Mis-spliced transcripts generate de novo proteins in TDP-43-related ALS/FTD.

Functional loss of TDP-43, an RNA binding protein genetically and pathologically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), leads to the inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote the degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. Here, we show that mRNA transcripts harboring cryptic exons generated de novo proteins in TDP-43-depleted human iPSC-derived neurons in vitro, and de novo peptides were found in cerebrospinal fluid (CSF) samples from patients with ALS or FTD. Using coordinated transcriptomic and proteomic studies of TDP-43-depleted human iPSC-derived neurons, we identified 65 peptides that mapped to 12 cryptic exons. Cryptic exons identified in TDP-43-depleted human iPSC-derived neurons were predictive of cryptic exons expressed in postmortem brain tissue from patients with TDP-43 proteinopathy. These cryptic exons produced transcript variants that generated de novo proteins. We found that the inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Last, we showed that 18 de novo peptides across 13 genes were present in CSF samples from patients with ALS/FTD spectrum disorders. The demonstration of cryptic exon translation suggests new mechanisms for ALS/FTD pathophysiology downstream of TDP-43 dysfunction and may provide a potential strategy to assay TDP-43 function in patient CSF.

Full-length direct RNA sequencing uncovers stress-granule dependent RNA decay upon cellular stress.

Cells react to stress by triggering response pathways, leading to extensive alterations in the transcriptome to restore cellular homeostasis. The role of RNA metabolism in shaping the cellular response to stress is vital, yet the global changes in RNA stability under these conditions remain unclear. In this work, we employ direct RNA sequencing with nanopores, enhanced by 5' end adaptor ligation, to comprehensively interrogate the human transcriptome at single-molecule and nucleotide resolution. By developing a statistical framework to identify robust RNA length variations in nanopore data, we find that cellular stress induces prevalent 5' end RNA decay that is coupled to translation and ribosome occupancy. Unlike typical RNA decay models in normal conditions, we show that stress-induced RNA decay is dependent on XRN1 but does not depend on removal of the poly(A) tail. We observed that RNAs undergoing decay are predominantly enriched in the stress granule transcriptome. Inhibition of stress granule formation via genetic ablation of G3BP1 and G3BP2 fully rescues RNA length and suppresses stress-induced decay. Our findings reveal RNA decay as a key determinant of RNA metabolism upon cellular stress and dependent on stress-granule formation.

Deep learning and direct sequencing of labeled RNA captures transcriptome dynamics.

Quantification of the dynamics of RNA metabolism is essential for understanding gene regulation in health and disease. Existing methods rely on metabolic labeling of nascent RNAs and physical separation or inference of labeling through PCR-generated mutations, followed by short-read sequencing. However, these methods are limited in their ability to identify transient decay intermediates or co-analyze RNA decay with cis-regulatory elements of RNA stability such as poly(A) tail length and modification status, at single molecule resolution. Here we use 5-ethynyl uridine (5EU) to label nascent RNA followed by direct RNA sequencing with nanopores. We developed RNAkinet, a deep convolutional and recurrent neural network that processes the electrical signal produced by nanopore sequencing to identify 5EU-labeled nascent RNA molecules. RNAkinet demonstrates generalizability to distinct cell types and organisms and reproducibly quantifies RNA kinetic parameters allowing the combined interrogation of RNA metabolism and cis-acting RNA regulatory elements.

Senescence suppresses the integrated stress response and activates a stress-enhanced secretory phenotype.

Senescence is a state of indefinite cell cycle arrest associated with aging, cancer, and age-related diseases. Here, using label-based mass spectrometry, ribosome profiling and nanopore direct RNA sequencing, we explore the coordinated interaction of translational and transcriptional programs of human cellular senescence. We find that translational deregulation and a corresponding maladaptive integrated stress response (ISR) is a hallmark of senescence that desensitizes senescent cells to stress. We present evidence that senescent cells maintain high levels of eIF2α phosphorylation, typical of ISR activation, but translationally repress production of the stress response transcription factor 4 (ATF4) by ineffective bypass of the inhibitory upstream open reading frames. Surprisingly, ATF4 translation remains inhibited even after acute proteotoxic and amino acid starvation stressors, resulting in a highly diminished stress response. Furthermore, absent a response, stress augments the senescence secretory phenotype, thus intensifying a proinflammatory state that exacerbates disease. Our results reveal a novel mechanism that senescent cells exploit to evade an adaptive stress response and remain viable.

Ribonucleic Acid-Mediated Control of Protein Translation Under Stress.

Significance: The need of cells to constantly respond to endogenous and exogenous stress has necessitated the evolution of pathways to counter the deleterious effects of stress and to restore cellular homeostasis. The inability to activate a timely and adequate response can lead to disease and is a hallmark of aging. Besides protein-coding genes, cells contain a plethora of noncoding regulatory elements that allow cells to respond rapidly and efficiently to external stimuli by activating highly specific and tightly controlled mechanisms. Many of these programs converge on the regulation of translation, one of the most energy-consuming processes in cells. Recent Advances: The noncoding dimension of translational regulation includes short and long noncoding ribonucleic acids (ncRNAs), as well as messenger RNA features, such as the sequence and modification status of the 5' and 3' untranslated regions (UTRs), that do not change the amino acid sequence of the produced protein. Critical Issues: In this review, we discuss the regulatory role of the nonprotein-coding components of translation under stress, particularly oxidative stress. We conclude that the regulation of translation through ncRNAs, UTRs, and nucleotide modifications is emerging as a critical component of the stress response. Future Directions: Further areas of study using long-read sequencing technologies will be discussed. Antioxid. Redox Signal. 39, 374-389.

The MOV10 RNA helicase is a dosage-dependent host restriction factor for LINE1 retrotransposition in mice.

Transposable elements constitute nearly half of the mammalian genome and play important roles in genome evolution. While a multitude of both transcriptional and post-transcriptional mechanisms exist to silence transposable elements, control of transposition in vivo remains poorly understood. MOV10, an RNA helicase, is an inhibitor of mobilization of retrotransposons and retroviruses in cell culture assays. Here we report that MOV10 restricts LINE1 retrotransposition in mice. Although MOV10 is broadly expressed, its loss causes only incomplete penetrance of embryonic lethality, and the surviving MOV10-deficient mice are healthy and fertile. Biochemically, MOV10 forms a complex with UPF1, a key component of the nonsense-mediated mRNA decay pathway, and primarily binds to the 3' UTR of somatically expressed transcripts in testis. Consequently, loss of MOV10 results in an altered transcriptome in testis. Analyses using a LINE1 reporter transgene reveal that loss of MOV10 leads to increased LINE1 retrotransposition in somatic and reproductive tissues from both embryos and adult mice. Moreover, the degree of LINE1 retrotransposition inhibition is dependent on the Mov10 gene dosage. Furthermore, MOV10 deficiency reduces reproductive fitness over successive generations. Our findings demonstrate that MOV10 attenuates LINE1 retrotransposition in a dosage-dependent manner in mice.

Gene body DNA hydroxymethylation restricts the magnitude of transcriptional changes during aging.

DNA hydroxymethylation (5hmC) is the most abundant oxidative derivative of DNA methylation (5mC) and is typically enriched at enhancers and gene bodies of transcriptionally active and tissue-specific genes. Although aberrant genomic 5hmC has been implicated in many age-related diseases, the functional role of the modification in aging remains largely unknown. Here, we report that 5hmC is stably enriched in multiple aged organs. Using the liver and cerebellum as model organs, we show that 5hmC accumulates in gene bodies associated with tissue-specific function and thereby restricts the magnitude of gene expression changes during aging. Mechanistically, we found that 5hmC decreases binding affinity of splicing factors compared to unmodified cytosine and 5mC, and is correlated with age-related alternative splicing events, suggesting RNA splicing as a potential mediator of 5hmC's transcriptionally restrictive function. Furthermore, we show that various age-related contexts, such as prolonged quiescence and senescence, are partially responsible for driving the accumulation of 5hmC with age. We provide evidence that this age-related function is conserved in mouse and human tissues, and further show that the modification is altered by regimens known to modulate lifespan. Our findings reveal that 5hmC is a regulator of tissue-specific function and may play a role in regulating longevity.

Mis-spliced transcripts generate de novo proteins in TDP-43-related ALS/FTD.

Functional loss of TDP-43, an RNA-binding protein genetically and pathologically linked to ALS and FTD, leads to inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. However, the possibility of de novo protein synthesis from cryptic exon transcripts has not been explored. Here, we show that mRNA transcripts harboring cryptic exons generate de novo proteins both in TDP-43 deficient cellular models and in disease. Using coordinated transcriptomic and proteomic studies of TDP-43 depleted iPSC-derived neurons, we identified numerous peptides that mapped to cryptic exons. Cryptic exons identified in iPSC models were highly predictive of cryptic exons expressed in brains of patients with TDP-43 proteinopathy, including cryptic transcripts that generated de novo proteins. We discovered that inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Finally, we showed that these de novo peptides were present in CSF from patients with ALS. The demonstration of cryptic exon translation suggests new mechanisms for ALS pathophysiology downstream of TDP-43 dysfunction and may provide a strategy for novel biomarker development.

Biology of Stress Responses in Aging.

On April 28th, 2022, a group of scientific leaders gathered virtually to discuss molecular and cellular mechanisms of responses to stress. Conditions of acute, high-intensity stress are well documented to induce a series of adaptive responses that aim to promote survival until the stress has dissipated and then guide recovery. However, high-intensity or persistent stress that goes beyond the cell's compensatory capacity are countered with resilience strategies that are not completely understood. These adaptative strategies, which are an essential component of the study of aging biology, were the theme of the meeting. Specific topics discussed included mechanisms of proteostasis, such as the unfolded protein response (UPR) and the integrated stress response (ISR), as well as mitochondrial stress and lysosomal stress responses. Attention was also given to regulatory mechanisms and associated biological processes linked to age-related conditions, such as muscle loss and regeneration, cancer, senescence, sleep quality, and degenerative disease, with a general focus on the relevance of stress responses to frailty. We summarize the concepts and potential future directions that emerged from the discussion and highlight their relevance to the study of aging and age-related chronic diseases.

SamQL: a structured query language and filtering tool for the SAM/BAM file format.

BACKGROUND: The Sequence Alignment/Map Format Specification (SAM) is one of the most widely adopted file formats in bioinformatics and many researchers use it daily. Several tools, including most high-throughput sequencing read aligners, use it as their primary output and many more tools have been developed to process it. However, despite its flexibility, SAM encoded files can often be difficult to query and understand even for experienced bioinformaticians. As genomic data are rapidly growing, structured, and efficient queries on data that are encoded in SAM/BAM files are becoming increasingly important. Existing tools are very limited in their query capabilities or are not efficient. Critically, new tools that address these shortcomings, should not be able to support existing large datasets but should also do so without requiring massive data transformations and file infrastructure reorganizations. RESULTS: Here we introduce SamQL, an SQL-like query language for the SAM format with intuitive syntax that supports complex and efficient queries on top of SAM/BAM files and that can replace commonly used Bash one-liners employed by many bioinformaticians. SamQL has high expressive power with no upper limit on query size and when parallelized, outperforms other substantially less expressive software. CONCLUSIONS: SamQL is a complete query language that we envision as a step to a structured database engine for genomics. SamQL is written in Go, and is freely available as standalone program and as an open-source library under an MIT license, https://github.com/maragkakislab/samql/ .

TERA-Seq: true end-to-end sequencing of native RNA molecules for transcriptome characterization.

Direct sequencing of single, native RNA molecules through nanopores has a strong potential to transform research in all aspects of RNA biology and clinical diagnostics. The existing platform from Oxford Nanopore Technologies is unable to sequence the very 5' ends of RNAs and is limited to polyadenylated molecules. Here, we develop True End-to-end RNA Sequencing (TERA-Seq), a platform that addresses these limitations, permitting more thorough transcriptome characterization. TERA-Seq describes both poly- and non-polyadenylated RNA molecules and accurately identifies their native 5' and 3' ends by ligating uniquely designed adapters that are sequenced along with the transcript. We find that capped, full-length mRNAs in human cells show marked variation of poly(A) tail lengths at the single molecule level. We report prevalent capping downstream of canonical transcriptional start sites in otherwise fully spliced and polyadenylated molecules. We reveal RNA processing and decay at single molecule level and find that mRNAs decay cotranslationally, often from their 5' ends, while frequently retaining poly(A) tails. TERA-Seq will prove useful in many applications where true end-to-end direct sequencing of single, native RNA molecules and their isoforms is desirable.

Modulation of Aub-TDRD interactions elucidates piRNA amplification and germplasm formation.

Aub guided by piRNAs ensures genome integrity by cleaving retrotransposons, and genome propagation by trapping mRNAs to form the germplasm that instructs germ cell formation. Arginines at the N-terminus of Aub (Aub-NTRs) interact with Tudor and other Tudor domain-containing proteins (TDRDs). Aub-TDRD interactions suppress active retrotransposons via piRNA amplification and form germplasm via generation of Aub-Tudor ribonucleoproteins. Here, we show that Aub-NTRs are dispensable for primary piRNA biogenesis but essential for piRNA amplification and that their symmetric dimethylation is required for germplasm formation and germ cell specification but largely redundant for piRNA amplification.

Amyotrophic Lateral Sclerosis associated FUS mutation shortens mitochondria and induces neurotoxicity.

Amyotrophic Lateral Sclerosis (ALS) is a lethal neurodegenerative disorder that primarily affects motor neurons. Dominant mutations in the RNA binding protein Fused in Sarcoma (FUS) have been identified as causative factors of ALS. Mutation, R495X, results in a premature stop codon and induces an aggressive disease phenotype by a largely unknown process. Here, we employ CLIP-Seq, RNA-Seq and Ribo-Seq in cultured neurons expressing R495X or wild-type FUS to identify the mutation effects on the FUS targetome and on the neuronal transcriptome at the expression and translation level, simultaneously. We report that, unlike wild-type FUS that binds on precursor mRNAs (pre-mRNAs), R495X binds mature mRNAs in the cytoplasm. R495X has a moderate effect on target mRNA expression and its binding induces only modest expression changes. In contrast, we find that R495X controls the translation of genes that are associated with mitochondria function and results in significant reduction of mitochondrial size. Importantly, we show that introduction of the 4FL mutation that alters binding of R495X to RNA, partially abrogates R495X-induced effects on mRNA translation, mitochondrial size and neurotoxicity. Our findings uncover a novel RNA-mediated pathway of FUS R495X-induced neurotoxicity that affects mitochondria morphology and provide insight to previous studies associating mitochondria dysfunction to ALS.

Ribothrypsis, a novel process of canonical mRNA decay, mediates ribosome-phased mRNA endonucleolysis.

mRNAs transmit the genetic information that dictates protein production and are a nexus for numerous pathways that regulate gene expression. The prevailing view of canonical mRNA decay is that it is mediated by deadenylation and decapping followed by exonucleolysis from the 3' and 5' ends. By developing Akron-seq, a novel approach that captures the native 3' and 5' ends of capped and polyadenylated RNAs, respectively, we show that canonical human mRNAs are subject to repeated cotranslational and ribosome-phased endonucleolytic cuts at the exit site of the mRNA ribosome channel, in a process that we term ribothrypsis. We uncovered RNA G quadruplexes among likely ribothrypsis triggers and show that ribothrypsis is a conserved process. Strikingly, we found that mRNA fragments are abundant in living cells and thus have important implications for the interpretation of experiments, such as RNA-seq, that rely on the assumption that mRNAs exist largely as full-length molecules in vivo.

cCLIP-Seq: Retrieval of Chimeric Reads from HITS-CLIP (CLIP-Seq) Libraries.

HITS-CLIP (High-Throughput Sequencing after in vivo Crosslinking and Immunoprecipitation, CLIP-Seq) libraries contain fragments of the RNA sequences bound in vivo by an RNA binding protein (RBP). Such fragments, especially if they represent RNA duplexes bound in vivo by the RBP, can occasionally be ligated together to form chimeric CLIP tags. Chimeric CLIP tags from Argonaute CLIP libraries can provide the exact base pairing profiles of small RNAs with their target RNA sequences, thus solving a critical problem in the field of post-transcriptional regulation. We recently reported an analysis of chimeric reads from the Drosophila Piwi protein Aubergine, which revealed a novel mechanism for mRNA entrapment within germ RNP granules. We term this novel approach chimeric CLIP (cCLIP) and present here the main steps that a researcher can take after the acquisition of the deep sequencing data, for the identification of candidate chimeric reads in Piwi CLIP libraries. Extending the scope beyond small-RNA binding proteins, we believe that cCLIP can be utilized to elucidate the in vivo functions of RNA-binding proteins in general, and especially those that modulate RNA secondary structures. We, therefore, also describe aspects of the generalized chimeric read identification problem, which can find use in the analysis of the CLIP libraries of any RNA-binding protein.

Kc167, a widely used Drosophila cell line, contains an active primary piRNA pathway.

PIWI family proteins bind to small RNAs known as PIWI-interacting RNAs (piRNAs) and play essential roles in the germline by silencing transposons and by promoting germ cell specification and function. Here we report that the widely used Kc167 cell line, derived from Drosophila melanogaster embryos, expresses piRNAs that are loaded to Aub and Piwi. Kc167 piRNAs are produced by a canonical, primary piRNA biogenesis pathway, from phased processing of precursor transcripts by the Zuc endonuclease, Armi helicase, and dGasz mitochondrial scaffold protein. Kc167 piRNAs derive from cytoplasmic transcripts, notably tRNAs and mRNAs, and their abundance correlates with that of parent transcripts. The expression of Aub is robust in Kc167, that of Piwi is modest, while Ago3 is undetectable, explaining the lack of transposon-related piRNA amplification by the Aub-Ago3, ping-pong mechanism. We propose that the default state of the primary piRNA biogenesis machinery is random transcript sampling to allow generation of piRNAs from any transcript, including newly acquired retrotransposons. This state is unmasked in Kc167, likely because they do not express piRNA cluster transcripts in sufficient amounts and do not amplify transposon piRNAs. We use Kc167 to characterize an inactive isoform of Aub protein. Since most Kc167 piRNAs are genic, they can be mapped uniquely to the genome, facilitating computational analyses. Furthermore, because Kc167 is a widely used and well-characterized cell line that is easily amenable to experimental manipulations, we expect that it will serve as an excellent system to study piRNA biogenesis and piRNA-related factors.

Sequence-dependent but not sequence-specific piRNA adhesion traps mRNAs to the germ plasm.

The conserved Piwi family of proteins and piwi-interacting RNAs (piRNAs) have a central role in genomic stability, which is inextricably linked to germ-cell formation, by forming Piwi ribonucleoproteins (piRNPs) that silence transposable elements. In Drosophila melanogaster and other animals, primordial germ-cell specification in the developing embryo is driven by maternal messenger RNAs and proteins that assemble into specialized messenger ribonucleoproteins (mRNPs) localized in the germ (pole) plasm at the posterior of the oocyte. Maternal piRNPs, especially those loaded on the Piwi protein Aubergine (Aub), are transmitted to the germ plasm to initiate transposon silencing in the offspring germ line. The transport of mRNAs to the oocyte by midoogenesis is an active, microtubule-dependent process; mRNAs necessary for primordial germ-cell formation are enriched in the germ plasm at late oogenesis via a diffusion and entrapment mechanism, the molecular identity of which remains unknown. Aub is a central component of germ granule RNPs, which house mRNAs in the germ plasm, and interactions between Aub and Tudor are essential for the formation of germ granules. Here we show that Aub-loaded piRNAs use partial base-pairing characteristics of Argonaute RNPs to bind mRNAs randomly in Drosophila, acting as an adhesive trap that captures mRNAs in the germ plasm, in a Tudor-dependent manner. Notably, germ plasm mRNAs in drosophilids are generally longer and more abundant than other mRNAs, suggesting that they provide more target sites for piRNAs to promote their preferential tethering in germ granules. Thus, complexes containing Tudor, Aub piRNPs and mRNAs couple piRNA inheritance with germline specification. Our findings reveal an unexpected function for piRNP complexes in mRNA trapping that may be generally relevant to the function of animal germ granules.

CLIPSeqTools--a novel bioinformatics CLIP-seq analysis suite.

Immunoprecipitation of RNA binding proteins (RBPs) after in vivo crosslinking, coupled with sequencing of associated RNA footprints (HITS-CLIP, CLIP-seq), is a method of choice for the identification of RNA targets and binding sites for RBPs. Compared with RNA-seq, CLIP-seq analysis is widely diverse and depending on the RBPs that are analyzed, the approaches vary significantly, necessitating the development of flexible and efficient informatics tools. In this study, we present CLIPSeqTools, a novel, highly flexible computational suite that can perform analysis from raw sequencing data with minimal user input. It contains a wide array of tools to provide an in-depth view of CLIP-seq data sets. It supports extensive customization and promotes improvization, a critical virtue, since CLIP-seq analysis is rarely well defined a priori. To highlight CLIPSeqTools capabilities, we used the suite to analyze Ago-miRNA HITS-CLIP data sets that we prepared from human brains.

The RNA helicase MOV10L1 binds piRNA precursors to initiate piRNA processing.

Piwi-piRNA (Piwi-interacting RNA) ribonucleoproteins (piRNPs) enforce retrotransposon silencing, a function critical for preserving the genome integrity of germ cells. The molecular functions of most of the factors that have been genetically implicated in primary piRNA biogenesis are still elusive. Here we show that MOV10L1 exhibits 5'-to-3' directional RNA-unwinding activity in vitro and that a point mutation that abolishes this activity causes a failure in primary piRNA biogenesis in vivo. We demonstrate that MOV10L1 selectively binds piRNA precursor transcripts and is essential for the generation of intermediate piRNA processing fragments that are subsequently loaded to Piwi proteins. Multiple analyses suggest an intimate coupling of piRNA precursor processing with elements of local secondary structures such as G quadruplexes. Our results support a model in which MOV10L1 RNA helicase activity promotes unwinding and funneling of the single-stranded piRNA precursor transcripts to the endonuclease that catalyzes the first cleavage step of piRNA processing.