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.

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.

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.

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.

FUS regulates genes coding for RNA-binding proteins in neurons by binding to their highly conserved introns.

Dominant mutations and mislocalization or aggregation of Fused in Sarcoma (FUS), an RNA-binding protein (RBP), cause neuronal degeneration in Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD), two incurable neurological diseases. However, the function of FUS in neurons is not well understood. To uncover the impact of FUS in the neuronal transcriptome, we used high-throughput sequencing of immunoprecipitated and cross-linked RNA (HITS-CLIP) of FUS in human brains and mouse neurons differentiated from embryonic stem cells, coupled with RNA-seq and FUS knockdowns. We report conserved neuronal RNA targets and networks that are regulated by FUS. We find that FUS regulates splicing of genes coding for RBPs by binding to their highly conserved introns. Our findings have important implications for understanding the impact of FUS in neurodegenerative diseases and suggest that perturbations of FUS can impact the neuronal transcriptome via perturbations of RBP transcripts.

Editing of Epstein-Barr virus-encoded BART6 microRNAs controls their dicer targeting and consequently affects viral latency.

Certain primary transcripts of miRNA (pri-microRNAs) undergo RNA editing that converts adenosine to inosine. The Epstein-Barr virus (EBV) genome encodes multiple microRNA genes of its own. Here we report that primary transcripts of ebv-miR-BART6 (pri-miR-BART6) are edited in latently EBV-infected cells. Editing of wild-type pri-miR-BART6 RNAs dramatically reduced loading of miR-BART6-5p RNAs onto the microRNA-induced silencing complex. Editing of a mutation-containing pri-miR-BART6 found in Daudi Burkitt lymphoma and nasopharyngeal carcinoma C666-1 cell lines suppressed processing of miR-BART6 RNAs. Most importantly, miR-BART6-5p RNAs silence Dicer through multiple target sites located in the 3'-UTR of Dicer mRNA. The significance of miR-BART6 was further investigated in cells in various stages of latency. We found that miR-BART6-5p RNAs suppress the EBNA2 viral oncogene required for transition from immunologically less responsive type I and type II latency to the more immunoreactive type III latency as well as Zta and Rta viral proteins essential for lytic replication, revealing the regulatory function of miR-BART6 in EBV infection and latency. Mutation and A-to-I editing appear to be adaptive mechanisms that antagonize miR-BART6 activities.

The DIANA-mirExTra web server: from gene expression data to microRNA function.

BACKGROUND: High-throughput gene expression experiments are widely used to identify the role of genes involved in biological conditions of interest. MicroRNAs (miRNA) are regulatory molecules that have been functionally associated with several developmental programs and their deregulation with diverse diseases including cancer. METHODOLOGY/PRINCIPAL FINDINGS: Although miRNA expression levels may not be routinely measured in high-throughput experiments, a possible involvement of miRNAs in the deregulation of gene expression can be computationally predicted and quantified through analysis of overrepresented motifs in the deregulated genes 3' untranslated region (3'UTR) sequences. Here, we introduce a user-friendly web-server, DIANA-mirExTra (www.microrna.gr/mirextra) that allows the comparison of frequencies of miRNA associated motifs between sets of genes that can lead to the identification of miRNAs responsible for the deregulation of large numbers of genes. To this end, we have investigated different approaches and measures, and have practically implemented them on experimental data. CONCLUSIONS/SIGNIFICANCE: On several datasets of miRNA overexpression and repression experiments, our proposed approaches have successfully identified the deregulated miRNA. Beyond the prediction of miRNAs responsible for the deregulation of transcripts, the web-server provides extensive links to DIANA-mirPath, a functional analysis tool incorporating miRNA targets in biological pathways. Additionally, in case information about miRNA expression changes is provided, the results can be filtered to display the analysis for miRNAs of interest only.