRxiv preprint

Frequently asked questions

Q1. What are the contributions in "Nematode small rna pathways in the absence of pirnas" ?

Claycomb et al. this paper found that C. elegans small RNAs are associated with diverse functions including repressing foreign invaders and mobile elements, transcriptional regulation, mRNA translation and degradation, DNA repair, chromatin regulation and epigenetic inheritance. 

Q2. What is the role of AsCSR-1 in spermatogenesis?

Plasticity of small RNA pathways during spermatogenesis AsNRDE-3 largely targets repetitive sequences in the female germline, early embryo, and early stages of spermatogenesis. 

Q3. What is the effect of AsCSR-1 on spermatogenesis?

The decrease in AsCSR-1 levels during the later stages of spermatogenesis could result in a reduction in licensing or protection from repression facilitating a decrease in AsCSR-1 target mRNAs. 

Q4. What are the main targets of Ascaris small RNAs?

Small RNAs that target mRNAs are generally antisense and fully complementary to mature mRNAs and associated with AsCSR-1, AsWAGO3, AsNRDE-3 (in the testis), and AsALG-4. 

Q5. What is the role of Ascaris Argonautes in spermatogenesis?

Several Ascaris Argonautes, including AsNRDE-3 and AsWAGO-3 targets are stage dependent altering their targets between mRNAs and repeats during spermatogenesis and development. 

Q6. What is the role of the RdRPs in the generation of secondary siRNAs?

Recent data suggest that in some C. elegans stages or compartments the CSR-1 slicer activity [22] may initially function to target and cleave mRNAs thereby serving to identify RNAs for RdRPs to initiate the generation of secondary siRNAs for CSR-1 [95]. 

Q7. What is the phylogenetic significance of AsALG-7?

Most of their phylogenetic analyses suggest that AsALG-5 and AsALG-7 appear related C. elegans ALG-3/4, while in a few cases, they cluster with either RDE-1 or ERGO-1. AsALG-7 expression is highest in early stages of spermatogenesis. 

Q8. What is the role of AsWAGO-3 in spermatogenesis?

Although AsWAGO-3 expression is in general low, in germline tissues AsWAGO-3 targets WAGO-repeats while in early embryos it targets mostly mRNAs (Figure 7B). 

Q9. What are the main components of C. elegans small RNAs?

Subsequent studies in C. elegans have revealed a diverse and complex set of small RNAs, pathways, and associated Argonautes [8-11].C. elegans small RNAs include miRNAs, 21U-RNAs (piRNAs) and small-interfering RNAs (siRNAs). 

Q10. What is the significance of the granules in C. briggsa?

CSR-1 antibodies did not identify P-granules in early embryos of C. briggsae [65] and P-granules have not been examined in other nematodes. 

Q11. What are the main functions of C. elegans secondary 22G-RNAs?

C. elegans secondary 22G-RNAs are amplified responses to targets identified by 21U- and 26G-RNAs and other primary siRNAs. 

Q12. What type of small RNAs are generated to mobile elements in Ascaris?

Although 22G-RNAs appear absent in Clades The author& II [90], other types of small RNAs are generated to mobile elements in these clades. 

Q13. What is the function of AsALG-4 and 26G-RNAs in spermati?

AsALG-4 and 26G-RNAs may be similar in function to pachytene piRNAs in mice that facilitate mRNA clearance in spermatids [96-99]. 

Q14. What are the common C. elegans small RNAs?

These C. elegans small RNAs are named based on their length and predominant first nucleotide of the RNA, e.g., 21U-, 22G- and 26G-RNAs.