mRNA Decay Research Articles

Drosophila Clueless ribonucleoprotein particles display novel dynamics that rely on the availability of functional protein and polysome equilibrium.

The cytoplasm is populated with many ribonucleoprotein (RNP) particles that post-transcriptionally regulate mRNAs. These membraneless organelles assemble and disassemble in response to stress, performing functions such as sequestering stalled translation pre-initiation complexes or mRNA storage, repression and decay. Drosophila Clueless (Clu) is a conserved multi-domain ribonucleoprotein essential for mitochondrial function that forms dynamic particles within the cytoplasm. Unlike well-known RNP particles, stress granules and Processing bodies, Clu particles completely disassemble under nutritional or oxidative stress. However, it is poorly understood how disrupting protein synthesis affects Clu particle dynamics, especially since Clu binds mRNA and ribosomes. Here, we capitalize on ex vivo and in vivo imaging of Drosophila female germ cells to determine what domains of Clu are necessary for Clu particle assembly, how manipulating translation using translation inhibitors affects particle dynamics, and how Clu particle movement relates to mitochondrial association. Using Clu deletion analysis and live and fixed imaging, we identified three protein domains in Clu, which are essential for particle assembly. In addition, we demonstrated that overexpressing functional Clu disassembled particles, while overexpression of deletion constructs did not. To examine how decreasing translation affects particle dynamics, we inhibited translation in Drosophila germ cells using cycloheximide and puromycin. In contrast to stress granules and Processing bodies, cycloheximide treatment did not disassemble Clu particles yet puromycin treatment did. Surprisingly, cycloheximide stabilized particles in the presence of oxidative and nutritional stress. These findings demonstrate that Clu particles have novel dynamics in response to altered ribosome activity compared to stress granules and Processing bodies and support a model where they function as hubs of translation whose assembly heavily depends on the dynamic availability of polysomes.

RNA Surveillance Factor SMG5 Is Essential for Mouse Embryonic Stem Cell Differentiation.

Nonsense-mediated mRNA decay (NMD) is a highly conserved post-transcriptional gene expression regulatory mechanism in eukaryotic cells. NMD eliminates aberrant mRNAs with premature termination codons to surveil transcriptome integrity. Furthermore, NMD fine-tunes gene expression by destabilizing RNAs with specific NMD features. Thus, by controlling the quality and quantity of the transcriptome, NMD plays a vital role in mammalian development, stress response, and tumorigenesis. Deficiencies of NMD factors result in early embryonic lethality, while the underlying mechanisms are poorly understood. SMG5 is a key NMD factor. In this study, we generated an Smg5 conditional knockout mouse model and found that Smg5-null results in early embryonic lethality before E13.5. Furthermore, we produced multiple lines of Smg5 knockout mouse embryonic stem cells (mESCs) and found that the deletion of Smg5 in mESCs does not compromise cell viability. Smg5-null delays differentiation of mESCs. Mechanistically, our study reveals that the c-MYC protein, but not c-Myc mRNA, is upregulated in SMG5-deficient mESCs. The overproduction of c-MYC protein could be caused by enhanced protein synthesis upon SMG5 loss. Furthermore, SMG5-null results in dysregulation of alternative splicing on multiple stem cell differentiation regulators. Overall, our findings underscore the importance of SMG5-NMD in regulating mESC cell-state transition.

An autoregulatory poison exon in Smndc1 is conserved across kingdoms and influences organism growth.

Many of the most highly conserved elements in the human genome are "poison exons," alternatively spliced exons that contain premature termination codons and permit post-transcriptional regulation of mRNA abundance through induction of nonsense-mediated mRNA decay (NMD). Poison exons are widely assumed to be highly conserved due to their presumed importance for organismal fitness, but this functional importance has never been tested in the context of a whole organism. Here, we report that a poison exon in Smndc1 is conserved across mammals and plants and plays a molecular autoregulatory function in both kingdoms. We generated mouse and A. thaliana models lacking this poison exon to find its loss leads to deregulation of SMNDC1 protein levels, pervasive alterations in mRNA processing, and organismal size restriction. Together, these models demonstrate the importance of poison exons for both molecular and organismal phenotypes that likely explain their extraordinary conservation.

METTL14-mediated m6A mRNA modification of G6PD promotes lung adenocarcinoma

METTL14 functions as an RNA methyltransferase involved in m6A modification, influencing mRNA biogenesis, decay, and translation processes. However, the specific mechanism by which METTL14 regulates glucose-6-phosphate dehydrogenase (G6PD) to promote the progression of lung adenocarcinoma (LUAD) is not well understood. Quantitative measurement and immunohistochemistry (IHC) analysis have demonstrated higher levels of m6A in LUAD tissues compared to adjacent normal tissues. Additionally, the expression of METTL14 was significantly increased in LUAD tissues. In LUAD cell lines, both METTL14 and m6A levels were elevated compared to normal human lung epithelial cells. Knockdown of METTL14 markedly reduced LUAD cell proliferation, migration, and invasion. Conversely, overexpression of METTL14, but not the mutant form, significantly enhanced these cellular processes in LUAD. In vivo studies using nude mice with subcutaneously transplanted LUAD cells demonstrated that stable METTL14 knockdown led to notably reduced tumor volume and weight, along with fewer Ki67-positive cells and lung metastatic sites. Importantly, METTL14 knockdown reduced glycolytic activity in LUAD cells. Through a combination of RNA sequencing and MeRIP-sequencing, we identified numerous altered genes and confirmed that IGF2BP2 enhances G6PD mRNA stability after METTL14-mediated m6A modification, thereby promoting tumor growth and metastasis. Moreover, LUAD patients with higher levels of G6PD had poorer overall survival (OS). In conclusion, our study indicates that METTL14 upregulates G6PD expression post-transcriptionally through an m6A-IGF2BP2-dependent mechanism, thereby stabilizing G6PD mRNA. These findings propose potential diagnostic biomarkers and effective targets for anti-metabolism therapy in LUAD.

UPF3B modulates endoplasmic reticulum stress through interaction with inositol-requiring enzyme-1α

The unfolded protein response (UPR) is a conserved and adaptive intracellular pathway that relieves the endoplasmic reticulum (ER) stress by activating ER transmembrane stress sensors. As a consequence of ER stress, the inhibition of nonsense-mediated mRNA decay (NMD) is due to an increase in the phosphorylation of eIF2α, which has the effect of inhibiting translation. However, the role of NMD in maintaining ER homeostasis remains unclear. In this study, we found that the three NMD factors, up-frameshift (UPF)1, UPF2, or UPF3B, were required to negate the UPR. Among these three NMD factors, only UPF3B interacted with inositol-requiring enzyme-1α (IRE1α). This interaction inhibited the kinase activity of IRE1α, abolished autophosphorylation, and reduced IRE1α clustering for ER stress. BiP and UPF3B jointly control the activation of IRE1α on both sides of the ER membrane. Under stress conditions, the phosphorylation of UPF3B was increased and the phosphorylated sites were identified. Both the UPF3BY160D genetic mutation and phosphorylation at Thr169 of UPF3B abolished its interaction with IRE1α and UPF2, respectively, leading to activation of ER stress and NMD dysfunction. Our study reveals a key physiological role for UPF3B in the reciprocal regulatory relationship between NMD and ER stress.

Membrane Localization of RNase Y Is Important for Global Gene Expression in Bacillus subtilis.

RNase Y is a key endoribonuclease that regulates global mRNA turnover and processing in Bacillus subtilis and likely many other bacteria. This enzyme is anchored to the cell membrane, creating a pseudo-compartmentalization that aligns with its role in initiating the decay of mRNAs primarily translated at the cell periphery. However, the reasons behind and the consequences of RNase Y's membrane attachment remain largely unknown. In our study, we examined a strain expressing wild-type levels of a cytoplasmic form of RNase Y from its chromosomal locus. This strain exhibits a slow-growth phenotype, similar to that of an RNase Y null mutant. Genome-wide data reveal a significant impact on the expression of hundreds of genes. While certain RNA substrates clearly depend on RNase Y's membrane attachment, others do not. We observed no correlation between mRNA stabilization in the mutant strains and the cellular location or function of the encoded proteins. Interestingly, the Y-complex, a specificity factor for RNase Y, also appears also recognize the cytoplasmic form of the enzyme, restoring wild-type levels of the corresponding transcripts. We propose that membrane attachment of RNase Y is crucial for its functional interaction with many coding and non-coding RNAs, limiting the cleavage of specific substrates, and potentially avoiding unfavorable competition with other ribonucleases like RNase J, which shares a similar evolutionarily conserved cleavage specificity.

A spatiotemporally resolved atlas of mRNA decay in the C. elegans embryo reveals differential regulation of mRNA stability across stages and cell types.

During embryonic development, cells undergo dynamic changes in gene expression that are required for appropriate cell fate specification. Although both transcription and mRNA degradation contribute to gene expression dynamics, patterns of mRNA decay are less well understood. Here, we directly measure spatiotemporally resolved mRNA decay rates transcriptome-wide throughout C. elegans embryogenesis by transcription inhibition followed by bulk and single-cell RNA sequencing. This allows us to calculate mRNA half-lives within specific cell types and developmental stages, and identify differentially regulated mRNA decay throughout embryonic development. We identify transcript features that are correlated with mRNA stability and find that mRNA decay rates are associated with distinct peaks in gene expression over time. Moreover, we provide evidence that, on average, mRNA is more stable in the germline than in the soma and in later embryonic stages than in earlier stages. This work suggests that differential mRNA decay across cell states and time helps to shape developmental gene expression, and it provides a valuable resource for studies of mRNA turnover regulatory mechanisms.

Human eRF1 Translation Regulation

Eukaryotic translation release factor eRF1 is an important cellular protein that plays a key role in translation termination, nonsense-mediated mRNA decay (NMD), and readthrough of stop codons. The amount of eRF1 in the cell influences all these processes. The mechanism of regulation of eRF1 translation through an autoregulatory NMD-dependent expression circuit has been described for plants and fungi, but the mechanisms of regulation of human eRF1 translation have not yet been studied. Using reporter constructs, we studied the effect of eRF1 mRNA elements on its translation in cell-free translation systems and HEK293 cell culture. Our data indicate the absence of an NMD-dependent autoregulatory circuit for human eRF1 expression. We found that the translation of the eRF1 coding sequence is most strongly influenced by the 5′ untranslated region of eRF1 mRNA and the start codon of the upstream open reading frame. According to the transcription start database, eRF1 mRNA is characterized by high heterogeneity of the transcription start and a variable 5' untranslated region in length. In addition, the start codon of the CDS in eRF1 mRNA is located within the known translational regulator of short 5' untranslated regions (TISU), which also stimulates mRNA transcription of genes with high transcription start heterogeneity. We hypothesize that regulation of human eRF1 synthesis occurs at both the transcriptional and translational levels. At the transcription level, the length of the eRF1 5' untranslated region and the number of the upstream open reading frames in it are regulated. This regulation in turn, regulates the production of eRF1 at the translation level.

TMK4-mediated FIP37 phosphorylation regulates auxin-triggered N6-methyladenosine modification of auxin biosynthetic genes in Arabidopsis

The dynamics of N6-methyladenosine (m6A) mRNA modification are tightly controlled by the m6A methyltransferase complex and demethylases. Here, we find that auxin treatment alters m6A modification on auxin-responsive genes. Mechanically, TRANSMEMBRANE KINASE 4 (TMK4), a component of the auxin signaling pathway, interacts with and phosphorylates FKBP12-INTERACTING PROTEIN 37 (FIP37), a core component of the m6A methyltransferase complex, in an auxin-dependent manner. Phosphorylation of FIP37 enhances its interaction with RNA, thereby increasing m6A modification on its target genes, such as NITRILASE 1 (NIT1), a gene involved in indole-3-acetic acid (IAA) biosynthesis. 1-Naphthalacetic acid (NAA) treatment accelerates the mRNA decay of NIT1, in a TMK4- and FIP37-dependent manner, which leads to inhibition of auxin biosynthesis. Our findings identify a regulatory mechanism by which auxin modulates m6A modification through the phosphorylation of FIP37, ultimately affecting mRNA stability and auxin biosynthesis in plants.

A homozygous nonsense variant in the alternatively spliced VLDLR exon 4 causes a neurodevelopmental disorder without features of VLDLR cerebellar hypoplasia.

VLDLR cerebellar hypoplasia is characterized by intellectual disability, non-progressive cerebellar ataxia, and seizures. The characteristic MRI findings include hypoplasia of the inferior portion of the cerebellar vermis and hemispheres, simplified cortical gyration, and a small brain stem. Biallelic VLDLR pathogenic variants cause loss-of-function of the encoded very low-density lipoprotein receptor. VLDLR exons 4 and 16 are alternatively spliced, resulting in the expression of four transcript variants, including two exon 4-lacking mRNAs expressed in the human brain. Previously reported VLDLR pathogenic variants affect all four transcript variants. Here we report on two sisters with facial dysmorphism, microcephaly, intellectual disability, and normal brain imaging. Exome sequencing in one patient identified the homozygous VLDLR nonsense variant c.376C>T; p.(Gln126*) in exon 4; her similarly affected sister also carried the homozygous variant and parents were heterozygous carriers. VLDLR transcript analysis identified mRNAs with and without exon 4 in patient fibroblasts, while exon 4-containing VLDLR mRNAs were predominantly detected in control fibroblasts. We found significantly reduced VLDLR mRNA levels in patient compared to control cells, likely caused by nonsense-mediated mRNA decay of exon 4-containing VLDLR transcripts. Expression of neuronal VLDLR isoforms produced from exon 4-lacking transcripts may have protected both patients from developing the cerebellar hypoplasia phenotype.

STAU1-mediated CNBP mRNA degradation by LINC00665 alters stem cell characteristics in ovarian cancer

BackgroundTo investigate the role of lncRNA LINC00665 in modulating ovarian cancer stemness and its influence on treatment resistance and cancer development.MethodsWe isolated ovarian cancer stem cells (OCSCs) from the COC1 cell line using a combination of chemotherapeutic agents and growth factors, and verified their stemness through western blotting and immunofluorescence for stem cell markers. Employing bioinformatics, we identified lncRNAs associated with ovarian cancer, with a focus on LINC00665 and its interaction with the CNBP mRNA. In situ hybridization, immunohistochemistry, and qPCR were utilized to examine their expression and localization, alongside functional assays to determine the effects of LINC00665 on CNBP.ResultsLINC00665 employs its Alu elements to interact with the 3’-UTR of CNBP mRNA, targeting it for degradation. This molecular crosstalk enhances stemness by promoting the STAU1-mediated decay of CNBP mRNA, thereby modulating the Wnt and Notch signaling cascades that are pivotal for maintaining CSC characteristics and driving tumor progression. These mechanistic insights were corroborated by a series of in vitro assays and validated in vivo using tumor xenograft models. Furthermore, we established a positive correlation between elevated CNBP levels and increased disease-free survival in patients with ovarian cancer, underscoring the prognostic value of CNBP in this context.ConclusionslncRNA LINC00665 enhances stemness in ovarian cancer by mediating the degradation of CNBP mRNA, thereby identifying LINC00665 as a potential therapeutic target to counteract drug resistance and tumor recurrence associated with CSCs.

Epilepsy due to potential loss of ATP6V1B2 function with mechanistic insight by a Drosophila Vha55 model.

ATP6V1B2 encodes the subunit of the vacuolar H+-ATPase, which is an enzyme responsible for the acidification of intracellular organelles and essential for cell signaling and neurotransmitter release. The aim of the study is to identify the correlation between ATP6V1B2 and epilepsy. Trio-exome sequencing was performed. Reverse Transcription-PCR and Quantitative real-time PCR analyses were carried out to determine whether this variant leads to nonsense-mediated mRNA decay (NMD). Drosophila models with knocked-down homologous genes of ATP6V1B2 were generated to study the causal relationship between the ATP6V1B2 and the phenotype of epilepsy. We described a 5-year-old male with a novel variant c.1163delT(p.Tyr389IlefsTer13) in ATP6V1B2, who presented with epilepsy. The expression level of the premature termination codon (PTC) transcript was normal in the patient, which indicated that NMD evasion existed in the PTC transcript. We generated an animal model using Drosophila to study the knock down effects of Vha55, which is the ATP6V1B2 ortholog in fly. The Vha55 knockdown flies show seizure-like behaviors and climbing defects. This study expands the variation spectrum of the ATP6V1B2 gene. Cross-species animal model demonstrates the causal relationship between ATP6V1B2 defect and epilepsy, and shed new insights into the disease mechanism caused by ATP6V1B2 LOF variants.

Decoding RNA Metabolism by RNA-linked CRISPR Screening in Human Cells.

RNAs undergo a complex choreography of metabolic processes in human cells that are regulated by thousands of RNA-associated proteins. While the effects of individual RNA-associated proteins on RNA metabolism have been extensively characterized, the full complement of regulators for most RNA metabolic events remain unknown. Here we present a massively parallel RNA-linked CRISPR (ReLiC) screening approach to measure the responses of diverse RNA metabolic events to knockout of 2,092 human genes encoding all known RNA-associated proteins. ReLiC screens highlight modular interactions between gene networks regulating splicing, translation, and decay of mRNAs. When combined with biochemical fractionation of polysomes, ReLiC reveals striking pathway-specific coupling between growth fitness and mRNA translation. Perturbing different components of the translation and proteostasis machineries have distinct effects on ribosome occupancy, while perturbing mRNA transcription leaves ribosome occupancy largely intact. Isoform-selective ReLiC screens capture differential regulation of intron retention and exon skipping by SF3b complex subunits. Chemogenomic screens using ReLiC decipher translational regulators upstream of mRNA decay and uncover a role for the ribosome collision sensor GCN1 during treatment with the anti-leukemic drug homoharringtonine. Our work demonstrates ReLiC as a versatile platform for discovering and dissecting regulatory principles of human RNA metabolism.

Poised PABP–RNA hubs implement signal-dependent mRNA decay in development

Signaling pathways drive cell fate transitions largely by changing gene expression. However, the mechanisms for rapid and selective transcriptome rewiring in response to signaling cues remain elusive. Here we use deep learning to deconvolve both the sequence determinants and the trans-acting regulators that trigger extracellular signal-regulated kinase (ERK)–mitogen-activated protein kinase kinase (MEK)-induced decay of the naive pluripotency mRNAs. Timing of decay is coupled to embryo implantation through ERK–MEK phosphorylation of LIN28A, which repositions pLIN28A to the highly A+U-rich 3′ untranslated region (3′UTR) termini of naive pluripotency mRNAs. Interestingly, these A+U-rich 3′UTR termini serve as poly(A)-binding protein (PABP)-binding hubs, poised for signal-induced convergence with LIN28A. The multivalency of AUU motifs determines the efficacy of pLIN28A–PABP convergence, which enhances PABP 3′UTR binding, decreases the protection of poly(A) tails and activates mRNA decay to enable progression toward primed pluripotency. Thus, the signal-induced convergence of LIN28A with PABP–RNA hubs drives the rapid selection of naive mRNAs for decay, enabling the transcriptome remodeling that ensures swift developmental progression.

DNE1 scissorhands: How the power of omics sheds light on the control of mRNA decay.

DNE1 scissorhands: How the power of omics sheds light on the control of mRNA decay.

The lethal homozygous variant in the ATP1A2 gene is associated with FARIMPD syndrome phenotypes in newborns.

FARIMPD (Fetal akinesia, respiratory insufficiency, microcephaly, polymicrogyria, and dysmorphic facies) syndrome is a severe condition caused by ATP1A2 gene variants. The syndrome's novelty and rarity have limited its clinical and molecular knowledge. This research tries to provide new insight by investigating the cause of the early deaths due to FARIMPD syndrome in a particular family and reviewing previous studies. DNA and RNA were extracted from the blood samples of newborns and their parents, followed by whole exome sequencing and segregation analysis. A pathogenic homozygous nonsense variant (c.1234C > T: p.Arg412*) in the ATP1A2 gene was found in newborns. This variant is reported as homozygous for the first time. The migraine symptoms were the result of the heterozygous state of this particular variant, which supported the dominant inheritance pattern of this disease. Real-time PCR was used to analyze ATP1A2 gene expression in the newborns compared to parents and control subjects. The expression analysis also showed significant mRNA degradation in the newborns compared to heterozygous and healthy individuals, due to Nonsense-mediated mRNA Decay phenomena. Our study describes an ATP1A2 nonsense variant (c.1234C > T) that appears compatible with infant survival in the heterozygous and compound heterozygous states but is lethal in the homozygous state.

A nonsense variant in KRT31 is associated with autosomal-dominant monilethrix.

Monilethrix is a rare hereditary hair disorder that is characterised by a beaded hair shaft structure and increased hair fragility. Patients may also present with keratosis pilaris and nail changes. Research has identified three genes for autosomal-dominant monilethrix (KRT81, KRT83, and KRT86), and one gene for the autosomal-recessive form (DSG4). To investigate the genetic basis of autosomal-dominant monilethrix in families with no pathogenic variants in any of the known monilethrix genes, and to understand the mechanistic basis of variant pathogenicity using a cellular model. Nine affected individuals from four unrelated families were included in this study. A clinical diagnosis of monilethrix was assigned based on clinical examination and/or trichoscopy. Exome sequencing (ES) was performed in six individuals to identify pathogenic variants, and Sanger sequencing was used for co-segregation and haplotype analyses. Cell culture experiments (immunoblotting, immunofluorescence, and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analyses) were used to confirm variant pathogenicity, to determine expression and subcellular localisation of proteins, and to identify a possible nonsense-mediated mRNA decay. In six affected individuals with clinically suggested monilethrix, ES led to the identification of the nonsense variant c.1081G>T; p.(Glu361*) in KRT31, which was subsequently identified in other affected members of these families by Sanger sequencing. This variant led to the abolition of both the last three amino acids of the 2B subdomain and the complete C-terminal tail domain of keratin 31. Immunoblotting demonstrated that when co-expressed with its binding partner keratin 85, the truncated keratin 31 was still expressed, albeit less abundantly than the wild type protein. Immunofluorescence revealed that p.(Glu361*) keratin 31 had an altered cytoskeletal localisation and formed vesicular-like structures in the cell cytoplasm near the cell membrane. RT-qPCR analysis did not generate evidence for a nonsense mediated decay of the mutant transcript. This study is the first to identify pathogenic variants in KRT31 as a cause of autosomal-dominant monilethrix. This highlights the importance of hair keratin proteins in hair biology, and will increase the molecular diagnostic yield for rare ectodermal phenotypes of hair and nail tissues.

The missing link: ARID1B non-truncating variants causing Coffin-Siris syndrome due to protein aggregation

ARID1B is the most frequently mutated gene in Coffin-Siris syndrome (CSS). To date, the vast majority of causative variants reported in ARID1B are truncating, leading to nonsense-mediated mRNA decay. In the absence of experimental data, only few ARID1B amino acid substitutions have been classified as pathogenic, mainly based on clinical data and their de novo occurrence, while most others are currently interpreted as variants of unknown significance. The present study substantiates the pathogenesis of ARID1B non-truncating/NMD-escaping variants located in the SMARCA4-interacting EHD2 and DNA-binding ARID domains. Overexpression assays in cell lines revealed that the majority of EHD2 variants lead to protein misfolding and formation of cytoplasmic aggresomes surrounded by vimentin cage-like structures and co-localizing with the microtubule organisation center. ARID domain variants exhibited not only aggresomes, but also nuclear aggregates, demonstrating robust pathological effects. Protein levels were not compromised, as shown by quantitative western blot analysis. In silico structural analysis predicted the exposure of amylogenic segments in both domains due to the nearby variants, likely causing this aggregation. Genome-wide transcriptome and methylation analysis in affected individuals revealed expression and methylome patterns consistent with those of the pathogenic haploinsufficiency ARID1B alterations in CSS cases. These results further support pathogenicity and indicate two approaches for disambiguation of such variants in everyday practice. The few affected individuals harbouring EHD2 non-truncating variants described to date exhibit mild CSS clinical traits. In summary, this study paves the way for the re-evaluation of previously unclear ARID1B non-truncating variants and opens a new era in CSS genetic diagnosis.

Ccr4-not ubiquitin ligase signaling regulates ribosomal protein homeostasis and inhibits 40S ribosomal autophagy

The Ccr4-Not complex contains the poorly understood Not4 ubiquitin ligase that functions in transcription, mRNA decay, translation, proteostasis, and endolysosomal nutrient signaling. To gain further insight into the in vivo functions of the ligase, we performed quantitative proteomics in Saccharomyces cerevisiae using yeast cells lacking Not4, or cells overexpressing wild-type Not4 or an inactive Not4 mutant. Herein, we provide evidence that balanced Not4 activity maintains ribosomal protein (RP) homeostasis independent of changes to RP mRNA or known Not4 ribosomal substrates. Intriguingly, we also find that Not4 loss activates 40S ribosomal autophagy independently of canonical Atg7-dependent macroautophagy, indicating that microautophagy is responsible. We previously demonstrated that Ccr4-Not stimulates target of rapamycin complex 1 (TORC1) signaling, which activates RP expression and inhibits autophagy, by maintaining vacuole V-ATPase H+ pump activity. Importantly, combining Not4 deficient cells with a mutant that blocks vacuole H+ export fully restores RP expression and increases 40S RP autophagy efficiency. In contrast, restoring TORC1 activity alone fails to rescue either process, indicating that Not4 loss disrupts additional endolysosomal functions that regulate RP expression and 40S autophagy. Analysis of the Not4 regulated proteome reveals increases in endolysosomal and autophagy-related factors that functionally interact with Not4 to control RP expression and affect 40S autophagy. Collectively, our data indicate that balanced Ccr4-Not ubiquitin ligase signaling maintains RP homeostasis and inhibits 40S autophagy via the ligase’s emerging role as an endolysosomal regulator.

Destabilization of mRNAs enhances competence to initiate meiosis in mouse spermatogenic cells.

The specialized cell cycle of meiosis transforms diploid germ cells into haploid gametes. In mammals, diploid spermatogenic cells acquire the competence to initiate meiosis in response to retinoic acid. Previous mouse studies revealed that MEIOC interacts with RNA-binding proteins YTHDC2 and RBM46 to repress mitotic genes and to promote robust meiotic gene expression in spermatogenic cells that have initiated meiosis. Here, we have used the enhanced resolution of scRNA-seq and bulk RNA-seq of developmentally synchronized spermatogenesis to define how MEIOC molecularly supports early meiosis in spermatogenic cells. We demonstrate that MEIOC mediates transcriptomic changes before meiotic initiation, earlier than previously appreciated. MEIOC, acting with YTHDC2 and RBM46, destabilizes its mRNA targets, including the transcriptional repressors E2f6 and Mga, in mitotic spermatogonia. MEIOC thereby derepresses E2F6- and MGA-repressed genes, including Meiosin and other meiosis-associated genes. This confers on spermatogenic cells the molecular competence to, in response to retinoic acid, fully activate the transcriptional regulator STRA8-MEIOSIN, which is required for the meiotic G1/S phase transition and for meiotic gene expression. We conclude that, in mice, mRNA decay mediated by MEIOC-YTHDC2-RBM46 enhances the competence of spermatogenic cells to initiate meiosis.