hnRNPM protects against the dsRNA-mediated interferon response by repressing LINE-associated cryptic splicing.

RNA splicing is pivotal in post-transcriptional gene regulation, yet the exponential expansion of intron length in humans poses a challenge for accurate splicing. Here, we identify hnRNPM as an essential RNA-binding protein that suppresses cryptic splicing through binding to deep introns, maintaining human transcriptome integrity. Long interspersed nuclear elements (LINEs) in introns harbor numerous pseudo splice sites. hnRNPM preferentially binds at intronic LINEs to repress pseudo splice site usage for cryptic splicing. Remarkably, cryptic exons can generate long dsRNAs through base-pairing of inverted ALU transposable elements interspersed among LINEs and consequently trigger an interferon response, a well-known antiviral defense mechanism. Significantly, hnRNPM-deficient tumors show upregulated interferon-associated pathways and elevated immune cell infiltration. These findings unveil hnRNPM as a guardian of transcriptome integrity by repressing cryptic splicing and suggest that targeting hnRNPM in tumors may be used to trigger an inflammatory immune response, thereby boosting cancer surveillance.

Identification of an ultra-rare Alu insertion in the CFTR gene: Pitfalls and challenges in genetic test interpretation.

Cystic fibrosis (CF) is a life-limiting genetic disorder characterized by defective chloride ion transport due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Early detection through newborn screening programs significantly improves outcomes for individuals with CF by enabling timely intervention. Here, we report the identification of an Alu element insertion within the exon 15 of CFTR gene, initially overlooked in standard next-generation sequencing analyses. However, using traditional molecular techniques, based on polymerase chain reaction and Sanger sequencing, allowed the identification of the Alu element and the reporting of a correct diagnosis. Our analysis, based on bioinformatics tools and molecular techniques, revealed that the Alu element insertion severely affects the gene expression, splicing patterns, and structure of CFTR protein. In conclusion, this study emphasizes the importance of how the integration of human expertise and modern technologies represents a pivotal step forward in genomic medicine, ensuring the delivery of precision healthcare to individuals affected by genetic diseases.

On the genetic basis of tail-loss evolution in humans and apes.

The loss of the tail is among the most notable anatomical changes to have occurred along the evolutionary lineage leading to humans and to the 'anthropomorphous apes'1-3, with a proposed role in contributing to human bipedalism4-6. Yet, the genetic mechanism that facilitated tail-loss evolution in hominoids remains unknown. Here we present evidence that an individual insertion of an Alu element in the genome of the hominoid ancestor may have contributed to tail-loss evolution. We demonstrate that this Alu element-inserted into an intron of the TBXT gene7-9-pairs with a neighbouring ancestral Alu element encoded in the reverse genomic orientation and leads to a hominoid-specific alternative splicing event. To study the effect of this splicing event, we generated multiple mouse models that express both full-length and exon-skipped isoforms of Tbxt, mimicking the expression pattern of its hominoid orthologue TBXT. Mice expressing both Tbxt isoforms exhibit a complete absence of the tail or a shortened tail depending on the relative abundance of Tbxt isoforms expressed at the embryonic tail bud. These results support the notion that the exon-skipped transcript is sufficient to induce a tail-loss phenotype. Moreover, mice expressing the exon-skipped Tbxt isoform develop neural tube defects, a condition that affects approximately 1 in 1,000 neonates in humans10. Thus, tail-loss evolution may have been associated with an adaptive cost of the potential for neural tube defects, which continue to affect human health today.

CircMTA1 promotes glioblastoma angiogenesis by encoding MTA1-134aa.

Glioblastoma multiforme (GBM) is the most malignant brain tumor with rapid angiogenesis. How to inhibit GBM angiogenesis is a key problem to be solved. To explore the targets of inhibiting GBM angiogenesis, this study confirmed that the expression of circMTA1 (hsa_circ_0033614) was significantly upregulated in human brain microvascular endothelial cells exposed to glioma cell-conditioned medium (GECs). The expression of circMTA1 in the cytoplasm was significantly higher than that in the nucleus. Upregulated circMTA1 in GECs can promote cell proliferation, migration, and tube formation. Further exploration of the circularization mechanism of circMTA1 confirmed that KHDRBS1 protein can bind to the upstream and downstream flanking sequences of circMTA1 and promote circMTA1 biogenesis by coordinating Alu element pairing. KHDRBS1 upregulated the proliferation, migration, and tube formation of GECs by promoting the biogenesis of circMTA1. CircMTA1 can encode the protein MTA1-134aa by internal ribosome entry site sequence-mediated translation mechanism, and promote the proliferation, migration, and tube formation of GECs through the encoded MTA1-134aa. This study provides a new target for inhibiting angiogenesis in brain GBM and a new strategy for improving the therapeutic efficacy of GBM.

Complementary Alu sequences mediate enhancer-promoter selectivity.

Enhancers determine spatiotemporal gene expression programs by engaging with long-range promoters1-4. However, it remains unknown how enhancers find their cognate promoters. We recently developed a RNA in situ conformation sequencing technology to identify enhancer-promoter connectivity using pairwise interacting enhancer RNAs and promoter-derived noncoding RNAs5,6. Here we apply this technology to generate high-confidence enhancer-promoter RNA interaction maps in six additional cell lines. Using these maps, we discover that 37.9% of the enhancer-promoter RNA interaction sites are overlapped with Alu sequences. These pairwise interacting Alu and non-Alu RNA sequences tend to be complementary and potentially form duplexes. Knockout of Alu elements compromises enhancer-promoter looping, whereas Alu insertion or CRISPR-dCasRx-mediated Alu tethering to unregulated promoter RNAs can create new loops to homologous enhancers. Mapping 535,404 noncoding risk variants back to the enhancer-promoter RNA interaction maps enabled us to construct variant-to-function maps for interpreting their molecular functions, including 15,318 deletions or insertions in 11,677 Alu elements that affect 6,497 protein-coding genes. We further demonstrate that polymorphic Alu insertion at the PTK2 enhancer can promote tumorigenesis. Our study uncovers a principle for determining enhancer-promoter pairing specificity and provides a framework to link noncoding risk variants to their molecular functions.

Evaluation of DNA Methylation Profiles of LINE-1, Alu and Ribosomal DNA Repeats in Human Cell Lines Exposed to Radiofrequency Radiation.

A large body of evidence indicates that environmental agents can induce alterations in DNA methylation (DNAm) profiles. Radiofrequency electromagnetic fields (RF-EMFs) are radiations emitted by everyday devices, which have been classified as "possibly carcinogenic"; however, their biological effects are unclear. As aberrant DNAm of genomic repetitive elements (REs) may promote genomic instability, here, we sought to determine whether exposure to RF-EMFs could affect DNAm of different classes of REs, such as long interspersed nuclear elements-1 (LINE-1), Alu short interspersed nuclear elements and ribosomal repeats. To this purpose, we analysed DNAm profiles of cervical cancer and neuroblastoma cell lines (HeLa, BE(2)C and SH-SY5Y) exposed to 900 MHz GSM-modulated RF-EMF through an Illumina-based targeted deep bisulfite sequencing approach. Our findings showed that radiofrequency exposure did not affect the DNAm of Alu elements in any of the cell lines analysed. Conversely, it influenced DNAm of LINE-1 and ribosomal repeats in terms of both average profiles and organisation of methylated and unmethylated CpG sites, in different ways in each of the three cell lines studied.

RNA editing underlies genetic risk of common inflammatory diseases.

A major challenge in human genetics is to identify the molecular mechanisms of trait-associated and disease-associated variants. To achieve this, quantitative trait locus (QTL) mapping of genetic variants with intermediate molecular phenotypes such as gene expression and splicing have been widely adopted1,2. However, despite successes, the molecular basis for a considerable fraction of trait-associated and disease-associated variants remains unclear3,4. Here we show that ADAR-mediated adenosine-to-inosine RNA editing, a post-transcriptional event vital for suppressing cellular double-stranded RNA (dsRNA)-mediated innate immune interferon responses5-11, is an important potential mechanism underlying genetic variants associated with common inflammatory diseases. We identified and characterized 30,319 cis-RNA editing QTLs (edQTLs) across 49 human tissues. These edQTLs were significantly enriched in genome-wide association study signals for autoimmune and immune-mediated diseases. Colocalization analysis of edQTLs with disease risk loci further pinpointed key, putatively immunogenic dsRNAs formed by expected inverted repeat Alu elements as well as unexpected, highly over-represented cis-natural antisense transcripts. Furthermore, inflammatory disease risk variants, in aggregate, were associated with reduced editing of nearby dsRNAs and induced interferon responses in inflammatory diseases. This unique directional effect agrees with the established mechanism that lack of RNA editing by ADAR1 leads to the specific activation of the dsRNA sensor MDA5 and subsequent interferon responses and inflammation7-9. Our findings implicate cellular dsRNA editing and sensing as a previously underappreciated mechanism of common inflammatory diseases.

Recombination of repeat elements generates somatic complexity in human genomes.

Non-allelic recombination between homologous repetitive elements contributes to evolution and human genetic disorders. Here, we combine short- and long-DNA read sequencing of repeat elements with a new bioinformatics pipeline to show that somatic recombination of Alu and L1 elements is widespread in the human genome. Our analysis uncovers tissue-specific non-allelic homologous recombination hallmarks; moreover, we find that centromeres and cancer-associated genes are enriched for retroelements that may act as recombination hotspots. We compare recombination profiles in human-induced pluripotent stem cells and differentiated neurons and find that the neuron-specific recombination of repeat elements accompanies chromatin changes during cell-fate determination. Finally, we report that somatic recombination profiles are altered in Parkinson's and Alzheimer's disease, suggesting a link between retroelement recombination and genomic instability in neurodegeneration. This work highlights a significant contribution of the somatic recombination of repeat elements to genomic diversity in health and disease.

Detection of a pathogenic Alu element insertion in PALB2 gene from targeted NGS diagnostic data.

Despite routine analysis of a large panel of genes, pathogenic variants are only detected in approximately 20% of families with hereditary breast and/or ovarian cancer. Mobile element insertions (MEI) are known to cause genetic diseases in humans, but remain challenging to detect. Retrospective analysis of targeted next-generation sequencing (NGS) data from 359 patients was performed using a dedicated MEI detection pipeline. We detected one MEI in exon 9 of the PALB2 gene in a woman with a family history of breast cancer. The pathogenic variant, c.2872_2888delins114AluL2, disrupts the PALB2 coding sequence and leads to the production of a truncated protein, p.(Gln958Valfs*38). This is the first report of a pathogenic MEI in PALB2. This study illustrates that MEI analysis may help to improve molecular diagnostic yield and can be performed from targeted NGS data used for routine diagnosis.

Alu RNA Structural Features Modulate Immune Cell Activation and A-to-I Editing of Alu RNAs Is Diminished in Human Inflammatory Bowel Disease.

Alu retrotransposons belong to the class of short interspersed nuclear elements (SINEs). Alu RNA is abundant in cells and its repetitive structure forms double-stranded RNAs (dsRNA) that activate dsRNA sensors and trigger innate immune responses with significant pathological consequences. Mechanisms to prevent innate immune activation include deamination of adenosines to inosines in dsRNAs, referred to as A-to-I editing, degradation of Alu RNAs by endoribonucleases, and sequestration of Alu RNAs by RNA binding proteins. We have previously demonstrated that widespread loss of Alu RNA A-to-I editing is associated with diverse human diseases including viral (COVID-19, influenza) and autoimmune diseases (multiple sclerosis). Here we demonstrate loss of A-to-I editing in leukocytes is also associated with inflammatory bowel diseases. Our structure-function analysis demonstrates that ability to activate innate immune responses resides in the left arm of Alu RNA, requires a 5'-PPP, RIG-I is the major Alu dsRNA sensor, and A-to-I editing disrupts both structure and function. Further, edited Alu RNAs inhibit activity of unedited Alu RNAs. Altering Alu RNA nucleotide sequence increases biological activity. Two classes of Alu RNAs exist, one class stimulates both IRF and NF-kB transcriptional activity and a second class only stimulates IRF transcriptional activity. Thus, Alu RNAs play important roles in human disease but may also have therapeutic potential.

The insertion and dysregulation of transposable elements in osteosarcoma and their association with patient event-free survival.

The dysregulation of transposable elements (TEs) has been explored in a variety of cancers. However, TE activities in osteosarcoma (OS) have not been extensively studied yet. By integrative analysis of RNA-seq, whole-genome sequencing (WGS), and methylation data, we showed aberrant TE activities associated with dysregulations of TEs in OS tumors. Specifically, expression levels of LINE-1 and Alu of different evolutionary ages, as well as subfamilies of SVA and HERV-K, were significantly up-regulated in OS tumors, accompanied by enhanced DNA repair responses. We verified the characteristics of LINE-1 mediated TE insertions, including target site duplication (TSD) length (centered around 15 bp) and preferential insertions into intergenic and AT-rich regions as well as intronic regions of longer genes. By filtering polymorphic TE insertions reported in 1000 genome project (1KGP), besides 148 tumor-specific somatic TE insertions, we found most OS patient-specific TE insertions (3175 out of 3326) are germline insertions, which are associated with genes involved in neuronal processes or with transcription factors important for cancer development. In addition to 68 TE-affected cancer genes, we found recurrent germline TE insertions in 72 non-cancer genes with high frequencies among patients. We also found that +/- 500 bps flanking regions of transcription start sites (TSS) of LINE-1 (young) and Alu showed lower methylation levels in OS tumor samples than controls. Interestingly, by incorporating patient clinical data and focusing on TE activities in OS tumors, our data analysis suggested that higher TE insertions in OS tumors are associated with a longer event-free survival time.

Genome-wide perturbations of Alu expression and Alu-associated post-transcriptional regulations distinguish oligodendroglioma from other gliomas.

Alu is a primate-specific repeat element in the human genome and has been increasingly appreciated as a regulatory element in many biological processes. But the appreciation of Alu has been limited in tumorigenesis, especially for brain tumor. To investigate the relevance of Alu to the gliomagenesis, we studied Alu element-associated post-transcriptional processes and the RNA expression of the element by performing RNA-seq for a total of 41 pairs of neurotypical and diverse glioma brain tissues. We find that A-to-I editing and circular RNA levels, as well as Alu RNA expression, are decreased overall in gliomas, compared to normal tissue. Interestingly, grade 2 oligodendrogliomas are least affected in A-to-I editing and circular RNA levels among gliomas, whereas they have a higher proportion of down-regulated Alu subfamilies, compared to the other gliomas. These findings collectively imply a unique pattern of Alu-associated transcriptomes in grade 2 oligodendroglioma, providing an insight to gliomagenesis from the perspective of an evolutionary genetic element.

Identification and Characterization of an Exonic Duplication in PALB2 in a Man with Synchronous Breast and Prostate Cancer.

PALB2 (partner and localizer of BRCA2), as indicated by its name, is a BRCA2-interacting protein that plays an important role in homologous recombination (HR) and DNA double-strand break (DSB) repair. While pathogenic variants of PALB2 have been well proven to confer an increased risk of breast cancer, data on its involvement in prostate cancer (PrC) have not been clearly demonstrated. We investigated, using targeted next generation sequencing (NGS), a 59-year-old Caucasian man who developed synchronous breast and prostate cancers. This genetic investigation allowed to identify an intragenic germline heterozygous duplication in PALB2, implicating intronic repetitive sequences spanning exon 11. This variant was confirmed by multiplex ligation probe amplification (MLPA), and genomic breakpoints have been identified and characterized at the nucleotide level (c.3114-811_3202-1756dup) using an approach based on walking PCR, long range PCR, and Sanger sequencing. RT-PCR using mRNA extracted from lymphocytes and followed by Sanger sequencing revealed a tandem duplication r.3114_3201dup; p.(Gly1068Glufs * 14). This duplication results in the synthesis of a truncated, and most-likely, non-functional protein. These findings expand the phenotypic spectrum of PALB2 variants and may improve the yield of genetic diagnoses in this field.

Association of a common genetic variant (insertion/deletion) in ACE gene with prostate cancer susceptibility in a Tunisian population.

BACKGROUND: Angiotensin-converting enzyme (ACE) plays a pivotal role in several pathologies including cancers. The association of insertion/deletion (I/D) polymorphism of the ACE gene with prostate cancer (PC) risk remains controversial. We aimed to investigate for the first time, to our Knowledge, in North Africa the potential relationship between ACE I/D polymorphism with PC susceptibility and clinical outcomes of PC patients. METHODS: This case-control study included 143 healthy individuals and 124 patients diagnosed with PC. Using genomic DNA, the samples were genotyped for ACE I/D polymorphism by polymerase chain reaction (PCR). RESULTS: We found that The D allele is significantly associated with an increased risk of PC and D/D + D/I genotypes were at 3 times increased risk of PC ([p = 0.005], OR = 2.95, IC 95% = 1.26-7.09) compared with I/I genotype (p = 0.003, OR = 0.3, IC 95% = 0.12-0.74). We observed an association between D/D and D/I genotypes with advanced age (≥70 years) (p = 0.014; r2  = 0.22). Furthermore, there is a significant prediction of advanced Gleason score ≥8 based on epidemiological parameters and ACE genotype (p = 0.000; R2  = 0.349), although no significant association was observed with stage and metastasis. CONCLUSION: The ACE I/D polymorphism is likely to predispose to PC and could play a role in PC progression and aggressiveness.

Circular RNA circPFKP promotes cell proliferation by activating IMPDH2 in prostate cancer.

Prostate cancer (PCa), especially castration-resistant PCa, is a common and fatal disease. circRNAs had been confirmed to affect the proliferation of a variety of malignant tumors. Exploring the role of circRNAs in PCa progression and discovering new therapeutic targets are of great importance for the treatment of PCa. In the present study, we found that the expression of circPFKP was significantly increased in PCa tissues compared with adjacent noncancerous prostate tissues, and was correlated with the D'Amico risk classification, N stage, and prognostic stage group of PCa. CircPFKP promotes the proliferation of PCa cells in vitro and in vivo. Suppressing circPFKP induced the G1/S arrest of PCa cells. Mechanistically, circPFKP interacted with IMPDH2, promoted the biogenesis of guanine nucleotides. Moreover, the replenishment of intracellular guanine nucleotides pool reverses the inhibitory effect of knocking-down circPFKP on PCa cell proliferation. hnRNPF might promote circPFKP generation by binding to flanking Alu elements. Our results identify a novel functional interaction of circPFKP with IMPDH2, which promotes the proliferation of PCa cells.

Identification and Characterization of New Alu Element Insertion in the BRCA1 Exon 14 Associated with Hereditary Breast and Ovarian Cancer.

Hereditary breast and ovarian cancer syndrome (HBOC) is an autosomal dominant cancer predisposition syndrome characterized by an increased risk of breast and ovarian cancers. Germline pathogenic variants in BRCA1 are found in about 7-10% of all familial breast cancers and 10% of ovarian cancers. Alu elements are the most abundant mobile DNA element in the human genome and are known to affect the human genome by different mechanisms leading to human disease. We report here the detection, by next-generation sequencing (NGS) analysis coupled with a suitable bioinformatics pipeline, of an AluYb8 element in exon 14 of the BRCA1 gene in a family with HBOC history first classified as BRCA-negative by Sanger sequencing and first NGS analysis. The c.4475_c.4476insAluYb8 mutation impacts splicing and induces the skipping of exon 14. As a result, the produced mRNA contains a premature stop, leading to the production of a short and likely non-functional protein (pAla1453Glyfs*10). Overall, our study allowed us to identify a novel pathogenic variant in BRCA1 and showed the importance of bioinformatics tool improvement and versioning.

Identification of fluoxetine as a direct NLRP3 inhibitor to treat atrophic macular degeneration.

The atrophic form of age-related macular degeneration (dry AMD) affects nearly 200 million people worldwide. There is no Food and Drug Administration (FDA)-approved therapy for this disease, which is the leading cause of irreversible blindness among people over 50 y of age. Vision loss in dry AMD results from degeneration of the retinal pigmented epithelium (RPE). RPE cell death is driven in part by accumulation of Alu RNAs, which are noncoding transcripts of a human retrotransposon. Alu RNA induces RPE degeneration by activating the NLRP3-ASC inflammasome. We report that fluoxetine, an FDA-approved drug for treating clinical depression, binds NLRP3 in silico, in vitro, and in vivo and inhibits activation of the NLRP3-ASC inflammasome and inflammatory cytokine release in RPE cells and macrophages, two critical cell types in dry AMD. We also demonstrate that fluoxetine, unlike several other antidepressant drugs, reduces Alu RNA-induced RPE degeneration in mice. Finally, by analyzing two health insurance databases comprising more than 100 million Americans, we report a reduced hazard of developing dry AMD among patients with depression who were treated with fluoxetine. Collectively, these studies identify fluoxetine as a potential drug-repurposing candidate for dry AMD.

ADAR and hnRNPC deficiency synergize in activating endogenous dsRNA-induced type I IFN responses.

Cytosolic double-stranded RNA (dsRNA) initiates type I IFN responses. Endogenous retroelements, notably Alu elements, constitute a source of dsRNA. Adenosine-to-inosine (A-to-I) editing by ADAR induces mismatches in dsRNA and prevents recognition by MDA5 and autoinflammation. To identify additional endogenous dsRNA checkpoints, we conducted a candidate screen in THP-1 monocytes and found that hnRNPC and ADAR deficiency resulted in synergistic induction of MDA5-dependent IFN responses. RNA-seq analysis demonstrated dysregulation of Alu-containing introns in hnRNPC-deficient cells via utilization of unmasked cryptic splice sites, including introns containing ADAR-dependent A-to-I editing clusters. These putative MDA5 ligands showed reduced editing in the absence of ADAR, providing a plausible mechanism for the combined effects of hnRNPC and ADAR. This study contributes to our understanding of the control of repetitive element-induced autoinflammation and suggests that patients with hnRNPC-mutated tumors might maximally benefit from ADAR inhibition-based immunotherapy.

Adenosine-to-inosine Alu RNA editing controls the stability of the pro-inflammatory long noncoding RNA NEAT1 in atherosclerotic cardiovascular disease.

Long non-coding RNAs (lncRNAs) have emerged as critical regulators in human disease including atherosclerosis. However, the mechanisms involved in the post-transcriptional regulation of the expression of disease-associated lncRNAs are not fully understood. Gene expression studies revealed that Nuclear Paraspeckle Assembly Transcript 1 (NEAT1) lncRNA expression was increased by >2-fold in peripheral blood mononuclear cells (PBMCs) derived from patients with coronary artery disease (CAD) or in carotid artery atherosclerotic plaques. We observed a linear association between NEAT1 lncRNA expression and prevalence of CAD which was independent of age, sex, cardiovascular traditional risk factors and renal function. NEAT1 expression was induced by TNF-α, while silencing of NEAT1 profoundly attenuated the TNF-α-induced vascular endothelial cell pro-inflammatory response as defined by the expression of CXCL8, CCL2, VCAM1 and ICAM1. Overexpression of the RNA editing enzyme adenosine deaminase acting on RNA-1 (ADAR1), but not of its editing-deficient mutant, upregulated NEAT1 levels. Conversely, silencing of ADAR1 suppressed the basal levels and the TNF-α-induced increase of NEAT1. NEAT1 lncRNA expression was strongly associated with ADAR1 in CAD and peripheral arterial vascular disease. RNA editing mapping studies revealed the presence of several inosines in close proximity to AU-rich elements within the AluSx3+/AluJo- double-stranded RNA complex. Silencing of the stabilizing RNA-binding protein AUF1 reduced NEAT1 levels while silencing of ADAR1 profoundly affected the binding capacity of AUF1 to NEAT1. Together, our findings propose a mechanism by which ADAR1-catalyzed A-to-I RNA editing controls NEAT1 lncRNA stability in ASCVD.