RNA Pathways Research Articles

GTSF1 is required for transposon silencing in the unicellular eukaryote Paramecium tetraurelia.

The PIWI-interacting RNA (piRNA) pathway is crucial for transposon repression and the maintenance of genomic integrity. Gametocyte-specific factor 1 (GTSF1), a PIWI-associated protein indispensable for transposon repression, has been recently shown to potentiate the catalytic activity of PIWI in many metazoans. Whether the requirement of GTSF1 extends to PIWI proteins beyond metazoans is unknown. In this study, we identified a homolog of GTSF1 in the unicellular eukaryote Paramecium tetraurelia (PtGtsf1) and found that its role as a PIWI-cofactor is conserved. PtGtsf1 interacts with PIWI (Ptiwi09) and Polycomb Repressive Complex 2 and is essential for PIWI-dependent DNA elimination of transposons during sexual development. PtGtsf1 is crucial for the degradation of PIWI-bound small RNAs that recognize the organism's own genomic sequences. Without PtGtsf1, self-matching small RNAs are not degraded and results in an accumulation of H3K9me3 and H3K27me3, which may disturb transposon recognition. Our results demonstrate that the PIWI-GTSF1 interaction also exists in unicellular eukaryotes with a role in transposon silencing.

Graphene quantum dots disrupt the mitochondrial potential of Trypanosoma brucei by interacting with the p18 subunit of ATP synthase F1 after endocytosis via the VSG recycling pathway

HypothesisTrypanosomiasis is one of the main threats to human and animal health in African countries. Trypanosoma brucei can evade the host immune recognition by rapidly altering its variant surface glycoprotein (VSG). The ATP synthase F1 subunit of the parasite exhibits extremely low similarity to that of its mammalian hosts, hypothetically making it an ideal target for the development of novel therapeutics. ExperimentsGraphene quantum dots (GQDs) were synthesized, and their adhesion to T. brucei surface and internalization was observed microscopically. The activity of ATP synthase and mitochondrial membrane potential of T. brucei were measured after exposure to GQDs. Proteomics, biolayer interferometry, and molecular dynamic simulations were utilized to evaluate the interaction between GQDs with the target proteins. FindingsGQDs specifically adhered to the VSG of T. brucei and were conveyed inside the parasite via the VSG internalization pathway. The GQDs promoted intracellular ROS production, interacted with, and inhibited the activity of the p18 subunit of ATP synthase, disrupted parasite mitochondrial membrane potential. Additionally, the GQDs caused a decrease in aminoacyl – tRNA biosynthesis, and upregulated RNA and protein degradation pathways. The findings of this study offer a novel avenue for the target-oriented discovery of anti-trypanosome drugs.

HERD-1 mediates multiphase condensate immiscibility to regulate small RNA-driven transgenerational epigenetic inheritance.

Biomolecular condensates, such as the nucleolus, stress granules/processing bodies and germ granules, are multiphase assemblages whose formation mechanisms and significance remain poorly understood. Here we identify protein constituents of the spatiotemporally ordered P, Z and M multiphase condensates in Caenorhabditis elegans germ granules using optimized TurboID-mediated proximity biotin labelling. These include 462, 41 and 86 proteins localizing to P, Z and M condensates, respectively, of which 522 were previously unknown protein constituents. Each condensate's proteins are enriched for distinct classes of structured and intrinsically disordered domains, suggesting divergent functions and assembly mechanisms. Through a functional screen, we identify a germ granule protein, HERD-1, which prevents the mixing of P, Z and M condensates. Mixing in herd-1 mutants correlates with disorganization of germline small RNA pathways and prolonged epigenetic inheritance of RNA interference-induced gene silencing. Forced mixing of these condensate components using a nanobody with specific binding activity against green fluorescent protein also extends epigenetic inheritance. We propose that active maintenance of germ granule immiscibility helps to organize and regulate small RNA-driven transgenerational epigenetic inheritance in C. elegans.

Distinct regulatory mechanisms by the nuclear Argonautes HRDE-1 and NRDE-3 in the soma of Caenorhabditis elegans.

This study examines the transcriptional consequences during the disruption of the nuclear RNAi silencing mechanism in C. elegans . Through microscopy and bioinformatic work, we demonstrate that although nuclear RNAi mutants exhibit significantly decondensed X chromosomes, chromosome-wide transcriptional de-repression is not detectable. Downstream analyses further explore the global influence of the nuclear RNAi pathway, indicating that the nuclear Argonautes HRDE-1 and NRDE-3 function through two distinct mechanisms.

Transposon-mediated genic rearrangements underlie variation in small RNA pathways

Transposable elements (TEs) can alter host gene structure and expression, whereas host organisms develop mechanisms to repress TE activities. In the nematode Caenorhabditis elegans , a small interfering RNA pathway dependent on the helicase ERI-6/7 primarily silences retrotransposons and recent genes of likely viral origin. By studying gene expression variation among wild C. elegans strains, we found that structural variants and transposon remnants likely underlie expression variation in eri-6/7 and the pathway targets. We further found that multiple insertions of the DNA transposons, Polintons, reshuffled the eri-6/7 locus and induced inversion of eri-6 in some wild strains. In the inverted configuration, gene function was previously shown to be repaired by unusual trans-splicing mediated by direct repeats. We identified that these direct repeats originated from terminal inverted repeats of Polintons . Our findings highlight the role of host-transposon interactions in driving rapid host genome diversification among natural populations and shed light on evolutionary novelty in genes and splicing mechanisms.

RNA-Binding Small Molecules in Drug Discovery and Delivery: An Overview from Fundamentals.

RNA molecules, similar to proteins, fold into complex structures to confer diverse functions in cells. The intertwining of functions with RNA structures offers a new therapeutic opportunity for small molecules to bind and manipulate disease-relevant RNA pathways, thus creating a therapeutic realm of RNA-binding small molecules. The ongoing interest in RNA targeting and subsequent screening campaigns have led to the identification of numerous compounds that can regulate RNAs from splicing, degradation to malfunctions, with therapeutic benefits for a variety of diseases. Moreover, along with the rise of RNA-based therapeutics, RNA-binding small molecules have expanded their application to the modification, regulation, and delivery of RNA drugs, leading to the burgeoning interest in this field. This Perspective overviews the emerging roles of RNA-binding small molecules in drug discovery and delivery, covering aspects from their action fundamentals to therapeutic applications, which may inspire researchers to advance the field.

Role of male gonad-enriched microRNAs in sperm production in Caenorhabditis elegans.

Germ cell development and gamete production in animals require small RNA pathways. While studies indicate that microRNAs (miRNAs) are necessary for normal sperm production and function, the specific roles for individual miRNAs are largely unknown. Here, we use small RNA sequencing (RNA-seq) of dissected gonads and functional analysis of new loss-of-function alleles to identify functions for miRNAs in the control of fecundity and sperm production in Caenorhabditis elegans males and hermaphrodites. We describe a set of 29 male gonad-enriched miRNAs and identify a set of individual miRNAs (mir-58.1 and mir-235) and a miRNA cluster (mir-4807-4810.1) that are required for optimal sperm production at 20°C and a set of miRNAs (mir-49, mir-57, mir-83, mir-261, and mir-357/358) that are required for sperm production at 25°C. We observed defects in meiotic progression in mutants missing mir-58.1, mir-83, mir-235, and mir-4807-4810.1, which may contribute to the observed defects in sperm production. Further, analysis of multiple mutants of these miRNAs suggested genetic interactions between these miRNAs. This study provides insights on the regulatory roles of miRNAs that promote optimal sperm production and fecundity in males and hermaphrodites.

Beyond RNAi: How the Dicer protein modulates the antiviral innate immune response in mammalian cells: Mammalian Dicer could regulate the innate immune response in an RNAi-independent manner as a result of losing long dsRNA processive activity.

While Dicer plays an important antiviral role through the RNAi pathway in plants and invertebrates, its contribution to antiviral immunity in vertebrates and more specifically mammals is more controversial. The apparent limited RNAi activity in mammalian cells has been attributed to the reduced long dsRNA processive activity of mammalian Dicer, as well as a functional incompatibility between the RNAi and IFN pathways. Why Dicer has lost this antiviral activity in the profit of the IFN pathway is still unclear. We propose that the primary direct antiviral activity of Dicer has been functionally replaced by other sensors in the IFN pathway, leading to its specialization toward microRNA maturation. As a result, Dicer can regulate the innate immune response and prevent basal activation of the IFN pathway in mammals. Here, we discuss this hypothesis, highlighting how the adaptation of the helicase domain of mammalian Dicer may be key to this process.

Combining RNA Interference and RIG-I Activation to Inhibit Hepatitis E Virus Replication.

Hepatitis E virus (HEV) poses a significant global health threat, with an estimated 20 million infections occurring annually. Despite being a self-limiting illness, in most cases, HEV infection can lead to severe outcomes, particularly in pregnant women and individuals with pre-existing liver disease. In the absence of specific antiviral treatments, the exploration of RNAi interference (RNAi) as a targeted strategy provides valuable insights for urgently needed therapeutic interventions against Hepatitis E. We designed small interfering RNAs (siRNAs) against HEV, which target the helicase domain and the open reading frame 3 (ORF3). These target regions will reduce the risk of viral escape through mutations, as they belong to the most conserved regions in the HEV genome. The siRNAs targeting the ORF3 efficiently inhibited viral replication in A549 cells after HEV infection. Importantly, the siRNA was also highly effective at inhibiting HEV in the persistently infected A549 cell line, which provides a suitable model for chronic infection in patients. Furthermore, we showed that a 5' triphosphate modification on the siRNA sense strand activates the RIG-I receptor, a cytoplasmic pattern recognition receptor that recognizes viral RNA. Upon activation, RIG-I triggers a signaling cascade, effectively suppressing HEV replication. This dual-action strategy, combining the activation of the adaptive immune response and the inherent RNAi pathway, inhibits HEV replication successfully and may lead to the development of new therapies.

DEAD-box RNA helicase RH20 positively regulates RNAi-based antiviral immunity in plants by associating with SGS3/RDR6 bodies.

RNA silencing plays a crucial role in defending against viral infections in diverse eukaryotic hosts. Despite extensive studies on core components of the antiviral RNAi pathway such as DCLs, AGOs and RDRs proteins, host factors involved in antiviral RNAi remain incompletely understood. In this study, we employed the proximity labelling approach to identify the host factors required for antiviral RNAi in Nicotiana benthamiana. Using the barley stripe mosaic virus (BSMV)-encoded γb, a viral suppressor of RNA silencing (VSR), as the bait protein, we identified the DEAD-box RNA helicase RH20, a broadly conserved protein in plants and animals with a homologous human protein known as DDX5. We demonstrated the interaction between RH20 and BSMV γb. Knockdown or knockout of RH20 attenuates the accumulation of viral small interfering RNAs, leading to increased susceptibility to BSMV, while overexpression of RH20 enhances resistance to BSMV, a process requiring the cytoplasmic localization and RNA-binding activity of RH20. In addition to BSMV, RH20 also negatively regulates the infection of several other positive-sense RNA viruses, suggesting the broad-spectrum antiviral activity of RH20. Mechanistic analysis revealed the colocalization and interaction of RH20 with SGS3/RDR6, and disruption of either SGS3 or RDR6 undermines the antiviral function of RH20, suggesting RH20 as a new component of the SGS3/RDR6 bodies. As a counter-defence, BSMV γb VSR subverts the RH20-mediated antiviral defence by interfering with the RH20-SGS3 interaction. Our results uncover RH20 as a new positive regulator of antiviral RNAi and provide new potential targets for controlling plant viral diseases.

Biomarkers related to m6A and succinic acid metabolism in papillary thyroid carcinoma

BackgroundStudies have shown that m6A modification is related to the occurrence and development of papillary thyroid carcinoma (PTC). The disorder of succinic acid metabolism is associated with the occurrence and development of various tumors. However, there are few studies based on m6A and succinate metabolism-related genes (SMRGs) in PTC.MethodsThe TCGA-Thyroid carcinoma (THCA), GSE33630, 1159 SMRGs, and 23 m6A regulatory factors were collected from the online databases. Subsequently, the differentially expressed genes (DEGs) were selected between PTC (Tumor) and Normal samples. The overlapping genes among the DEGs, m6A, and SMRGs were applied to screen the biomarkers. Using the 3 machine-learning algorithms, the biomarkers were determined based on the overlapping genes. Next, the biomarkers were evaluated by the ROC curve and expression analysis in TCGA-THCA and GSE33630. Then, the overall survival (OS) differences were compared between the high-and low-expression biomarkers. Finally, immune infiltration analysis, molecular regulatory network, and drug prediction were performed based on the biomarkers.ResultsIn TCGA-THCA, there were 2800 DEGs between and Normal samples, and then 7 overlapping genes were obtained. Importantly, ADK, TNFRSF10B, CYP7B1, FGFR2, and CPQ were determined as biomarkers with excellent diagnostic efficiency (AUC > 0.7). In PTC samples, ADK and TNFRSF10B were high-expressed while CYP7B1, FGFR2, and CPQ were low-expressed. Especially, the high-expression groups of ADK had a better prognosis, while the high-expression groups of CYP7B1, FGFR2, and CPQ had a worse prognosis. Afterward, immune infiltration analysis found that 16 immune cells had infiltration differences between the Tumor and Normal samples. Finally, transcription factor SP1 could regulate CYP7B1 and TNFRSF10B. Moreover, Navitoclax was a potential drug for PTC patients.ConclusionOverall, we described 5 biomarkers associated with adverse prognosis of PTC, including ADK, TNFRSF10B, CYP7B1, FGFR2, and CPQ. All these biomarkers were involved in succinate metabolism and m6A modification of RNA. This set of biomarkers should be explored further for their diagnostic value in PTC. Investigations into the mechanistic role of alteration of succinate metabolism and m6A modification of RNA pathways in the pathophysiology of PTC are warranted.

51 Epigenetic regulation of ribosomal RNA transcription and processing by the tumor suppressor SETD2

Abstract Background The methyltransferase SETD2 was originally identified as a tumor suppressor in clear cell renal cell carcinoma (ccRCC), and ccRCC with SETD2 mutations have increased metastatic potential and poor progression free survival. A known epigenetic regulator, SETD2 interacts with actively transcribing RNA polymerase II (RNAPII) and tri-methylates Histone 3 at Lysine 36 (H3K36me3), a modification that correlates with active gene transcription and is recognized by mRNA methylation and splicing factors. Despite these known roles in mRNA transcription, loss of SETD2 does not alter genic transcription, raising the question of whether or how SETD2 regulates RNA biology. Methods Given that SETD2 influences RNA processing, we sought to identify RNA-protein complexes that are regulated by SETD2. To do this in an unbiased and high-throughput manner, we used orthogonal organic phase separation (OOPS) coupled with mass spectrometry to identify changes in RNA binding proteins and pathways between control and SETD2-knockout cells. Results Corroborating previous findings, we identified that RNA binding proteins governing mRNA splicing and cellular metabolism were dysregulated in SETD2 mutant cells. However, the most robust changes in SETD2 knockout cells occurred in pathways regulated ribosome biogenesis (See Figure, orange highlights), resulting in a significant decrease in binding of ribosomal RNA (rRNA) processing factors to RNA. We confirm these alterations lead to defects in rRNA transcription, processing, and ribosome biogenesis. We identify that loss of SETD2 catalytic activity phenocopies these rRNA processing defects, suggesting H3K36me3 is essential for rRNA processing. Disruptions occur to both the large (60S) and small (40S) ribosome subunits, modifying protein translation. Lastly, we identify synthetic lethality between SETD2 deletion or inhibition and small molecules that interfere with rRNA transcription. Conclusions While the majority of research emphasizes the role SETD2 in the regulation of mRNA, our data identify that SETD2 and its catalytic activity play a critical role in the transcription and processing of rRNA. Importantly, rRNA comprises 80-90% of cellular RNA yet is typically overlooked and understudied. Next-generation RNA sequencing or transcriptional assays investigate mRNA or genic transcription while ignoring rRNA, demonstrating why SETD2-mediated rRNA processing has been unexplored. This may also have important implications for prospective clinical trials that utilized RNA-sequencing to identify optimal therapeutic regimens. Our work also identified that SETD2-mutated cells are more sensitive to compounds that inhibit rRNA transcription, potentially uncovering a new therapeutic vulnerability of ccRCC with mono- and bi-allelic loss of SETD2. Alterations in ribosome biogenesis leads to epithelial-to-mesenchymal transition and metastasis in many tumor types, and our future goal will be to investigate the role of ribosomes and rRNA transcription in transformation and metastasis in ccRCC as well as the molecular mechanisms connecting SETD2 to ribosome fidelity. DOD CDMRP Funding: yes

Caenorhabditis elegans SynMuv B gene activity is down-regulated during a viral infection to enhance RNA interference.

Small RNA pathways regulate eukaryotic antiviral defense. Many of the Caenorhabditis elegans mutations that were identified based on their enhanced RNAi, the synMuv B genes, also emerged from unrelated genetic screens for increased growth factor signaling. The dozen synMuv B genes encode homologues of the mammalian dREAM complex found in nearly all animals and plants, which includes the lin-35/retinoblastoma oncogene. We show that a set of highly induced mRNAs in synMuv B mutants is congruent with mRNAs induced by Orsay RNA virus infection of C. elegans. In wild type animals, a combination of a synMuv A mutation and a synMuv B mutation are required for the Muv phenotype of increased growth factor signaling. But we show that Orsay virus infection of a single synMuv A mutant can induce a Muv phenotype, unlike the uninfected single synMuv A mutant. This suggests that decreased synMuv B activity, which activates the antiviral RNAi pathway, is a defense response to viral infection. Small RNA deep sequencing analysis of various dREAM complex mutants uncovers distinct siRNA profiles indicative of such an siRNA response. We conclude that the synMuv B mutants maintain an antiviral readiness state even in the absence of actual infection. The enhanced RNAi and conservation of the dREAM complex mutants suggests new therapeutic avenues to boost antiviral defenses.

Evolutionary adaptation of an HP1-protein chromodomain integrates chromatin and DNA sequence signals.

Members of the diverse heterochromatin protein 1 (HP1) family play crucial roles in heterochromatin formation and maintenance. Despite the similar affinities of their chromodomains for di- and tri-methylated histone H3 lysine 9 (H3K9me2/3), different HP1 proteins exhibit distinct chromatin-binding patterns, likely due to interactions with various specificity factors. Previously, we showed that the chromatin-binding pattern of the HP1 protein Rhino, a crucial factor of the Drosophila PIWI-interacting RNA (piRNA) pathway, is largely defined by a DNA sequence-specific C2H2 zinc finger protein named Kipferl (Baumgartner et al., 2022). Here, we elucidate the molecular basis of the interaction between Rhino and its guidance factor Kipferl. Through phylogenetic analyses, structure prediction, and in vivo genetics, we identify a single amino acid change within Rhino's chromodomain, G31D, that does not affect H3K9me2/3 binding but disrupts the interaction between Rhino and Kipferl. Flies carrying the rhinoG31D mutation phenocopy kipferl mutant flies, with Rhino redistributing from piRNA clusters to satellite repeats, causing pronounced changes in the ovarian piRNA profile of rhinoG31D flies. Thus, Rhino's chromodomain functions as a dual-specificity module, facilitating interactions with both a histone mark and a DNA-binding protein.

Effect of benralizumab on histopathology and inflammatory signatures in a clinical cohort of eosinophilic esophagitis.

A preliminary report from the recent phase 3 trial of benralizumab, a monoclonal antibody that binds to interleukin-5 receptor alpha (IL5Rα), in patients with EoE revealed that medication use led to tissue eosinophil eradication but did not meet the clinical endpoint of symptom resolution. Here, we characterized the clinical, endoscopic, histologic, and transcriptional changes in patients with active EoE following benralizumab treatment. We retrospectively examined patients with EoE treated with benralizumab at the University of Utah (n =11) and reviewed reported clinical symptoms, circulating and tissue eosinophilia, and endoscopic and histologic scores. Gene expression profiles from available esophageal tissue from benralizumab-treated patients were compared to those from patients with remission EoE (n =5), active EoE (n =10), and controls (n =22). Benralizumab treatment resulted in partial symptom improvement and significant reduction in tissue eosinophilia, and endoscopic and histologic disease scoring (P < 0.01). Histologic score reductions were driven by eosinophil feature scores, while scores for epithelial features (basal cell hyperplasia and dilated intercellular spaces) were similar to those in active EoE. The gene signatures in benralizumab-treated patients mimicked those of active EoE (e.g. upregulation of POSTN, CDH26, CCL26, and downregulation of DSG1). RNA profiles and pathways support histologic findings of impaired epithelial function that persists despite benralizumab treatment. In conclusion, despite eosinophil eradication, patients treated with benralizumab had persistent epithelial injury at the histologic and transcriptional level. In this cohort, benralizumab therapy failed to eradicate inflammation and epithelial dysfunction showing that interleukin-5 receptor alpha blockade monotherapy is insufficient to control EoE.

The Upstream Sequence Transcription Complex dictates nucleosome positioning and promoter accessibility at piRNA genes in the C. elegans germ line.

The piRNA pathway is a conserved germline-specific small RNA pathway that ensures genomic integrity and continued fertility. In C. elegans and other nematodes, Type-I piRNAs are expressed from >10,000 independently transcribed genes clustered within two discrete domains of 1.5 and 3.5 MB on Chromosome IV. Clustering of piRNA genes contributes to their germline-specific expression, but the underlying mechanisms are unclear. We analyze isolated germ nuclei to demonstrate that the piRNA genomic domains are located in a heterochromatin-like environment. USTC (Upstream Sequence Transcription Complex) promotes strong association of nucleosomes throughout piRNA clusters, yet organizes the local nucleosome environment to direct the exposure of individual piRNA genes. Localization of USTC to the piRNA domains depends upon the ATPase chromatin remodeler ISW-1, which maintains high nucleosome density across piRNA clusters and ongoing production of piRNA precursors. Overall, this work provides insight into how chromatin states coordinate transcriptional regulation over large genomic domains, with implications for global genome organization.

Small RNA-mediated genetic switches coordinate ALG-3/4 small RNA pathway function.

Coordination of gene regulatory networks is necessary for proper execution of cellular programs throughout development. RNA interference (RNAi) is an essential regulatory mechanism in all metazoans. Proper RNAi-mediated gene regulation requires coordination of several RNAi branches to ensure homeostasis. For example, in Caenorhabditis elegans, the Argonautes, ALG-3 and ALG-4, are expressed specifically during spermatogenesis (L4 stage) and bind small interfering RNAs (siRNAs) complementary to sperm-enriched genes. We find that alg-3 and alg-4 are regulated by siRNAs. Our work shows that gene switches are operated via these siRNAs to regulate the Argonautes' expression in a temporal manner. This RNAi-to-RNAi regulatory cascade is essential for coordinating ALG-3/4 pathway function, particularly during heat stress, to provide thermotolerant sperm-based fertility. This work provides insight into one regulatory motif used to maintain RNAi homeostasis, across developmental stages, despite environmental stressors. As RNAi pathways are evolutionarily conserved, other species likely use similar regulatory architectures to maintain RNAi homeostasis.

Roles of long non‑coding RNAs in esophageal cell squamous carcinoma (Review).

Esophageal squamous cell carcinoma (ESCC) is a prevalent and deadly malignancy of the digestive tract. Recent research has identified long non‑coding RNAs (lncRNAs) as crucial regulators in the pathogenesis of ESCC. These lncRNAs, typically exceeding 200 nucleotides, modulate gene expression through various mechanisms, including the competing endogenous RNA (ceRNA) pathway and RNA‑protein interactions. The current study reviews the multifaceted roles of lncRNAs in ESCC, highlighting their involvement in processes such as proliferation, migration, invasion, epithelial‑mesenchymal transition, cell cycle progression, resistance to radiotherapy and chemotherapy, glycolysis, apoptosis, angiogenesis, autophagy, tumor growth, metastasis and the maintenance of cancer stem cells. Specific lncRNAs like HLA complex P5, LINC00963 and non‑coding repressor of NFAT have been shown to enhance resistance to radio‑ and chemotherapy by modulating pathways such as AKT signaling and microRNA interaction, which promote cell survival and proliferation under therapeutic stress. Furthermore, lncRNAs like family with sequence similarity 83, member A antisense RNA 1, zinc finger NFX1‑type containing 1 antisense RNA 1 and taurine upregulated gene 1 are implicated in enhancing invasive and proliferative capabilities of ESCC cells through the ceRNA mechanism, while interactions with RNA‑binding proteins further influence cancer cell behavior. The comprehensive analysis underscores the potential of lncRNAs as biomarkers for prognosis and therapeutic targets in ESCC, suggesting avenues for future research focused on elucidating the detailed molecular mechanisms and clinical applications of lncRNAs in ESCC management.

Precise measurement of molecular phenotypes with barcode-based CRISPRi systems.

Genome-wide CRISPR-Cas9 screens have untangled regulatory networks and revealed the genetic underpinnings of diverse biological processes. Their success relies on experimental designs that interrogate specific molecular phenotypes and distinguish key regulators from background effects. Here, we realize these goals with a generalizable platform for CRISPR interference with barcoded expression reporter sequencing (CiBER-seq) that dramatically improves the sensitivity and scope of genome-wide screens. We systematically address technical factors that distort phenotypic measurements by normalizing expression reporters against closely-matched control promoters, integrated together into the genome at single copy. To test our ability to capture post-transcriptional and post-translational regulation through sequencing, we screened for genes that affected nonsense-mediated mRNA decay and Doa10-mediated cytosolic protein decay. Our optimized CiBER-seq screens accurately capture the known components of well-studied RNA and protein quality control pathways with minimal background. These results demonstrate the precision and versatility of CiBER-seq for dissecting the genetic networks controlling cellular behaviors.

Escalation of genome defense capacity enables control of an expanding meiotic driver.

From RNA interference to chromatin silencing, diverse genome defense pathways silence selfish genetic elements to safeguard genome integrity 1,2 . Despite their diversity, different defense pathways share a modular organization, where numerous specificity factors identify diverse targets and common effectors silence them. In the PIWI-interacting RNA (piRNA) pathway, which controls selfish elements in the metazoan germline, diverse target RNAs are first identified by complementary base pairing with piRNAs and then silenced by PIWI-clade nucleases via enzymatic cleavage 1,3 . Such a binary architecture allows the defense systems to be readily adaptable, where new targets can be captured via the innovation of new specificity factors 4,5 . Thus, our current understanding of genome defense against lineage-specific selfish genes has been largely limited to the evolution of specificity factors, while it remains poorly understood whether other types of innovations are required. Here, we describe a new type of innovation, which escalates the defense capacity of the piRNA pathway to control a recently expanded selfish gene in Drosophila melanogaster . Through an in vivo RNAi screen for repressors of Stellate -a recently evolved and expanded selfish meiotic driver 6-8 -we discovered a novel defense factor, Trailblazer. Trailblazer is a transcription factor that promotes the expression of two PIWI-clade nucleases, Aub and AGO3, to match Stellate in abundance. Recent innovation in the DNA-binding domain of Trailblazer enabled it to drastically elevate Aub and AGO3 expression in the D. melanogaster lineage, thereby escalating the silencing capacity of the piRNA pathway to control expanded Stellate and safeguard fertility. As copy-number expansion is a recurrent feature of diverse selfish genes across the tree of life 9-12 , we envision that augmenting the defense capacity to quantitatively match selfish genes is likely a repeatedly employed defense strategy in evolution.