Post-transcriptional Regulation of De Novo Lipogenesis by mTORC1-S6K1-SRPK2 Signaling.

mTORC1 is a signal integrator and master regulator of cellular anabolic processes linked to cell growth and survival. Here, we demonstrate that mTORC1 promotes lipid biogenesis via SRPK2, a key regulator of RNA-binding SR proteins. mTORC1-activated S6K1 phosphorylates SRPK2 at Ser494, which primes Ser497 phosphorylation by CK1. These phosphorylation events promote SRPK2 nuclear translocation and phosphorylation of SR proteins. Genome-wide transcriptome analysis reveals that lipid biosynthetic enzymes are among the downstream targets of mTORC1-SRPK2 signaling. Mechanistically, SRPK2 promotes SR protein binding to U1-70K to induce splicing of lipogenic pre-mRNAs. Inhibition of this signaling pathway leads to intron retention of lipogenic genes, which triggers nonsense-mediated mRNA decay. Genetic or pharmacological inhibition of SRPK2 blunts de novo lipid synthesis, thereby suppressing cell growth. These results thus reveal a novel role of mTORC1-SRPK2 signaling in post-transcriptional regulation of lipid metabolism and demonstrate that SRPK2 is a potential therapeutic target for mTORC1-driven metabolic disorders.

Targeted pseudouridylation: An approach for suppressing nonsense mutations in disease genes.

Nonsense mutations create premature termination codons (PTCs), activating the nonsense-mediated mRNA decay (NMD) pathway to degrade most PTC-containing mRNAs. The undegraded mRNA is translated, but translation terminates at the PTC, leading to no production of the full-length protein. This work presents targeted PTC pseudouridylation, an approach for nonsense suppression in human cells. Specifically, an artificial box H/ACA guide RNA designed to target the mRNA PTC can suppress both NMD and premature translation termination in various sequence contexts. Targeted pseudouridylation exhibits a level of suppression comparable with that of aminoglycoside antibiotic treatments. When targeted pseudouridylation is combined with antibiotic treatment, a much higher level of suppression is observed. Transfection of a disease model cell line (carrying a chromosomal PTC) with a designer guide RNA gene targeting the PTC also leads to nonsense suppression. Thus, targeted pseudouridylation is an RNA-directed gene-specific approach that suppresses NMD and concurrently promotes PTC readthrough.

PTBP1-activated co-transcriptional splicing controls epigenetic status of pluripotent stem cells.

Many spliceosomal introns are excised from nascent transcripts emerging from RNA polymerase II (RNA Pol II). The extent of cell-type-specific regulation and possible functions of such co-transcriptional events remain poorly understood. We examined the role of the RNA-binding protein PTBP1 in this process using an acute depletion approach followed by the analysis of chromatin- and RNA Pol II-associated transcripts. We show that PTBP1 activates the co-transcriptional excision of hundreds of introns, a surprising effect given that this protein is known to promote intron retention. Importantly, some co-transcriptionally activated introns fail to complete their splicing without PTBP1. In a striking example, retention of a PTBP1-dependent intron triggers nonsense-mediated decay of transcripts encoding DNA methyltransferase DNMT3B. We provide evidence that this regulation facilitates the natural decline in DNMT3B levels in developing neurons and protects differentiation-specific genes from ectopic methylation. Thus, PTBP1-activated co-transcriptional splicing is a widespread phenomenon mediating epigenetic control of cellular identity.

CRISPR-free, programmable RNA pseudouridylation to suppress premature termination codons.

Nonsense mutations, accounting for >20% of disease-associated mutations, lead to premature translation termination. Replacing uridine with pseudouridine in stop codons suppresses translation termination, which could be harnessed to mediate readthrough of premature termination codons (PTCs). Here, we present RESTART, a programmable RNA base editor, to revert PTC-induced translation termination in mammalian cells. RESTART utilizes an engineered guide snoRNA (gsnoRNA) and the endogenous H/ACA box snoRNP machinery to achieve precise pseudouridylation. We also identified and optimized gsnoRNA scaffolds to increase the editing efficiency. Unexpectedly, we found that a minor isoform of pseudouridine synthase DKC1, lacking a C-terminal nuclear localization signal, greatly improved the PTC-readthrough efficiency. Although RESTART induced restricted off-target pseudouridylation, they did not change the coding information nor the expression level of off-targets. Finally, RESTART enables robust pseudouridylation in primary cells and achieves functional PTC readthrough in disease-relevant contexts. Collectively, RESTART is a promising RNA-editing tool for research and therapeutics.

DNA damage promotes HLA class I presentation by stimulating a pioneer round of translation-associated antigen production.

Antigen presentation by the human leukocyte antigen (HLA) on the cell surface is critical for the transduction of the immune signal toward cytotoxic T lymphocytes. DNA damage upregulates HLA class I presentation; however, the mechanism is unclear. Here, we show that DNA-damage-induced HLA (di-HLA) presentation requires an immunoproteasome, PSMB8/9/10, and antigen-transporter, TAP1/2, demonstrating that antigen production is essential. Furthermore, we show that di-HLA presentation requires ATR, AKT, mTORC1, and p70-S6K signaling. Notably, the depletion of CBP20, a factor initiating the pioneer round of translation (PRT) that precedes nonsense-mediated mRNA decay (NMD), abolishes di-HLA presentation, suggesting that di-antigen production requires PRT. RNA-seq analysis demonstrates that DNA damage reduces NMD transcripts in an ATR-dependent manner, consistent with the requirement for ATR in the initiation of PRT/NMD. Finally, bioinformatics analysis identifies that PRT-derived 9-mer peptides bind to HLA and are potentially immunogenic. Therefore, DNA damage signaling produces immunogenic antigens by utilizing the machinery of PRT/NMD.

AKT constitutes a signal-promoted alternative exon-junction complex that regulates nonsense-mediated mRNA decay.

Despite a long appreciation for the role of nonsense-mediated mRNA decay (NMD) in destroying faulty, disease-causing mRNAs and maintaining normal, physiologic mRNA abundance, additional effectors that regulate NMD activity in mammalian cells continue to be identified. Here, we describe a haploid-cell genetic screen for NMD effectors that has unexpectedly identified 13 proteins constituting the AKT signaling pathway. We show that AKT supersedes UPF2 in exon-junction complexes (EJCs) that are devoid of RNPS1 but contain CASC3, defining an unanticipated insulin-stimulated EJC. Without altering UPF1 RNA binding or ATPase activity, AKT-mediated phosphorylation of the UPF1 CH domain at T151 augments UPF1 helicase activity, which is critical for NMD and also decreases the dependence of helicase activity on ATP. We demonstrate that upregulation of AKT signaling contributes to the hyperactivation of NMD that typifies Fragile X syndrome, as exemplified using FMR1-KO neural stem cells derived from induced pluripotent stem cells.

NMD is required for timely cell fate transitions by fine-tuning gene expression and regulating translation.

Cell fate transitions depend on balanced rewiring of transcription and translation programs to mediate ordered developmental progression. Components of the nonsense-mediated mRNA decay (NMD) pathway have been implicated in regulating embryonic stem cell (ESC) differentiation, but the exact mechanism is unclear. Here we show that NMD controls expression levels of the translation initiation factor Eif4a2 and its premature termination codon-encoding isoform (Eif4a2PTC ). NMD deficiency leads to translation of the truncated eIF4A2PTC protein. eIF4A2PTC elicits increased mTORC1 activity and translation rates and causes differentiation delays. This establishes a previously unknown feedback loop between NMD and translation initiation. Furthermore, our results show a clear hierarchy in the severity of target deregulation and differentiation phenotypes between NMD effector KOs (Smg5 KO > Smg6 KO > Smg7 KO), which highlights heterodimer-independent functions for SMG5 and SMG7. Together, our findings expose an intricate link between mRNA homeostasis and mTORC1 activity that must be maintained for normal dynamics of cell state transitions.

Identification of a localized nonsense-mediated decay pathway at the endoplasmic reticulum.

Nonsense-mediated decay (NMD) is a translation-dependent RNA quality control mechanism that occurs in the cytoplasm. However, it is unknown how NMD regulates the stability of RNAs translated at the endoplasmic reticulum (ER). Here, we identify a localized NMD pathway dedicated to ER-translated mRNAs. We previously identified NBAS, a component of the Syntaxin 18 complex involved in Golgi-to-ER trafficking, as a novel NMD factor. Furthermore, we show that NBAS fulfills an independent function in NMD. This ER-NMD pathway requires the interaction of NBAS with the core NMD factor UPF1, which is partially localized at the ER in the proximity of the translocon. NBAS and UPF1 coregulate the stability of ER-associated transcripts, in particular those associated with the cellular stress response. We propose a model where NBAS recruits UPF1 to the membrane of the ER and activates an ER-dedicated NMD pathway, thus providing an ER-protective function by ensuring quality control of ER-translated mRNAs.

Recurrent SRSF2 mutations in MDS affect both splicing and NMD.

Oncogenic mutations in the RNA splicing factors SRSF2, SF3B1, and U2AF1 are the most frequent class of mutations in myelodysplastic syndromes and are also common in clonal hematopoiesis, acute myeloid leukemia, chronic lymphocytic leukemia, and a variety of solid tumors. They cause genome-wide splicing alterations that affect important regulators of hematopoiesis. Several mRNA isoforms promoted by the various splicing factor mutants comprise a premature termination codon (PTC) and are therefore potential targets of nonsense-mediated mRNA decay (NMD). In light of the mechanistic relationship between splicing and NMD, we sought evidence for a specific role of mutant SRSF2 in NMD. We show that SRSF2 Pro95 hot spot mutations elicit enhanced mRNA decay, which is dependent on sequence-specific RNA binding and splicing. SRSF2 mutants enhance the deposition of exon junction complexes (EJCs) downstream from the PTC through RNA-mediated molecular interactions. This architecture then favors the association of key NMD factors to elicit mRNA decay. Gene-specific blocking of EJC deposition by antisense oligonucleotides circumvents aberrant NMD promoted by mutant SRSF2, restoring the expression of PTC-containing transcript. Our study uncovered critical effects of SRSF2 mutants in hematologic malignancies, reflecting the regulation at multiple levels of RNA metabolism, from splicing to decay.

A transcription-independent mechanism determines rapid periodic fluctuations of BRCA1 expression.

BRCA1 expression is highly regulated to prevent genomic instability and tumorigenesis. Dysregulation of BRCA1 expression correlates closely with sporadic basal-like breast cancer and ovarian cancer. The most significant characteristic of BRCA1 regulation is periodic expression fluctuation throughout the cell cycle, which is important for the orderly progression of different DNA repair pathways throughout the various cell cycle phases and for further genomic stability. However, the underlying mechanism driving this phenomenon is poorly understood. Here, we demonstrate that RBM10-mediated RNA alternative splicing coupled to nonsense-mediated mRNA decay (AS-NMD), rather than transcription, determines the periodic fluctuations in G1/S-phase BRCA1 expression. Furthermore, AS-NMD broadly regulates the expression of period genes, such as DNA replication-related genes, in an uneconomical but more rapid manner. In summary, we identified an unexpected posttranscriptional mechanism distinct from canonical processes that mediates the rapid regulation of BRCA1 as well as other period gene expression during the G1/S-phase transition and provided insights into potential targets for cancer therapy.

Translation-coupled mRNA quality control mechanisms.

mRNA surveillance pathways are essential for accurate gene expression and to maintain translation homeostasis, ensuring the production of fully functional proteins. Future insights into mRNA quality control pathways will enable us to understand how cellular mRNA levels are controlled, how defective or unwanted mRNAs can be eliminated, and how dysregulation of these can contribute to human disease. Here we review translation-coupled mRNA quality control mechanisms, including the non-stop and no-go mRNA decay pathways, describing their mechanisms, shared trans-acting factors, and differences. We also describe advances in our understanding of the nonsense-mediated mRNA decay (NMD) pathway, highlighting recent mechanistic findings, the discovery of novel factors, as well as the role of NMD in cellular physiology and its impact on human disease.

Systematic analysis of variants escaping nonsense-mediated decay uncovers candidate Mendelian diseases.

Protein-truncating variants (PTVs) near the 3' end of genes may escape nonsense-mediated decay (NMD). PTVs in the NMD-escape region (PTVescs) can cause Mendelian disease but are difficult to interpret given their varying impact on protein function. Previously, PTVesc burden was assessed in an epilepsy cohort, but no large-scale analysis has systematically evaluated these variants in rare disease. We performed a retrospective analysis of 29,031 neurodevelopmental disorder (NDD) parent-offspring trios referred for clinical exome sequencing to identify PTVesc de novo mutations (DNMs). We identified 1,376 PTVesc DNMs and 133 genes that were significantly enriched (binomial p < 0.001). The PTVesc-enriched genes included those with PTVescs previously described to cause dominant Mendelian disease (e.g., SEMA6B, PPM1D, and DAGLA). We annotated ClinVar variants for PTVescs and identified 948 genes with at least one high-confidence pathogenic variant. Twenty-two known Mendelian PTVesc-enriched genes had no prior evidence of PTVesc-associated disease. We found 22 additional PTVesc-enriched genes that are not well established to be associated with Mendelian disease, several of which showed phenotypic similarity between individuals harboring PTVesc variants in the same gene. Four individuals with PTVesc mutations in RAB1A had similar phenotypes including NDD and spasticity. PTVesc mutations in IRF2BP1 were found in two individuals who each had severe immunodeficiency manifesting in NDD. Three individuals with PTVesc mutations in LDB1 all had NDD and multiple congenital anomalies. Using a large-scale, systematic analysis of DNMs, we extend the mutation spectrum for known Mendelian disease-associated genes and identify potentially novel disease-associated genes.

Single-Molecule Imaging Uncovers Rules Governing Nonsense-Mediated mRNA Decay.

Nonsense-mediated decay (NMD) is a surveillance system that degrades mRNAs containing a premature termination codon (PTC) and plays important roles in protein homeostasis and disease. The efficiency of NMD is variable, impacting the clinical outcome of genetic mutations. However, limited resolution of bulk analyses has hampered the study of NMD efficiency. Here, we develop an assay to visualize NMD of individual mRNA molecules in real time. We find that NMD occurs with equal probability during each round of translation of an mRNA molecule. However, this probability is variable and depends on the exon sequence downstream of the PTC, the PTC-to-intron distance, and the number of introns both upstream and downstream of the PTC. Additionally, a subpopulation of mRNAs can escape NMD, further contributing to variation in NMD efficiency. Our study uncovers real-time dynamics of NMD, reveals key mechanisms that influence NMD efficiency, and provides a powerful method to study NMD.

The broader sense of nonsense.

The term 'nonsense-mediated mRNA decay' (NMD) was initially coined to describe the translation-dependent degradation of mRNAs harboring premature termination codons (PTCs), but it is meanwhile known that NMD also targets many canonical mRNAs with numerous biological implications. The molecular mechanisms determining on which RNAs NMD ensues are only partially understood. Considering the broad range of NMD-sensitive RNAs and the variable degrees of their degradation, we highlight here the hallmarks of mammalian NMD and point out open questions. We review the links between NMD and disease by summarizing the role of NMD in cancer, neurodegeneration, and viral infections. Finally, we describe strategies to modulate NMD activity and specificity as potential therapeutic approaches for various diseases.

Noncoding snoRNA host genes are a distinct subclass of long noncoding RNAs.

Mammalian genomes are pervasively transcribed into different noncoding (nc)RNA classes, each one with its own hallmarks and exceptions. Some of them are nested into each other, such as host genes for small nucleolar RNAs (snoRNAs), which were long believed to simply act as molecular containers strictly facilitating snoRNA biogenesis. However, recent findings show that noncoding snoRNA host genes (ncSNHGs) display features different from those of 'regular' long ncRNAs (lncRNAs) and, more importantly, they can exert independent and unrelated functions to those of the encoded snoRNAs. Here, we review and summarize past and recent evidence that ncSNHGs form a defined subclass among the plethora of lncRNAs, and discuss future research that can further elucidate their biological relevance.

Mammalian UPF3A and UPF3B can activate nonsense-mediated mRNA decay independently of their exon junction complex binding.

Nonsense-mediated mRNA decay (NMD) is governed by the three conserved factors-UPF1, UPF2, and UPF3. While all three are required for NMD in yeast, UPF3B is dispensable for NMD in mammals, and its paralog UPF3A is suggested to only weakly activate or even repress NMD due to its weaker binding to the exon junction complex (EJC). Here, we characterize the UPF3A/B-dependence of NMD in human cell lines deleted of one or both UPF3 paralogs. We show that in human colorectal cancer HCT116 cells, NMD can operate in a UPF3B-dependent and -independent manner. While UPF3A is almost dispensable for NMD in wild-type cells, it strongly activates NMD in cells lacking UPF3B. Notably, NMD remains partially active in cells lacking both UPF3 paralogs. Complementation studies in these cells show that EJC-binding domain of UPF3 paralogs is dispensable for NMD. Instead, the conserved "mid" domain of UPF3 paralogs is consequential for their NMD activity. Altogether, our results demonstrate that the mammalian UPF3 proteins play a more active role in NMD than simply bridging the EJC and the UPF complex.

Human UPF3A and UPF3B enable fault-tolerant activation of nonsense-mediated mRNA decay.

The paralogous human proteins UPF3A and UPF3B are involved in recognizing mRNAs targeted by nonsense-mediated mRNA decay (NMD). UPF3B has been demonstrated to support NMD, presumably by bridging an exon junction complex (EJC) to the NMD factor UPF2. The role of UPF3A has been described either as a weak NMD activator or an NMD inhibitor. Here, we present a comprehensive functional analysis of UPF3A and UPF3B in human cells using combinatory experimental approaches. Overexpression or knockout of UPF3A as well as knockout of UPF3B did not substantially change global NMD activity. In contrast, the co-depletion of UPF3A and UPF3B resulted in a marked NMD inhibition and a transcriptome-wide upregulation of NMD substrates, demonstrating a functional redundancy between both NMD factors. In rescue experiments, UPF2 or EJC binding-deficient UPF3B largely retained NMD activity. However, combinations of different mutants, including deletion of the middle domain, showed additive or synergistic effects and therefore failed to maintain NMD. Collectively, UPF3A and UPF3B emerge as fault-tolerant, functionally redundant NMD activators in human cells.

An alternative UPF1 isoform drives conditional remodeling of nonsense-mediated mRNA decay.

The nonsense-mediated mRNA decay (NMD) pathway monitors translation termination in order to degrade transcripts with premature stop codons and regulate thousands of human genes. Here, we show that an alternative mammalian-specific isoform of the core NMD factor UPF1, termed UPF1LL , enables condition-dependent remodeling of NMD specificity. Previous studies indicate that the extension of a conserved regulatory loop in the UPF1LL helicase core confers a decreased propensity to dissociate from RNA upon ATP hydrolysis relative to UPF1SL , the major UPF1 isoform. Using biochemical and transcriptome-wide approaches, we find that UPF1LL can circumvent the protective RNA binding proteins PTBP1 and hnRNP L to preferentially bind and down-regulate transcripts with long 3'UTRs normally shielded from NMD. Unexpectedly, UPF1LL supports induction of NMD on new populations of substrate mRNAs in response to activation of the integrated stress response and impaired translation efficiency. Thus, while canonical NMD is abolished by moderate translational repression, UPF1LL activity is enhanced, offering the possibility to rapidly rewire NMD specificity in response to cellular stress.

Transcript-specific induction of stop codon readthrough using a CRISPR-dCas13 system.

Stop codon readthrough (SCR) is the process where translation continues beyond a stop codon on an mRNA. Here, we describe a strategy to enhance or induce SCR in a transcript-selective manner using a CRISPR-dCas13 system. Using specific guide RNAs, we target dCas13 to the region downstream of canonical stop codons of mammalian AGO1 and VEGFA mRNAs, known to exhibit natural SCR. Readthrough assays reveal enhanced SCR of these mRNAs (both exogenous and endogenous) caused by the dCas13-gRNA complexes. This effect is associated with ribosomal pausing, which has been reported for several SCR events. Our data show that CRISPR-dCas13 can also induce SCR across premature termination codons (PTCs) in the mRNAs of green fluorescent protein and TP53. We demonstrate the utility of this strategy in the induction of readthrough across the thalassemia-causing PTC in HBB mRNA and hereditary spherocytosis-causing PTC in SPTA1 mRNA. Thus, CRISPR-dCas13 can be programmed to enhance or induce SCR in a transcript-selective and stop codon-specific manner.

Coupled protein quality control during nonsense-mediated mRNA decay.

Translation of mRNAs containing premature termination codons (PTCs) results in truncated protein products with deleterious effects. Nonsense-mediated decay (NMD) is a surveillance pathway responsible for detecting PTC containing transcripts. Although the molecular mechanisms governing mRNA degradation have been extensively studied, the fate of the nascent protein product remains largely uncharacterized. Here, we use a fluorescent reporter system in mammalian cells to reveal a selective degradation pathway specifically targeting the protein product of an NMD mRNA. We show that this process is post-translational and dependent on the ubiquitin proteasome system. To systematically uncover factors involved in NMD-linked protein quality control, we conducted genome-wide flow cytometry-based screens. Our screens recovered known NMD factors but suggested that protein degradation did not depend on the canonical ribosome-quality control (RQC) pathway. A subsequent arrayed screen demonstrated that protein and mRNA branches of NMD rely on a shared recognition event. Our results establish the existence of a targeted pathway for nascent protein degradation from PTC containing mRNAs, and provide a reference for the field to identify and characterize required factors.