The Caenorhabditis elegans HEN1 Ortholog, HENN-1, Methylates and Stabilizes Select Subclasses of Germline Small RNAs

Small RNAs regulate diverse biological processes by directing effector proteins called Argonautes to silence complementary mRNAs. Maturation of some classes of small RNAs involves terminal 2′-O-methylation to prevent degradation. This modification is catalyzed by members of the conserved HEN1 RNA methyltransferase family. In animals, Piwi-interacting RNAs (piRNAs) and some endogenous and exogenous small interfering RNAs (siRNAs) are methylated, whereas microRNAs are not. However, the mechanisms that determine animal HEN1 substrate specificity have yet to be fully resolved. In Caenorhabditis elegans, a HEN1 ortholog has not been studied, but there is evidence for methylation of piRNAs and some endogenous siRNAs. Here, we report that the worm HEN1 ortholog, HENN-1 (HEN of Nematode), is required for methylation of C. elegans small RNAs. Our results indicate that piRNAs are universally methylated by HENN-1. In contrast, 26G RNAs, a class of primary endogenous siRNAs, are methylated in female germline and embryo, but not in male germline. Intriguingly, the methylation pattern of 26G RNAs correlates with the expression of distinct male and female germline Argonautes. Moreover, loss of the female germline Argonaute results in loss of 26G RNA methylation altogether. These findings support a model wherein methylation status of a metazoan small RNA is dictated by the Argonaute to which it binds. Loss of henn-1 results in phenotypes that reflect destabilization of substrate small RNAs: dysregulation of target mRNAs, impaired fertility, and enhanced somatic RNAi. Additionally, the henn-1 mutant shows a weakened response to RNAi knockdown of germline genes, suggesting that HENN-1 may also function in canonical RNAi. Together, our results indicate a broad role for HENN-1 in both endogenous and exogenous gene silencing pathways and provide further insight into the mechanisms of HEN1 substrate discrimination and the diversity within the Argonaute family

Context-dependent modulation of Pol II CTD phosphatase SSUP-72 regulates alternative polyadenylation in neuronal development

Alternative polyadenylation (APA) is widespread in neuronal development and activity-mediated neural plasticity. However, the underlying molecular mechanisms are largely unknown. We used systematic genetic studies and genome-wide surveys of the transcriptional landscape to identify a context-dependent regulatory pathway controlling APA in the Caenorhabditis elegans nervous system. Loss of function in ssup-72, a Ser5 phosphatase for the RNA polymerase II (Pol II) C-terminal domain (CTD), dampens transcription termination at a strong intronic polyadenylation site (PAS) in unc-44/ankyrin yet promotes termination at the weak intronic PAS of the MAP kinase dlk-1. A nuclear protein, SYDN-1, which regulates neuronal development, antagonizes the function of SSUP-72 and several nuclear polyadenylation factors. This regulatory pathway allows the production of a neuron-specific isoform of unc-44 and an inhibitory isoform of dlk-1. Dysregulation of the unc-44 and dlk-1 mRNA isoforms in sydn-1 mutants impairs neuronal development. Deleting the intronic PAS of unc-44 results in increased pre-mRNA processing of neuronal ankyrin and suppresses sydn-1 mutants. These results reveal a mechanism by which regulation of CTD phosphorylation controls coding region APA in the nervous system

A Conserved Upstream Motif Orchestrates Autonomous, Germline-Enriched Expression of <em>Caenorhabditis elegans</em> piRNAs

<div><p>Piwi-interacting RNAs (piRNAs) fulfill a critical, conserved role in defending the genome against foreign genetic elements. In many organisms, piRNAs appear to be derived from processing of a long, polycistronic RNA precursor. Here, we establish that each <i>Caenorhabditis elegans</i> piRNA represents a tiny, autonomous transcriptional unit. Remarkably, the minimal <i>C. elegans</i> piRNA cassette requires only a 21 nucleotide (nt) piRNA sequence and an ∼50 nt upstream motif with limited genomic context for expression. Combining computational analyses with a novel, in vivo transgenic system, we demonstrate that this upstream motif is necessary for independent expression of a germline-enriched, Piwi-dependent piRNA. We further show that a single nucleotide position within this motif directs differential germline enrichment. Accordingly, over 70% of <i>C. elegans</i> piRNAs are selectively expressed in male or female germline, and comparison of the genes they target suggests that these two populations have evolved independently. Together, our results indicate that <i>C. elegans</i> piRNA upstream motifs act as independent promoters to specify which sequences are expressed as piRNAs, how abundantly they are expressed, and in what germline. As the genome encodes well over 15,000 unique piRNA sequences, our study reveals that the number of transcriptional units encoding piRNAs rivals the number of mRNA coding genes in the <i>C. elegans</i> genome.</p> </div

Descriptions of transgenic alleles and features of the transgenes.

<p>Both high-copy and MosSCI transgenes used in this study are listed with a short description, sequence characteristics, integration information, and strain notation. Full transgene data are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003392#s4" target="_blank">Materials and Methods</a>. Bolded letters indicate mutated nucleotides. Eight nt core upstream motifs are capitalized while motif positions are underlined. N/D, not determined.</p><p>IV. The ♀Min2502 transgene also expresses from a single-copy insertion on chromosome II.</p

Variation in the core upstream motif correlates with 21U RNA germline enrichment.

<p>(A) Spacer lengths follow expected distribution for all enrichment classifications. Dotted lines: canonical spacer length range (35–42 nt). (B) Male, but not female, 21U RNA loci show enrichment for core motifs with 5′ cytidines. Significantly fewer female 21U RNAs exhibit a GTTTC-containing core motif than male. Top: Weblogo plots illustrate core motif differences. Bottom: Pie charts depict proportions of 21U RNAs with GTTTC-containing core motifs indicating the 5′ nt (colors) or with no GTTTC-containing core motif (NM, no motif, dark grey). (C) Core motif variations correlate with male 21U RNA abundance in 5′-monophosphate-dependent libraries. Average 21U RNA abundance was calculated based on the 5′ nt of the core motif. Error bars: ±1 standard error of the mean (SEM). (D) Core motif variations do not correlate with female 21U RNA abundance in 5′-monophosphate-dependent libraries. Average 21U RNA abundance was calculated as in (C).</p

Over 70% of 21U RNAs show distinct germline enrichment.

<p>(A) Pipeline for computational identification of male and female 21U RNAs. A majority of 21U RNAs are classified as male or female germline-enriched. Pie chart depicts classification as proportion of 13,711 21U RNAs analyzed. (B,C) Male 21U RNAs are more highly expressed in male animals, and female 21U RNAs are more highly expressed in female animals. Relative expression of representative 21U RNAs was assayed by Taqman RT-qPCR in <i>him-8(e1489)</i> (B) and <i>fog-2(q71)</i> (C) male versus <i>fem-1(hc17)</i> female animals and normalized to non-enriched 21U RNA 21UR-1. Error bars: ±1 standard deviation (SD) of two biological replicates. AU: arbitrary units.</p

HEN1 Stabilizes ERGO-1 Class, but Not ALG-3/ALG-4 Class, 26G RNAs.

<p>A) Loss of <i>henn-1</i> impairs ERGO-1 class 26G RNA accumulation at all stages. Levels of ERGO-1 class 26G RNA 26G-O3 were assayed by Taqman qPCR across development of wild-type and <i>henn-1(tm4477)</i> mutant animals at 25°C. Standard deviation is shown for biological triplicates. Taqman qPCR data for seven additional ERGO-1 class 26G RNAs are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002617#pgen.1002617.s008" target="_blank">Figure S8</a>. B) ALG-3/ALG-4 class 26G RNAs are <i>henn-1</i>-independent. Levels of ALG-3/ALG-4 class 26G RNA 26G-S5 were assayed across the period of development in which ALG-3/ALG-4 class 26G RNAs are readily detectable. Standard deviation is shown for biological triplicates. Taqman qPCR data for two additional ALG-3/ALG-4 class 26G RNAs are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002617#pgen.1002617.s009" target="_blank">Figure S9</a>. C) Loss of <i>henn-1</i> may result in modest, sporadic defects in ERGO-1 class 26G RNA target silencing. Levels of eight target and two non-target mRNAs were assayed across development of wild-type and <i>henn-1(tm4477)</i> mutant animals at 25°C and normalized to <i>eft-2</i>. Expression in the <i>henn-1(tm4477)</i> mutant relative to wild-type is represented according to the red-green color scheme indicated in the right panel. Raw data is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002617#pgen.1002617.s010" target="_blank">Figure S10</a>. E, embryo.</p

The <i>henn-1</i> Mutant Exhibits Opposite RNAi Sensitivity Phenotypes in Soma and Germline.

<p>A) <i>henn-1(tm4477)</i> mutant animals exhibit mildly enhanced somatic RNAi. Animals of the indicated genotype were plated as L1 larvae on <i>lir-1</i> feeding RNAi diluted 1∶1 with empty vector (1/2 strength) and grown for 70 hours at 20°C. Data is quantified in part B. RNAi sensitivity data for knockdown of two additional somatic transcripts are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002617#pgen.1002617.s011" target="_blank">Figure S11</a>. B) Endogenous expression of <i>henn-1::gfp</i> from <i>xkSi1</i> rescues somatic RNAi sensitivity. Percent of animals reaching full size on <i>lir-1</i> feeding RNAi of the indicated strength at 70 hours is plotted. N = 8 plates of >50 animals per strain. Standard deviation is shown. C) <i>henn-1(tm4477)</i> mutant animals exhibit defective germline RNAi. Brood size of animals plated at 20°C as L1 larvae on <i>pos-1</i> feeding RNAi diluted 1∶2 with empty vector is plotted. N≥13 animals per strain. Mean and standard deviation are shown. RNAi sensitivity data for knockdown of four additional germline transcripts are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002617#pgen.1002617.s012" target="_blank">Figure S12</a>. Alleles used in this figure: <i>eri-1(mg366)</i>, <i>prg-1(tm872)</i>, <i>rde-4(ne301)</i>.</p

21U RNA sequences are specified by the genomic positions of upstream core motifs.

<p>(A) Schematic of transgenes with 5′ nt of 21U RNA mutated. (B–C) Mutation of the 5′ genomic thymidine disrupts expression of 21UR-synth by northern blot (B) and Taqman assay (C). (D) 21U RNA abundances correlate with distances downstream of core motifs. Miniclustered 21U RNAs with 37–40 nt spacer lengths are more abundant than solitary 21U RNAs. Asterisks indicate Welch's <i>t</i>-tests, p<0.05. Error bars: ±1 SEM. (E) Optimal downstream windows are more thymidine-rich for shared core motifs than non-shared (Welch's <i>t</i>-test, p = 2.5e-46). The number of genomic thymidines located 35–42 nt downstream of each GTTTC-containing motif was counted. (F) 21U RNA miniclusters are significantly biased for being composed of 21U RNAs with the same, as opposed to opposite, germline enrichment than expected if the same 21U RNAs were randomly paired.</p

Methylation of 21U RNAs Requires <i>C. elegans</i> HEN1 Ortholog HENN-1.

<p>A) HENN-1 is required for 21U RNA methylation. Endogenous (<i>xkSi1</i>) and germline-specific (<i>xkSi2</i>) expression of <i>henn-1::gfp</i> rescue 21U RNA methylation in <i>henn-1(tm4477)</i> mutant embryo. Total embryo RNA of the indicated genotypes was β-eliminated (βe +) or control treated (βe −) and probed for piRNA 21UR-4292. <i>prg-1(tm872)</i> lacks 21U RNAs and is included as a negative control. Below, ethidium bromide staining of 5.8S rRNA is shown. Additional 21U RNA northern blots are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002617#pgen.1002617.s003" target="_blank">Figure S3A</a>. B) <i>C. elegans</i> miRNAs are unmethylated. Total embryo RNA was probed for miR-1. Variable intensity of 5.8S rRNA bands in embryo indicates unequal loading.</p