A secreted endoribonuclease ENDU-2 from the soma protects germline immortality in C. elegans

Multicellular organisms coordinate tissue specific response to environmental information via both cell-autonomous and non-autonomous mechanisms. In addition to secreted ligands, secreted small RNAs have recently been reported to regulate gene expression across tissue boundaries. Here we show that the conserved poly-U specific endoribonuclease ENDU-2 is secreted from the soma and taken-up by the germline to ensure germline immortality at elevated temperature in C. elegans. ENDU-2 binds to mature mRNAs and negatively regulates mRNA abundance both in the soma and the germline. While ENDU-2 promotes RNA decay in the soma directly via its endoribonuclease activity, ENDU-2 prevents misexpression of soma-specific genes in the germline and preserves germline immortality independent of its RNA-cleavage activity. In summary, our results suggest that the secreted RNase ENDU-2 transmits environmental information across tissue boundaries and contributes to maintenance of stem cell immortality probably via retaining a stem cell specific program of gene expression.

). We speculate that the 128 N-terminal 3xFlag fusion may prevent secretion by impairing binding of the secretion signal 129 peptide by signal recognition particle (SRP), thus allowing detection of the weak expression in the either neurons or muscles of heat-stressed animals resulted in ENDU-2::EGFP localization in the 138 pharynx (Supplemental Fig. S4B), suggesting that either secretion or uptake of ENDU-2 in these 139 tissues could be modulated by temperature.

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Secretion of ENDU-2 from the soma to the gonad protects germline immortality 141 It is generally accepted that multi-copy extrachromosomal arrays are silenced and not expressed 142 in the C. elegans germline (Kelly et al., 1997). However, we found that extrachromosomal endu-143 2::EGFP transgenes rescued the Mrt germline phenotype of endu-2(lf) animals (Fig. 3A), 144 suggesting that somatic ENDU-2 might preserve germline immortality across tissue boundaries. 145 Our hypothesis was that secreted ENDU-2 could be endocytosed by the gonad. To test this 146 assumption, we first tested whether ENDU-2 protein could be detected in the germline. All of our 147 endu-2::EGFP transgenic reporters displayed weak expression level unless secretion was blocked. 148 Therefore, we could only occasionally observe faint ENDU-2::EGFP in few oocytes 149 (Supplemental Fig. S4C). Via GFP antibody staining we detected ENDU-2::EGFP in punctate  Table S3). Taken together, these results implicate that 160 somatically expressed ENDU-2 protein can enter the gonad via its secretion and uptake. 161 Next, we asked from which tissue ENDU-2 ensures germline immortality. Expressing ENDU-2 162 specifically in the intestine or the neurons, but not the muscle or somatic gonad, were sufficient to 163 rescue the Mrt phenotype, indicating a non-cell-autonomous ENDU-2 signal from the neurons and 164 intestine to the germline (Fig. 3B). In addition, expressing the secretion deficient ∆ssENDU-165 2::EGFP failed to rescue the Mrt phenotype (Fig. 3C). Furthermore, SSsel-1∷ ∆ssENDU-2::EGFP 166 rescued the Mrt phenotype (Fig. 3C), suggesting that guiding ENDU-2 into ER-Golgi secretory 167 pathway rather than the first 19 amino acids of ENDU-2 is essential for its function in the germline.

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To directly test whether loss of somatic endu-2 expression is sufficient to induce a mortal germline, 169 we performed endu-2 RNAi knock-down in a ppw-1 mutant background in which germline RNAi  To know whether ENDU-2 affects oocytes or sperm to ensure germline immortality, we tested the 180 parental contribution of ENDU-2 during sexual reproduction. For this purpose, we crossed endu-2(+/+) wild type parents with either males or hermaphrodites of endu-2(-/-) that had been grown 182 at 25°C for five to seven generations and displayed strongly reduced brood size and high 183 percentage of sterile phenotype (Fig. 3D). Whereas the heterozygous progeny of endu-2(+/+) 184 mothers had brood sizes with typically more than 100 F2 animals, the brood size of F1 cross-185 progeny derived from endu-2(-/-) mothers was similarly low as that of their mothers. We conclude 186 that ENDU-2 function is required predominantly in the oocytes to preserve germline immortality.

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The temperature dependent Mrt phenotype in endu-2 mutants resembles that of hrde-1 mutants in 188 the nuclear RNAi pathway (Buckley et al., 2012;Ni et al., 2016). In addition to loss of germline 189 immortality, hrde-1 is also defective in multigenerational inheritance of germline RNAi. To know 190 whether ENDU-2 acts in the nuclear RNAi pathway, we examined oma-1 RNAi inheritance, which 191 leads to suppression of the embryonic lethality of the oma-1 gain-of-function mutant for several 192 generations (Alcazar et al., 2008). Unlike the hrde-1 mutants that lost inheritance of oma-1 RNAi 193 within two generations, effect of oma-1 RNAi knock-down persisted for 5-6 generations both in 194 endu-2(lf) and wild type animals (Supplemental Fig. S6B), suggesting that ENDU-2 does not act 195 in the nuclear RNAi pathway to control transgenerational inheritance of germline RNAi. 196 Therefore, maintenance of germline immortality by ENDU-2 probably functions via a distinct 197 mechanism from that of the nuclear RNAi pathway. recently that ENDU-2 might also be an RNA-binding protein (Ujisawa et al., 2018). To identify 203 the candidate RNAs associated with ENDU-2, we precipitated ENDU-2::EGFP and analyzed co-204 immunoprecipitated RNAs by deep-sequencing (RIP-Seq). We also reasoned that, since wild type 205 ENDU-2 would potentially cleave its RNA targets, this might prevent enrichment of intact RNA 206 targets for identification. Although we had no direct proof of an RNA cleaving activity of ENDU-207 2 yet, we thought that generation of an ENDU-2 variant that maintains RNA-binding, but loses 208 cleavage activity, should facilitate RNA target detection. Xenopus XendoU mutants with E to Q RIP-Seq with these two strains in additional to wild type ENDU-2::EGFP. By plotting normalized 215 reads (RPM) of each transcript we were able to investigate RNA binding affinity of ENDU-2 under 216 different conditions. In general, all three ENDU-2 variants tested showed stronger RNA-binding 217 affinity at 15°C than at 25°C (Fig. 4A). E460Q showed weaker RNA binding already at 15°C than 218 wild type ENDU-2 and almost completely lost RNA-binding capacity at 25°C. In addition, ENDU-219 2(E454Q) displayed stronger RNA binding activity than ENDU-2(wt) at 15°C. Therefore, we used 220 RIP-Seq data of ENDU-2(E454Q) at 15°C for detecting RNAs bound by ENDU-2. A total of 5,920 221 transcripts were co-immunoprecipitated with ENDU-2(E454Q) (Supplemental Table S1). Most of 222 them (>99%) were protein coding transcripts except few non-coding RNAs (5 snoRNAs, 27 223 pseudogenes, 5 ncRNAs and 1 lincRNA). In addition, reads distribution analysis showed > 99% 224 of sequenced reads were mapped to exonic regions, suggesting that ENDU-2(E454Q) primarily 225 bound to processed mRNAs (Fig. 4B). We performed in vitro mRNA binding assays with recombinant ENDU-2 proteins and two selected mRNA targets from the RIP-Seq data and could 227 confirm that both ENDU-2 and ENDU-2(E454Q) directly interacts with these mRNAs (Fig. 4C). but not of ENDU-2(E460Q), fully rescued the Mrt phenotype of endu-2(lf) at 25°C (Fig. 4E). We 237 conclude that mRNA-binding, rather than -cleavage activity of ENDU-2, is essential in the 238 germline to maintain stem cell immortality at elevated temperature.  Table S2). qPCR quantifications of two selected genes cav-1 and trsc-247 1 confirmed our microarray result and revealed that their expression levels strongly decreased in 248 endu-2 mutants compared with wild type animals at 25°C (Supplemental Fig. S10C and S10D).
However, neither a germline expressed cav-1::GFP reporter nor smFISH staining of trcs-1 250 confirmed significantly altered germline expression in situ (Supplemental Fig. S10A, S10B and 251 S10D). As the gonads of endu-2(lf) mutants at 25°C became significantly smaller (Supplemental 252 Fig. S9B), we speculate that apparent down-regulation of germline mRNA within the 253 transcriptome data set may be caused by reduced sample size of the gonad in endu-2 mutants.

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Therefore, we put our focus on genes that were up-regulated in endu-2(lf) back ground. 32% (76 255 out of 237) of these transcripts were candidates for direct ENDU-2 targets, since they had been co-256 immunoprecipitated with ENDU-2 (Supplemental Tab. S1). In addition, the vast majority of them 257 (62 out of 76) did not have increased expression in the gonad of endu-2(lf) animals (see the next 258 chapter, Supplemental Table S1). This suggests that, at minimum, these 62 transcripts are down-259 regulated by ENDU-2 in the somatic tissues. GO term analyses implicated these somatic ENDU-260 2 targets in regulation of various metabolic processes ( Fig. 5A and Fig. S9C). Consistently, we 261 found loss of endu-2 resulted in increased lipid content at 25°C but not at 15°C (Fig. 5B). Taken 262 together, these results indicate a putative regulatory role of ENDU-2 in limiting abundance of the 263 mRNAs that are primarily involved in metabolic functions in the soma.

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As some aspects of the endu-2(lf) phenotype were temperature dependent, we asked whether 265 ENDU-2 regulates mRNA abundance in response to temperature alterations. To test this, we 266 performed qPCR to quantitate ten selected mRNAs which were bound and down-regulated by 267 ENDU-2. We found that mRNA levels of these targets were up-regulated in endu-2 mutant 268 background, compared to wild type, already at standard growth temperature (20°C) and were even 269 more abundant at 25°C (Fig. 5C). Increased temperature, on the other hand, did not strongly affect 270 the abundance of most of these mRNAs in wild type background. These results suggest a 271 temperature dependent negative influence of ENDU-2 on the levels of these mRNA targets. We also performed smFISH to inspect the influence of ENDU-2 on the mRNA level of yet another 273 target fat-7. fat-7 mRNA was only detected in the intestine and endu-2(-) animals had higher fat-274 7 transcript levels than wild type at both 20°C and 25°C (Fig. 5D). Moreover, we used a fat-7::GFP 275 translational fusion reporter to monitor FAT-7::GFP protein level. Wild type animals showed 276 reduced FAT-7::GFP expression at 25°C vs. 15°C (Fig. 5E). endu-2(lf) displayed stronger FAT-277 7::GFP expression at both temperatures. Furthermore, transgenic expression of endu-2(wt) but not 278 the endu-2(E454Q) transgene (that has lost RNA cleavage activity) restored decreased FAT-279 7::GFP expression (Fig. 5F). We conclude that ENDU-2 mediated mRNA-cleavage is required for 280 decreasing expression of at least some of its somatic target genes, such as fat-7.

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To investigate germline transcriptomes regulated by ENDU-2, we isolated gonads from wild type 283 and endu-2(lf) animals which had been grown at 25°C from L1 stage, and sequenced two biological Chi-Square test) were co-immunoprecipitated with ENDU-2. We considered them as direct targets 293 of ENDU-2 and grouped them in two classes, depending on whether they are up-(class I) or down-294 regulated (class II) in endu-2(lf) background (Fig. 5B). Expression of a class I target is repressed while class II genes are activated by wild type ENDU-2 activity. Consistently, we noticed that 296 most of the class I, but not the class II target genes were not, or very lowly, expressed in the gonad S3). We compared, for this analysis, both RNA and protein levels of ENDU-2 as well as different 340 ENDU-2 variants with truncations in the N-terminal secretion signal peptide (ΔssENDU-2 and SSsel-1:: ΔssENDU-2). We could demonstrate that the tissues containing ENDU-2 protein differ 342 from those that harbor endu-2 mRNA and presence of a secretion signal peptide is necessary and 343 sufficient to target ENDU-2 to the secretory pathway to reach distant cells.

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Our data strongly suggests that ENDU-2 production and function may occur in different tissues, 345 and that secretion of ENDU-2 allows the control of mRNA abundance in the distance. A simple 346 assumption would be that ENDU-2 has similar functions in any of its target tissues, no matter from 347 which cells it is expressed. This seems to be the case for some activities mediated by ENDU-2.

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For example, the egg-laying defect observed as a consequence of abnormal vulva development in 349 an endu-2(lf) mutant is rescued by transgenic endu-2 expression in the muscle, intestine or neuron 350 (data not shown). In contrast, only neuronally or intestinally expressed ENDU-2 was able to rescue 351 germline immortality (Fig. 3C), indicating existence of functional difference of ENDU-2 from 352 distinct origins. Tissue-specific interactors or modifiers of ENDU-2 are probably crucial to specify 353 its individual activities. It is currently not known whether there are isoform-specific functions of 354 ENDU-2, or whether ENDU-2 protein is modulated by protein modifications. 355 We distinguish two activities of ENDU-2: mRNA-binding and mRNA-cleavage. RIP-Seq and 356 transcriptomic results demonstrate that the levels of only about 10% of the mRNAs to which 357 ENDU-2 binds change in endu-2(lf) mutants at elevated temperature ( Fig. 4-6), suggesting that 358 ENDU-2 is able to discriminate its regulated targets from the bound transcripts. Such scenarios 359 have also been proposed for SMG-2/UPF1, the core factor of the non-sense mediated decay  while it utilizes primarily RNA-binding but not cleavage activity to prevent misexpression of 363 soma-specific genes in the germline, suggesting rather an indirect mechanism of ENDU-2 in the germline to restrict its target mRNA abundance. Why such different layers of regulatory 365 mechanisms exist is currently unknown. We speculate that one reason might be association of 366 ENDU-2 with distinct protein complexes in the soma and germline. Future study focusing on 367 dissecting tissue specific mechanisms will help to elucidate how ENDU-2 coordinates gene 368 expression in the soma and germline upon environmental stimuli.        Table S4). Wild type and endu-2(tm4977) L4 animals were selected and dissected 24 hours later 598 for smFISH staining using a published protocol procedure (Lee et al., 2016). Images were acquired 599 with an Image Z1 fluorescence microscope. Exposure times and acquisition settings were identical 600 between replicates. mRNA puncta were quantified by using ImageJ 1.51s cell counter plugin.

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Regions of interest for acquisition were defined by nuclei DAPI staining.