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Cancer therapies that target epigenetic repressors can mediate their effects by activating retroelements within the human genome. Retroelement transcripts can form double-stranded RNA (dsRNA) that activates the MDA5 pattern recognition receptor 1 – 6 . This state of viral mimicry leads to loss of cancer cell fitness and stimulates innate and adaptive immune responses 7 , 8 . However, the clinical efficacy of epigenetic therapies has been limited. To find targets that would synergize with the viral mimicry response, we sought to identify the immunogenic retroelements that are activated by epigenetic therapies. Here we show that intronic and intergenic SINE elements, specifically inverted-repeat Alus, are the major source of drug-induced immunogenic dsRNA. These inverted-repeat Alus are frequently located downstream of ‘orphan’ CpG islands 9 . In mammals, the ADAR1 enzyme targets and destabilizes inverted-repeat Alu dsRNA 10 , which prevents activation of the MDA5 receptor 11 . We found that ADAR1 establishes a negative-feedback loop, restricting the viral mimicry response to epigenetic therapy. Depletion of ADAR1 in patient-derived cancer cells potentiates the efficacy of epigenetic therapy, restraining tumour growth and reducing cancer initiation. Therefore, epigenetic therapies trigger viral mimicry by inducing a subset of inverted-repeats Alus, leading to an ADAR1 dependency. Our findings suggest that combining epigenetic therapies with ADAR1 inhibitors represents a promising strategy for cancer treatment. Inverted-repeat Alu elements are the main source of drug-induced immunogenic double-stranded RNAs, which are destabilized by the RNA deaminase ADAR1, thereby limiting activation of the immune response.

Enhancers determine spatiotemporal gene expression programs by engaging with long-range promoters 1 – 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 RNAs 5 , 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. Using RNA in situ conformation sequencing technology, the role of Alu elements in mediating the interaction between enhancers and promoters is shown.

A sequence that is frequently found in Alu elements drives the localization of some long RNAs to the nucleus in human cells. Signposting lncRNAs to the nucleus Messenger RNA (mRNA) can contain localization signals that determine whether the mRNA ends up primarily in the cytoplasm or in the nucleus of cells. Long noncoding RNAs (lncRNAs) typically localize to the nucleus, but the reasons for this preference were unclear. Yoav Lubelsky and Igor Ulitsky show that the presence of a binding site for the nuclear protein HNRNPK in lncRNAs can promote the nuclear accumulation of these RNA molecules in human cells. This sequence is found in Alu elements, which are present in many transcripts and may be widespread drivers of nuclear localization across species. Long noncoding RNAs (lncRNAs) are emerging as key parts of multiple cellular pathways 1 , but their modes of action and how these are dictated by sequence remain unclear. lncRNAs tend to be enriched in the nuclear fraction, whereas most mRNAs are overtly cytoplasmic 2 , although several studies have found that hundreds of mRNAs in various cell types are retained in the nucleus 3 , 4 . It is thus conceivable that some mechanisms that promote nuclear enrichment are shared between lncRNAs and mRNAs. Here, to identify elements in lncRNAs and mRNAs that can force nuclear localization, we screened libraries of short fragments tiled across nuclear RNAs, which were cloned into the untranslated regions of an efficiently exported mRNA. The screen identified a short sequence derived from Alu elements and bound by HNRNPK that increased nuclear accumulation. Binding of HNRNPK to C-rich motifs outside Alu elements is also associated with nuclear enrichment in both lncRNAs and mRNAs, and this mechanism is conserved across species. Our results thus identify a pathway for regulation of RNA accumulation and subcellular localization that has been co-opted to regulate the fate of transcripts with integrated Alu elements.

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 adopted 1 , 2 . However, despite successes, the molecular basis for a considerable fraction of trait-associated and disease-associated variants remains unclear 3 , 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 responses 5 , 6 , 7 , 8 , 9 , 10 – 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 inflammation 7 , 8 – 9 . Our findings implicate cellular dsRNA editing and sensing as a previously underappreciated mechanism of common inflammatory diseases. cis -RNA editing quantitative trait loci, which are associated with immunogenic double-stranded RNAs, underlie genome-wide association study variants in common autoimmune and inflammatory diseases.

Among epigenetic modifiers, telomeres represent attractive modulators of the genome in part through position effects. Telomere Position Effect‐Over Long Distances (TPE‐OLD) modulates gene expression by changes in telomere‐dependent long‐distance loops. To gain insights into the molecular mechanisms of TPE‐OLD, we performed a genome‐wide transcriptome and methylome analysis in proliferative fibroblasts and myoblasts or differentiated myotubes with controlled telomere lengths. By integrating omics data, we identified a common TPE‐OLD dependent cis‐acting motif that behaves as an insulator or enhancer. Next, we uncovered trans partners that regulate these activities and observed the consistent depletion of one candidate factor, RBPJ, at TPE‐OLD associated loci upon telomere shortening. Importantly, we confirmed our findings by unbiased comparisons to recent Human transcriptomic studies, including those from the Genotype‐Tissue Expression (GTEx) project. We concluded that TPE‐OLD acts at the genome‐wide level and can be relayed by RBPJ bridging Alu‐like elements to telomeres. In response to physiological (i.e., aging) or pathological cues, TPE‐OLD might coordinate the genome‐wide impact of telomeres through recently evolved Alu elements acting as enhancers in association with RBPJ. Proposed TPE‐OLD model. TPE‐OLD genes are defined by a progressive modulation of gene expression following telomere attrition. In our study, focusing on the molecular mechanism of TPE‐OLD, we found that the TPE‐OLD loop requires a cis‐signature corresponding to a newly evolved Alu repeats (AluY). The Alu repeat can be either in an intergenic region, as the sequence holds the same properties as an enhancer/insulator, or positioned within the gene, thus potentially affecting splicing. The DNA‐loop is maintained by trans partners, including SMCHD1, TZAP, TRF2, and more specifically RBPJ. As telomere shortens and DNA methylation associated with the Alu element decreases, the distance between the TPE‐OLD gene and telomeres increases and induces a modulation of transcription. We conclude that TPE‐OLD is regulated at three levels: (i) at the telomere through its associated length, (ii) at the Alu element through its associated methylation, and (iii) at the telomere‐associated proteins levels and their localization (SMCHD1, TZAP, TRF2, RBPJ). According to our work, RBPJ (directly or indirectly) along with TRF2 (directly) are the most important proteins for the maintenance of the TPE‐OLD loop.

LINE-1 and Alu retrotransposons are components of the human genome and have been implicated in many human diseases. These elements can influence human transcriptome plasticity in various mechanisms. Chimeric transcripts derived from LINE-1 and Alu can also impact the human transcriptome, such as exonization and post-transcriptional modification. However, its specific role in ASD neuropathology remains unclear, particularly in the cerebellum tissues. We performed RNA-sequencing of post-mortem cerebellum tissues from ASD and unaffected individuals for transposable elements profiling and chimeric transcript identification. The majority of free transcripts of transposable elements were not changed in the cerebellum tissues of ASD compared with unaffected individuals. Nevertheless, we observed that chimeric transcripts derived from LINE-1 and Alu were embedded in the transcripts of differentially expressed genes in the cerebellum of ASD, and these genes were related to developments and abnormalities of the cerebellum. In addition, the expression levels of these genes were correlated with the significantly decreased thickness of the molecular layer in the cerebellum of ASD. We also found that global methylation and expression of LINE-1 and Alu elements were not changed in ASD, but observed in the ASD sub-phenotypes. Our findings showed associations between transposable elements and cerebellar abnormalities in ASD, particularly in distinct phenotypic subgroups. Further investigations using appropriate models are warranted to elucidate the structural and functional implications of LINE-1 and Alu elements in ASD neuropathology.

Alu elements are transposable elements that can influence gene regulation through several mechanisms; nevertheless, it remains unclear whether dysregulation of Alu elements contributes to the neuropathology of autism spectrum disorder (ASD). In this study, we characterized transposable element expression profiles and their sequence characteristics in the prefrontal cortex tissues of ASD and unaffected individuals using RNA-sequencing data. Our results showed that most of the differentially expressed transposable elements belong to the Alu family, with 659 loci of Alu elements corresponding to 456 differentially expressed genes in the prefrontal cortex of ASD individuals. We predicted cis- and trans-regulation of Alu elements to host/distant genes by conducting correlation analyses. The expression level of Alu elements correlated significantly with 133 host genes (cis-regulation, adjusted p < 0.05) associated with ASD as well as the cell survival and cell death of neuronal cells. Transcription factor binding sites in the promoter regions of differentially expressed Alu elements are conserved and associated with autism candidate genes, including RORA. COBRA analyses of postmortem brain tissues showed significant hypomethylation in global methylation analyses of Alu elements in ASD subphenotypes as well as DNA methylation of Alu elements located near the RNF-135 gene (p < 0.05). In addition, we found that neuronal cell density, which was significantly increased (p = 0.042), correlated with the expression of genes associated with Alu elements in the prefrontal cortex of ASD. Finally, we determined a relationship between these findings and the ASD severity (i.e., ADI-R scores) of individuals with ASD. Our findings provide a better understanding of the impact of Alu elements on gene regulation and molecular neuropathology in the brain tissues of ASD individuals, which deserves further investigation.

Alu elements are primate-specific short interspersed elements (SINEs), over 1 million copies of which are present in the human genome; thus, Alu elements are useful targets for detecting human cells. However, previous Alu-based techniques for detecting human genomic DNA do not reach the theoretical limits of sensitivity and specificity. In this study, we developed a highly sensitive and specific Alu-based real-time PCR method for discriminating human cells from rodent cells, using a primer and probe set carefully designed to avoid possible cross-reactions with rodent genomes. From 100 ng of mixed human and rodent genomes, 1 fg of human genome, equivalent to 1 human cell in 100 million rodent cells, was detectable. Furthermore, in vivo mouse subrenal capsule xenotransplantation assays revealed that 10 human cells per mouse organ were detectable. In addition, after intravenous injection of human mesenchymal stem cells into NOD/SCID mice via tail vein, the biodistribution of human cells was trackable in the mouse lungs and kidneys for at least 1 week. Our findings indicate that our primer and probe set is applicable for the quantitative detection of tiny amounts of human cells, such as xenotransplanted human cancer or stem cells, in rodents.

PIWI-interacting RNAs (piRNAs) are the most diverse small RNAs in animals. These small RNAs have been known to play an important role in the suppression of transposable elements (TEs). Protein-coding genes (PCGs) are the most well-recognized functional genes in genomes. In the present study, we designed and performed a set of statistics-based evolutionary analyses to reveal nonrandom phenomena in the evolution of human piRNA-PCG targeting relationships. Through analyzing the occurrence of single nucleotide variants (SNVs) in potential piRNA target sites in human PCGs, we provide evidence that there exists a mutational force biased to strengthen piRNA-PCG targeting relationships. Through analyzing the allele frequencies of SNVs in potential piRNA target sites in human PCGs, we provide evidence that there exists a piRNA-dependent selective force acting on potential piRNA target sites in human PCGs. Because of these nonrandom evolutionary forces, human piRNAs and their potential target sites in PCGs are not independent in evolution. Additionally, we found evidence that potential piRNA target sites in human PCGs are particularly likely to be present in regions derived from Alu elements. This finding suggests that the aforementioned evolutionary forces acting on piRNA-PCG targeting relationships could be particularly prone to affect Alu -derived regions in human PCGs. Collectively, our findings provide new insights into the evolutionary interplay between piRNAs, PCGs, and Alu elements in the evolution of the human genome.

The human genome is under constant invasion by retrotransposable elements. The most successful of these are the Alu elements; with a copy number of over a million, they occupy about 10 % of the entire genome. Interestingly, the vast majority of these Alu insertions are located in gene-rich regions, and one-third of all human genes contains an Alu insertion. Alu sequences are often embedded in gene sequence encoding pre-mRNAs and mature mRNAs, usually as part of their intron or UTRs. Once transcribed, they can regulate gene expression as well as increase the number of RNA isoforms expressed in a tissue or a species. They also regulate the function of other RNAs, like microRNAs, circular RNAs, and potentially long non-coding RNAs. Mechanistically, Alu elements exert their effects by influencing diverse processes, such as RNA editing, exonization, and RNA processing. In so doing, they have undoubtedly had a profound effect on human evolution.