Q&A with Jun Wei Pek and Amanda Yunn Ee Ng.

Investigator Jun Wei Pek (J.P.) and graduate student Amanda Yunn Ee Ng (A.Y.) spoke to Cell Reports about their scientific journeys and love of science, their work on gene expression regulation during reproductive development, and challenges encountered during the pandemic.

Genetic compensation between ribosomal protein paralogs mediated by a cognate circular RNA.

Inter-regulation between related genes, such as ribosomal protein (RP) paralogs, has been observed to be important for genetic compensation and paralog-specific functions. However, how paralogs communicate to modulate their expression levels is unknown. Here, we report a circular RNA involved in the inter-regulation between RP paralogs RpL22 and RpL22-like during Drosophila spermatogenesis. Both paralogs are mutually regulated by the circular stable intronic sequence RNA (sisRNA) circRpL22(NE,3S) produced from the RpL22 locus. RpL22 represses itself and RpL22-like. Interestingly, circRpL22 binds to RpL22 to repress RpL22-like, but not RpL22, suggesting that circRpL22 modulates RpL22's function. circRpL22 is in turn controlled by RpL22-like, which regulates RpL22 binding to circRpL22 to indirectly modulate RpL22. This circRpL22-centric inter-regulatory circuit enables the loss of RpL22-like to be genetically compensated by RpL22 upregulation to ensure robust male germline development. Thus, our study identifies sisRNA as a possible mechanism of genetic crosstalk between paralogous genes.

Nutrient-dependent regulation of a stable intron modulates germline mitochondrial quality control.

Mitochondria are inherited exclusively from the mothers and are required for the proper development of embryos. Hence, germline mitochondrial quality is highly regulated during oogenesis to ensure oocyte viability. How nutrient availability influences germline mitochondrial quality control is unclear. Here we find that fasting leads to the accumulation of mitochondrial clumps and oogenesis arrest in Drosophila. Fasting induces the downregulation of the DIP1-Clueless pathway, leading to an increase in the expression of a stable intronic sequence RNA called sisR-1. Mechanistically, sisR-1 localizes to the mitochondrial clumps to inhibit the poly-ubiquitination of the outer mitochondrial protein Porin/VDAC1, thereby suppressing p62-mediated mitophagy. Alleviation of the fasting-induced high sisR-1 levels by either sisR-1 RNAi or refeeding leads to mitophagy, the resumption of oogenesis and an improvement in oocyte quality. Thus, our study provides a possible mechanism by which fasting can improve oocyte quality by modulating the mitochondrial quality control pathway. Of note, we uncover that the sisR-1 response also regulates mitochondrial clumping and oogenesis during protein deprivation, heat shock and aging, suggesting a broader role for this mechanism in germline mitochondrial quality control.

Distinct biogenesis pathways may have led to functional divergence of the human and Drosophila Arglu1 sisRNA.

Stable intronic sequence RNAs (sisRNAs) are stable, long noncoding RNAs containing intronic sequences. While sisRNAs have been found across diverse species, their level of conservation remains poorly understood. Here we report that the biogenesis and functions of a sisRNA transcribed from the highly conserved Arglu1 locus are distinct in human and Drosophila melanogaster. The Arglu1 genes in both species show similar exon-intron structures where the intron 2 is orthologous and positionally conserved. In humans, Arglu1 sisRNA retains the entire intron 2 and promotes host gene splicing. Mechanistically, Arglu1 sisRNA represses the splicing-inhibitory activity of ARGLU1 protein by binding to ARGLU1 protein and promoting its localization to nuclear speckles, away from the Arglu1 gene locus. In contrast, Drosophila dArglu1 sisRNA forms via premature cleavage of intron 2 and represses host gene splicing. This repression occurs through a local accumulation of dARGLU1 protein and inhibition of telescripting by U1 snRNPs at the dArglu1 locus. We propose that distinct biogenesis of positionally conserved Arglu1 sisRNAs in both species may have led to functional divergence.

Passing down maternal dietary memories through lncRNAs.

Parental diet is known to influence the offspring in an intergenerational manner, and this has been implicated in species adaptation and general health. Recent studies highlight the role of maternal long noncoding RNAs (lncRNAs) in serving as one of the 'memories' of maternal diet in regulating offspring development and predisposition to metabolic disease.

Maternal starvation primes progeny response to nutritional stress.

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.

Circular sisRNA identification and characterisation.

Stable Intronic Sequence RNA (sisRNA) is a relatively new class of non-coding RNA. Found in many organisms, these sisRNA produced from their host genes are generally involved in regulatory roles, controlling gene expression at multiple levels through active involvement in regulatory feedback loops. Large scale identification of sisRNA via genome-wide RNA sequencing has been difficult, largely in part due to its low abundance. Done on its own, RNA sequencing often yields a large mass of information that is ironically uninformative; the potential sisRNA reads being masked by other highly abundant RNA species like ribosomal RNA and messenger RNA. In this review, we present a practical workflow for the enrichment of circular sisRNA through the use of transcriptionally quiescent systems, rRNA-depletion, and RNase R treatment prior to deep sequencing. This workflow allows circular sisRNA to be reliably detected. We also present various methods to experimentally validate the circularity and stability of the circular sisRNA identified, as well as a few methods for further functional characterisation.

Maternally inherited intron coordinates primordial germ cell homeostasis during Drosophila embryogenesis.

Primordial germ cells (PGCs) give rise to the germline stem cells (GSCs) in the adult Drosophila gonads. Both PGCs and GSCs need to be tightly regulated to safeguard the survival of the entire species. During larval development, a non-cell autonomous homeostatic mechanism is in place to maintain PGC number in the gonads. Whether such germline homeostasis occurs during early embryogenesis before PGCs reach the gonads remains unclear. We have previously shown that the maternally deposited sisRNA sisR-2 can influence GSC number in the female progeny. Here we uncover the presence of a homeostatic mechanism regulating PGCs during embryogenesis. sisR-2 represses PGC number by promoting PGC death. Surprisingly, increasing maternal sisR-2 leads to an increase in PGC death, but no drop in PGC number was observed. This is due to ectopic division of PGCs via the de-repression of Cyclin B, which is governed by a genetic pathway involving sisR-2, bantam and brat. We propose a cell autonomous model whereby germline homeostasis is achieved by preserving PGC number during embryogenesis.

SON protects nascent transcripts from unproductive degradation by counteracting DIP1.

Gene expression involves the transcription and splicing of nascent transcripts through the removal of introns. In Drosophila, a double-stranded RNA binding protein Disco-interacting protein 1 (DIP1) targets INE-1 stable intronic sequence RNAs (sisRNAs) for degradation after splicing. How nascent transcripts that also contain INE-1 sequences escape degradation remains unknown. Here we observe that these nascent transcripts can also be bound by DIP1 but the Drosophila homolog of SON (Dsn) protects them from unproductive degradation in ovaries. Dsn localizes to the satellite body where active decay of INE-1 sisRNAs by DIP1 occurs. Dsn is a repressor of DIP1 posttranslational modifications (primarily sumoylation) that are assumed to be required for efficient DIP1 activity. Moreover, the pre-mRNA destabilization caused by Dsn depletion is rescued in DIP1 or Sumo heterozygous mutants, suggesting that Dsn is a negative regulator of DIP1. Our results reveal that under normal circumstances nascent transcripts are susceptible to DIP1-mediated degradation, however intronic sequences are protected by Dsn until intron excision has taken place.

Stable Intronic Sequence RNAs (sisRNAs): An Expanding Universe.

Intronic sequences are often regarded as 'nonsense' transcripts that are rapidly degraded. We highlight here recent studies on intronic sequences that play regulatory roles as long noncoding RNAs (lncRNAs) which are classified as sisRNAs. Interestingly, sisRNAs come in different forms and are produced via a variety of ways. They regulate genes at the DNA, RNA, and protein levels, and frequently engage in autoregulatory feedback loops to ensure cellular homeostasis under normal and stress conditions. Future directions, evolutionary insights, and potential implications of dysregulated sisRNAs are also discussed, especially in relation to human pathogenesis.

Generation of Drosophila sisRNAs by Independent Transcription from Cognate Introns.

Although stable intronic sequence RNAs (sisRNAs) are conserved in plants and animals, their functional significance is still unclear. We identify a pool of polyadenylated maternally deposited sisRNAs in Drosophila melanogaster. These sisRNAs can be generated by independent transcription from the cognate introns. The ovary-specific poly(A) polymerase Wispy mediates the polyadenylation of maternal sisRNAs and confers their stability as maternal transcripts. A developmentally regulated sisRNA sisR-3 represses the expression of a long noncoding RNA CR44148 and is required during development. Our results expand the pool of sisRNAs and suggest that sisRNAs perform regulatory functions during development in Drosophila.

A sisRNA/miRNA Axis Prevents Loss of Germline Stem Cells during Starvation in Drosophila.

Animal reproduction responds to nutritional status. During starvation, Drosophila and Caenorhabditis elegans enter a period of reproductive diapause with increase apoptosis, while maintaining a stable pool of germline stem cells (GSCs). How GSCs are protected is not understood. Here, we show that a sisRNA/miRNA axis maintains ovarian GSCs during starvation in Drosophila. Starvation induces the expression of an ovary-enriched sisRNA sisR-2, which negatively regulates GSC maintenance via a fatty acid metabolism gene dFAR1. sisR-2 promotes the expression of bantam, which in turn inhibits the activity of sisR-2, forming a negative feedback loop. Therefore, bantam acts as a buffer to counteract sisR-2 activity to prevent GSC loss during starvation. We propose that the sisR-2/bantam axis confers robustness to GSCs in Drosophila.

Germline Stem Cell Heterogeneity Supports Homeostasis in Drosophila.

Adult and embryonic stem cells exhibit fluctuating gene expression; however, the biological significance of stem cell heterogeneity is not well understood. We show that, in Drosophila, female germline stem cells (GSCs) exhibit heterogeneous expression of a GSC differentiation-promoting factor Regena (Rga). The Drosophila homolog of human SON, dsn, is required to maintain GSC heterogeneity by suppressing sustained high levels of Rga. Reducing the expression of Rga in dsn mutants restores GSC heterogeneity and self-renewal. Thus, GSC heterogeneity is linked to GSC homeostasis.

Stable Intronic Sequence RNAs Engage in Feedback Loops.

Stable intronic sequence RNAs (sisRNAs) are conserved in various organisms. Recent observations in Drosophila suggest that sisRNAs often engage in regulatory feedback loops to control the expression of their parental genes. The use of sisRNAs as mediators for local feedback control may be a general phenomenon.

DIP1 modulates stem cell homeostasis in Drosophila through regulation of sisR-1.

Stable intronic sequence RNAs (sisRNAs) are by-products of splicing and regulate gene expression. How sisRNAs are regulated is unclear. Here we report that a double-stranded RNA binding protein, Disco-interacting protein 1 (DIP1) regulates sisRNAs in Drosophila. DIP1 negatively regulates the abundance of sisR-1 and INE-1 sisRNAs. Fine-tuning of sisR-1 by DIP1 is important to maintain female germline stem cell homeostasis by modulating germline stem cell differentiation and niche adhesion. Drosophila DIP1 localizes to a nuclear body (satellite body) and associates with the fourth chromosome, which contains a very high density of INE-1 transposable element sequences that are processed into sisRNAs. DIP1 presumably acts outside the satellite bodies to regulate sisR-1, which is not on the fourth chromosome. Thus, our study identifies DIP1 as a sisRNA regulatory protein that controls germline stem cell self-renewal in Drosophila.Stable intronic sequence RNAs (sisRNAs) are by-products of splicing from introns with roles in embryonic development in Drosophila. Here, the authors show that the RNA binding protein DIP1 regulates sisRNAs in Drosophila, which is necessary for germline stem cell homeostasis.

Maternally Inherited Stable Intronic Sequence RNA Triggers a Self-Reinforcing Feedback Loop during Development.

Maternally inherited noncoding RNAs (ncRNAs) can regulate zygotic gene expression across generations [1-4]. Recently, many stable intronic sequence RNAs (sisRNAs), which are byproducts of pre-mRNA splicing, were found to be maternally deposited and persist till zygotic transcription in Xenopus and Drosophila [5-7]. In various organisms, sisRNAs can be in linear or circular conformations, and they have been suggested to regulate host gene expression [5-10]. It is unknown whether maternally deposited sisRNAs can regulate zygotic gene expression in the embryos. Here, we show that a maternally inherited sisRNA (sisR-4) from the deadpan locus is important for embryonic development in Drosophila. Mothers, but not fathers, mutant for sisR-4 produce embryos that fail to hatch. During embryogenesis, sisR-4 promotes transcription of its host gene (deadpan), which is essential for development. Interestingly, sisR-4 functions by activating an enhancer present in the intron where sisR-4 is encoded. We propose that a maternal sisRNA triggers expression of its host gene via a positive feedback loop during embryogenesis.

Stable intronic sequence RNAs (sisRNAs): a new layer of gene regulation.

Upon splicing, introns are rapidly degraded. Hence, RNAs derived from introns are commonly deemed as junk sequences. However, the discoveries of intronic-derived small nucleolar RNAs (snoRNAs), small Cajal body associated RNAs (scaRNAs) and microRNAs (miRNAs) suggested otherwise. These non-coding RNAs are shown to play various roles in gene regulation. In this review, we highlight another class of intron-derived RNAs known as stable intronic sequence RNAs (sisRNAs). sisRNAs have been observed since the 1980 s; however, we are only beginning to understand their biological significance. Recent studies have shown or suggested that sisRNAs regulate their own host's gene expression, function as molecular sinks or sponges, and regulate protein translation. We propose that sisRNAs function as an additional layer of gene regulation in the cells.

Stable intronic sequence RNAs have possible regulatory roles in Drosophila melanogaster.

Stable intronic sequence RNAs (sisRNAs) have been found in Xenopus tropicalis, human cell lines, and Epstein-Barr virus; however, the biological significance of sisRNAs remains poorly understood. We identify sisRNAs in Drosophila melanogaster by deep sequencing, reverse transcription polymerase chain reaction, and Northern blotting. We characterize a sisRNA (sisR-1) from the regena (rga) locus and show that it can be processed from the precursor messenger RNA (pre-mRNA). We also document a cis-natural antisense transcript (ASTR) from the rga locus, which is highly expressed in early embryos. During embryogenesis, ASTR promotes robust rga pre-mRNA expression. Interestingly, sisR-1 represses ASTR, with consequential effects on rga pre-mRNA expression. Our results suggest a model in which sisR-1 modulates its host gene expression by repressing ASTR during embryogenesis. We propose that sisR-1 belongs to a class of sisRNAs with probable regulatory activities in Drosophila.