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Maternal starvation primes progeny response to nutritional stress

['Kelly Voo', 'Singapore Polytechnic', 'Singapore', 'Jeralyn Wen Hui Ching', 'Ngee Ann Polytechnic', 'Joseph Wee Hao Lim', 'Seow Neng Chan', 'Temasek Life Sciences Laboratory', 'National University Of Singapore', 'Amanda Yunn Ee Ng']

Date: 2022-01 

Organisms adapt to environmental changes in order to survive. Mothers exposed to nutritional stresses can induce an adaptive response in their offspring. However, the molecular mechanisms behind such inheritable links are not clear. Here we report that in Drosophila, starvation of mothers primes the progeny against subsequent nutritional stress. We found that RpL10Ab represses TOR pathway activity by genetically interacting with TOR pathway components TSC2 and Rheb. In addition, starved mothers produce offspring with lower levels of RpL10Ab in the germline, which results in higher TOR pathway activity, conferring greater resistance to starvation-induced oocyte loss. The RpL10Ab locus encodes for the RpL10Ab mRNA and a stable intronic sequence RNA (sisR-8), which collectively repress RpL10Ab pre-mRNA splicing in a negative feedback mechanism. During starvation, an increase in maternally deposited RpL10Ab and sisR-8 transcripts leads to the reduction of RpL10Ab expression in the offspring. Our study suggests that the maternally deposited RpL10Ab and sisR-8 transcripts trigger a negative feedback loop that mediates intergenerational adaptation to nutritional stress as a starvation response.

In the wild, animals need to adapt to frequent changes in the environment. Mothers who are exposed to nutritional stresses are known to produce offspring which are preconditioned to adapt to the mothers’ environment. However, it is unclear how such maternal “memory” is being passed on to the offspring. Here we show that Drosophila mothers exposed to starvation produce offspring which are more resistant to starvation during oogenesis. This process is mediated by maternally inherited RpL10Ab mRNA and a stable intronic sequence RNA (sisR-8), which collectively repress the splicing of RpL10Ab pre-mRNA, leading to lower RpL10Ab expression in the offspring ovaries. As a consequence, lower RpL10Ab expression results in higher TOR pathway activity, conferring greater resistance to starvation during oogenesis. Hence, maternally inherited transcripts may play a role as mediators in conferring intergenerational adaption to starvation.

Here we report that in Drosophila, starvation of mothers primes the progeny to better handle subsequent nutritional stress. The RpL10Ab locus encodes for the RpL10Ab mRNA and a sisRNA (sisR-8), which collectively repress RpL10Ab pre-mRNA splicing in a negative feedback mechanism. During starvation, an increase in maternally deposited RpL10Ab and sisR-8 transcripts leads to reduction of RpL10Ab expression in the offspring. We found that RpL10Ab represses TOR pathway activity by genetically interacting with TOR pathway components TSC2 and Rheb. Lower levels of RpL10Ab in the progeny germline also lead to higher TOR pathway activity, which confers greater resistance to starvation-induced oocyte loss. Our study suggests that the maternally deposited RpL10Ab and sisR-8 trigger a negative feedback loop that mediates intergenerational adaptation to nutritional stress as a starvation response.

Although sisRNAs have been observed across diverse organisms, it remains unclear if they are generated from conserved genetic loci. Because some sisRNAs have been proposed to regulate their cognate genes in cis, finding sisRNAs from conserved genetic loci may suggest important auto-regulatory functions [ 14 ]. The RpL10A gene is under the control of an autoregulatory feedback loop, which is conserved in C. elegans and human [ 23 ]. The RpL10A protein binds to a conserved stretch of intron and inhibits splicing of the RpL10A pre-mRNA in a negative feedback mechanism. Interestingly, overexpression of RpL10Ab in Drosophila germline cells triggered apoptosis of stage 8/9 egg chambers, reminiscent of a starvation phenotype [ 24 ]. It is unknown whether the RpL10Ab locus in Drosophila generates any sisRNAs that are involved in the conserved autoregulatory feedback loop, or its potential involvement in the maternal transmission of starvation memory to the offspring.

Stable intronic sequence RNAs (sisRNAs) belong to a class of long noncoding RNAs that are stable and contain intronic sequences [ 9 – 11 ]. They can be produced in various ways such as canonical splicing and alternative splicing (via intron retention). They are also stabilized as linear and circular (or lariat) forms, and are able to regulate transcription, mRNAs and other noncoding RNAs [ 12 – 18 ]. In yeast, starvation or TOR inhibition leads to widespread increase in sisRNAs [ 19 , 20 ]. In Drosophila, starvation has been found to lead to an increase in the amount of maternally inherited sisRNA sisR-2, which regulates the homeostasis of primordial germ cells in the progeny [ 21 ]. Another maternally inherited sisRNA sisR-4 has also been shown to influence the development of the offspring during embryogenesis in Drosophila [ 16 ]. Starvation leads to a dramatic decrease in stage 14 oocyte production, and it is unknown whether maternal starvation has any impact on offspring adaptation to starvation [ 22 ]. Thus, it is unknown whether maternally deposited sisRNAs may provide a molecular link between maternal starvation and the progeny’s adaptive response.

In Caenorhabditis elegans (C. elegans), maternal environmental factors such as nutrient availability and osmotic conditions lead to changes in the maternal inheritance of small RNA, insulin signalling, vitellogenin and sugars, which protect the progeny against subsequent stressful conditions [ 3 – 7 ]. In human, starvation of the mothers had been proposed to lead to inappropriate adaptation, which subsequently resulted in a higher incidence of metabolic syndrome such as diabetes and obesity in the next generation [ 8 ]. However, the molecular mechanisms behind such an inheritable link are generally unknown.

Studies in various organisms have suggested an adaptive function for maternal effects [ 1 ]. The egg contains abundant materials such as proteins, metabolites, organelles, mRNA, and noncoding RNA inherited from the mother that can potentially influence the fitness of the offspring [ 2 ]. Equally important is the effect of epigenetic modification of the oocyte DNA that can serve as a memory of maternal experience [ 2 ].

Results

Identification of a sisRNA sisR-8 from Drosophila RpL10Ab locus To search for potentially conserved sisRNA from the Drosophila RpL10Ab locus, we compared the gene structures of human and Drosophila RpL10Ab (Fig 1A). Previously, Drosophila RpL10Ab intron 2 was reported to be orthologous to human RpL10Ab intron 3. A short stretch of 40 nucleotides within the orthologous intron (Fig 1A, yellow) is highly conserved from C. elegans to human. Furthermore, a portion of the orthologous intron (Fig 1A, yellow) can be included as an exon by alternative splicing using a downstream 5’ splice site. Taken together, these observations suggest that a portion of intron 2 may be retained as a sisRNA via alternative splicing. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 1. Identification of a sisRNA sisR-8 from Drosophila RpL10Ab locus. (a) Exon-intron structures of RpL10A/RpL10Ab in fly (D. melanogaster) and human (H. sapiens). Gene annotations were retrieved from publicly available databases such as UCSC genome browser and FlyBase. Orthologous introns between the two species were highlighted in grey. (b) A drawing of an ovariole showing germline cells at different stages of oogenesis. To detect sisRNAs, unfertilized eggs were used as they are transcriptionally quiescent, hence no contamination of pre-mRNAs and unstable mRNAs that undergo nonsense mediate decay. (c) Genome browser view showing the presence of sisRNAs (reads mapping to intron 2) from RpL10Ab in Drosophila unfertilized eggs. Samples were enriched for poly(A) plus and minus RNA. An annotation of sisR-8 derived from our experiments was shown in blue below. Arrows indicate the positions and directions of the primers used in D and E. (d, e) Agarose gels showing RT-PCR detecting the presence of sisR-8 in Drosophila unfertilized eggs. A PCR product of the expected size was amplified using P1 + P2 showing the presence of RpL10Ab mRNA as a positive control. PCR products were not amplified using P3 + P4 and P3 + P5 although the primers can amplify bands of the expected sizes using genomic DNA as positive controls. PCR products of the expected size was amplified using P3 + P6, P3 + P7 and P3 + P8, consistent with sisR-8 having a shorter 3’ end than the mRNA. In (E) RT+ bands were shorter than those from gDNA due to splicing, excluding gDNA contamination. g = genomic DNA as positive controls. https://doi.org/10.1371/journal.pgen.1009932.g001 To determine whether intron 2 of Drosophila RpL10Ab produces a sisRNA, we examined previously published RNA sequencing data from unfertilized eggs [12]. The unfertilized egg is an excellent system to unambiguously identify sisRNAs because it is transcriptionally quiescent and contains a pool of stable RNA (Fig 1B). Reads mapping to the alternatively spliced intron 2 were detected in poly(A)-plus but not poly(A)-minus samples, suggesting the presence of polyadenylated sisRNA (Fig 1C). To confirm this, we performed RT-PCR on RNA from unfertilized eggs. As a positive control, primers P1 and P2 detected the presence of RpL10Ab mRNA. However, using primers P3 (specific to intron 2) and P4 (complementary to the extreme 3’ end of RpL10Ab mRNA), we did not detect any PCR product, despite the fact that they can amplify a product from genomic DNA (Fig 1D). To verify the length of the sisRNA, we designed reverse primers that span the 3’ end of the transcript (P5-8). All of the reverse primers (except P5) were able to amplify a PCR product with P3, confirming that sisR-8 has a shorter 3’ end compared to RpL10Ab mRNA (Fig 1C, blue, and 1E).

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[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1009932

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