Alu Elements Research Articles

Abstract IA020: Targeting transposable elements with epigenetic modulators to reverse ovarian cancer immune evasion

Abstract Transposable elements (TEs) comprise the majority of the human genome. In most somatic tissues, TEs are silenced by DNA methylation and other epigenetic modifications. Tumors exhibit changes in DNA methylation including global loss of methylation at regions that are silenced for genome stability, like TEs, and gain of methylation at the promoter regions of tumor suppressor genes. We showed that low doses of DNMT inhibitors (DNMTis) upregulate immune signaling in solid tumor cell lines and patient samples. DNMTis activate type I interferon signaling by reducing methylation and increasing expression of endogenous retroviruses (ERVs), a type of TE, that activate dsRNA sensors. DNMTis plus HDACis (histone deacetylase inhibitors, which increase histone acetylation) increase TEs in a mouse model of ovarian cancer, activating interferon signaling and recruiting CD8+ T cells to kill cancer cells and sensitize to immune checkpoint blockade therapy. We performed a genome-wide analysis of the response to DNMTi, HDACi, and combination HDACi/DNMTi in four ovarian cancer cell lines (RNA-Seq, global methylome profiling, ATAC-Seq, and P53 chIP-Seq). The addition of HDACis to DNMTis augments the upregulation of specific ERVs and the resulting downstream interferon response in human ovarian cancer cell lines. Other TE species, including LINE1 and Alu elements, are increased by DNMTi and HDACi and may contribute to the interferon response. Alu elements, which can directly bind to the dsRNA sensor MDA5, are the most significantly demethylated TEs after DNMTi treatment. Importantly, we find considerable overlap between TEs upregulated by DNMTi in cell lines and TEs upregulated by DNMTi in ovarian cancer patients from a DNMTi clinical trial. We observed that ovarian cancer cell lines with TP53 mutations exhibited significantly fewer upregulated TEs with epigenetic therapy than wild-type TP53 cell lines. This observation was validated using isogenic cell lines; the CRISPR-edited TP53-mutant cell line had significantly higher baseline expression of TEs but upregulated fewer upon epigenetic treatment. p53 bound directly to TE DNA by chIP-Seq analysis and upregulated TEs in wild type but not mutant cell lines. In addition, our RNA-sequencing analysis indicated that A-to-I RNA editing of TEs by ADAR1 was increased after DNMTi treatment. This A-to-I editing makes TE RNA less immunogenic and inhibits the immune response to DNMTi. We find that combining ADAR1 knockdown and DNMTi treatment significantly increases type I interferon signaling, recruitment and activation of host immune cells, and survival in a murine model of ovarian cancer. These data thus give a comprehensive, genome-wide picture of TE chromatin and transcription-related changes in ovarian cancer after epigenetic treatment and implicate novel regulators (p53 and ADAR1) in response to epigenetic therapies for cancer. Citation Format: Katherine B. Chiappinelli. Targeting transposable elements with epigenetic modulators to reverse ovarian cancer immune evasion [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr IA020.

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.

Characterizing PALB2 intragenic duplication breakpoints in a triple-negative breast cancer case using long-read sequencing.

Accurate identification and characterization of Large Genomic Rearrangements (LGR), especially duplications, are crucial for precise diagnosis and risk assessment. In this report, we characterized an intragenic duplication breakpoint of PALB2 to determine its pathogenicity significance. A 52-year-old female with triple-negative breast cancer was diagnosed with a novel PALB2 LGR. An efficient and accurate methodology was applied, combining long-read sequencing and transcript analysis for the rapid characterization of the duplication. Duplication of exons 5 and 6 of PALB2 was validated by transcript analysis. Long-read sequencing enabled the localization of breakpoints within Alu elements, providing insights into the mechanism of duplication via non-allelic homologous recombination. Using our combined methodology, we reclassified the PALB2 duplication as a pathogenic variant. This reclassification suggests a possible causative link between this specific genetic alteration and the aggressive phenotype of the patient.

Cryo-EMstructure of SRP68/72 reveals an extended dimerization domain with RNA-binding activity.

The signal recognition particle (SRP) is a critical component in protein sorting pathways in all domains of life. Human SRP contains six proteins bound to the 7S RNA and their structures and functions have been mostly elucidated. The SRP68/72 dimer is the largest SRP component and is essential for SRP function. Although the structures of the SRP68/72 RNA binding and dimerization domains have been previously reported, the structure and function of large portions of the SRP68/72 dimer remain unknown. Here, we analyse full-length SRP68/72 using cryo-EM and report that SRP68/72 depend on each other for stability and form an extended dimerization domain. This newly observed dimerization domain is both a protein- and RNA-binding domain. Comparative analysis with current structural models suggests that this dimerization domain undergoes dramatic translocation upon SRP docking onto SRP receptor and eventually comes close to the Alu domain. We propose that the SRP68/72 dimerization domain functions by binding and detaching the Alu domain and SRP9/14 from the ribosomal surface, thus releasing elongation arrest upon docking onto the ER membrane.

LDLR gene rearrangements in Czech FH patients likely arise from one mutational event

BackgroundLarge deletions and duplications within the low-density lipoprotein receptor (LDLR) gene make up approximately 10% of LDLR pathogenic variants found in Czech patients with familial hypercholesterolemia. The goal of this study was to test the hypothesis that all probands with each rearrangement share identical breakpoints inherited from a common ancestor and to determine the role of Alu repetitive elements in the generation of these rearrangements.MethodsThe breakpoint sequence was determined by PCR amplification and Sanger sequencing. To confirm the breakpoint position, an NGS analysis was performed. Haplotype analysis of common LDLR variants was performed using PCR and Sanger sequencing.ResultsThe breakpoints of 8 rearrangements within the LDLR gene were analysed, including the four most common LDLR rearrangements in the Czech population (number of probands ranging from 8 to 28), and four less common rearrangements (1–4 probands). Probands with a specific rearrangement shared identical breakpoint positions and haplotypes associated with the rearrangement, suggesting a shared origin from a common ancestor. All breakpoints except for one were located inside an Alu element. In 6 out of 8 breakpoints, there was high homology (≥ 70%) between the two Alu repeats in which the break occurred.ConclusionsThe most common rearrangements of the LDLR gene in the Czech population likely arose from one mutational event. Alu elements likely played a role in the generation of the majority of rearrangements inside the LDLR gene.

Alternative polyadenylation determines the functional landscape of inverted Alu repeats

Inverted Alu repeats (IRAlus) are abundantly found in the transcriptome, especially in introns and 3' untranslated regions (UTRs). Yet, the biological significance of IRAlus embedded in 3' UTRs remains largely unknown. Here, we find that 3' UTR IRAlus silences genes involved in essential signaling pathways. We utilize J2 antibody to directly capture and map the double-stranded RNA structure of 3' UTR IRAlus in the transcriptome. Bioinformatic analysis reveals alternative polyadenylation as a major axis of IRAlus-mediated gene regulation. Notably, the expression of mouse double minute 2 (MDM2), an inhibitor of p53, is upregulated by the exclusion of IRAlus during UTR shortening, which is exploited to silence p53 during tumorigenesis. Moreover, the transcriptome-wide UTR lengthening in neural progenitor cells results in the global downregulation of genes associated with neurodegenerative diseases, including amyotrophic lateral sclerosis, via IRAlus inclusion. Our study establishes the functional landscape of 3' UTR IRAlus and its role in human pathophysiology.

Machine learning to detect the SINEs of cancer.

We previously described an approach called RealSeqS to evaluate aneuploidy in plasma cell-free DNA through the amplification of ~350,000 repeated elements with a single primer. We hypothesized that an unbiased evaluation of the large amount of sequencing data obtained with RealSeqS might reveal other differences between plasma samples from patients with and without cancer. This hypothesis was tested through the development of a machine learning approach called Alu Profile Learning Using Sequencing (A-PLUS) and its application to 7615 samples from 5178 individuals, 2073 with solid cancer and the remainder without cancer. Samples from patients with cancer and controls were prespecified into four cohorts used for model training, analyte integration, and threshold determination, validation, and reproducibility. A-PLUS alone provided a sensitivity of 40.5% across 11 different cancer types in the validation cohort, at a specificity of 98.5%. Combining A-PLUS with aneuploidy and eight common protein biomarkers detected 51% of the cancers at 98.9% specificity. We found that part of the power of A-PLUS could be ascribed to a single feature-the global reduction of AluS subfamily elements in the circulating DNA of patients with solid cancer. We confirmed this reduction through the analysis of another independent dataset obtained with a different approach (whole-genome sequencing). The evaluation of Alu elements may therefore have the potential to enhance the performance of several methods designed for the earlier detection of cancer.

Detection of a novel gross deletion in the UNC13D gene ends the diagnostic odyssey for a family with familial hemophagocytic lymphohistiocytosis 3

BackgroundFamilial hemophagocytic lymphohistiocytosis (FHL) is an immunological disorder characterized by overactivation of macrophages and T lymphocytes. This autosomal recessive condition has been characterized into multiple types depending on the genetic etiology. FHL type 3 is associated with bi-allelic pathogenic variants in the UNC13D gene.Case presentationWe present a 12-year diagnostic odyssey for a family with FHL that signifies the advances of FHL genetic testing in a clinical genetic diagnostic laboratory setting. We describe the first case of a large UNC13D gross deletion in trans to a nonsense variant in a family with FHL3, which may have been mediated by Alu elements within introns 12 and 25 of the UNC13D gene.ConclusionsThis case highlights the importance of re-evaluating past genetic testing for a patient and family as test technology evolves in order to end a diagnostic odyssey.

Multi-Omics Factor Analysis (MOFA) Identifies Transposable Element Expression As a Risk Factor and Inflammaging As a Protective Factor in Myelodysplastic Syndromes

Introduction Myelodysplastic syndromes (MDS) are haematological diseases characterised by clonal proliferation due to genetic and epigenetic alterations within haematopoietic stem and progenitor cells. Thus far, the prediction of a patient's overall survival (OS) and event-free survival (EFS) has been dependent predominantly on the degree of peripheral blood cytopenias, bone marrow blast percentage, cytogenetics, and genetic features. However, this may overlook other biological phenotypes, including, for example, the expression of transposable elements (TEs), ageing, and immunology profile. In this study, we present a comprehensive analysis of three data modalities (clinical, genotypic, and transcriptomic) and eight different views derived from these modalities to identify the hidden factors that significantly impact MDS prognosis. Methods Three (immunology, ageing, and cellular composition) out of eight views of the data were derived from RNA-seq by applying singscore, a gene set scoring tool developed by Foroutan et al., 2018, on RNA-seq gene expression from bone marrow samples of 94 MDS patients obtained from Shiozawa et al., 2017 (Fig 1.A). Each of these three views was carried forward in the workflow by a number of gene sets. The other five views were clinical numeric data, clinical categorical data, TE expression, genotype data, and known AML/MDS antigens (obtained from TANTIGEN 2.0), expressed in at least 10% of MDS samples in the study. We then employed Multi-Omics Factor Analysis (MOFA), a computational framework developed by Argelaguet et al., 2018, to uncover hidden phenotypic states within a multi-omic dataset. Through MOFA, we generated factors representing specific biological data modalities, establishing connections between features across these different categories. The total variance ( R2) that can be attributed to specific factors is also exhibited by MOFA. Results Factors were ranked based on their explained variance. We present the 10 factors with a threshold of variance explained of 2% from the 15 default factors generated by MOFA (Fig 1.A). Factor 1 was one of the factors that linked the most categories of data: immunology, cellular composition, ageing, and TEs. Most notably, Factor 1 was aligned with features of less differentiated cells (more HSC-like and less GMP-like). We observed that samples represented by high Factor 1 scores also had a higher expression of SINE:Alu elements. (Fig 1.A). Moreover, Factor 1 aligns the following immunology features: increased T-helper 1 (Th1) cells, macrophages, and T regulatory cells. The relationship between Th1 and SINE elements is particularly interesting, given the role of Th1 cell activation in host defence against infection that may be modulated by SINE activation. Factor 2 comprised of ageing and clinical categorical data, which showed that less inflammaging may lead to a poorer EFS ( P=0.00023) (Fig 1.B). Factor 4 linked immunology, cellular decomposition, ageing and clinical categorical data. The following features mainly influenced Factor 4: increased HSC-like cells, decreased CD8+ T cells, decreased immunosenescence and increased inflammatory chemokines. Using Kaplan-Meier plots to predict OS, we observed that patients with more HSC-like cells, fewer leukocytes and T cells, and less immunosenescence and inflammaging were linked to poorer EFS ( P=0.0001) and OS ( P=0.00017, data not shown). Furthermore, Factor 9 comprised solely of the TEs category and exhibited a correlation between increased TE expression and poorer EFS ( P=0.0074) and OS ( P=0.0065, data not shown) (Fig 1.B). Conclusions Our analysis using MOFA on a diverse set of biological data modalities uncovered hidden factors in MDS, providing insights into the complex interplay between various biological layers. Notably, we identified TE expression as a risk factor and inflammaging as a protective factor in MDS. HSC-like cells and specific immunological profiles were also associated with significantly poor OS and EFS. This study contributes to a deeper understanding of MDS pathogenesis and identifies potential prognostic markers for this disease. It also elucidates the importance of considering the relationships between different pathways, markers, and mutations in predicting patient outcomes, highlighting the efficacy of a comprehensive approach that goes beyond all the scoring systems described thus far for MDS.

Deciphering the role of extrachromosomal circular DNA in adipose stem cells from old and young donors

BackgroundThe functional impairment of adipose stem cells (ASCs) during aging limits their clinical transformation. Studies have shown that extrachromosomal circular DNAs (eccDNAs) are associated with tumor progression and cell aging, but the roles of eccDNAs in ASCs remain unknown.MethodWe conducted Circle sequencing (Circle-seq) to identify eccDNAs in ASCs isolated from young and old donors. The differentially expressed eccDNAs were calculated, annotated and validated via polymerase chain reaction.ResultsThousands of eccDNAs were identified and comprehensively characterized. Most of them were GC-rich, < 1000 base pairs in size, and were enriched on chromosome 19 and 17 with a high density of Alu elements and genes, 2 kb upstream/downstream of genes and satellites. In total, 3025 eccDNAs were differentially expressed among the two ASC groups. Conjoint analysis of the Circle-seq results and previous RNA-seq results revealed that 73 eccDNAs and 55 genes exhibited the same differential expression between the two groups. KEGG and GO analyses revealed that genes encoding differentially expressed eccDNAs were enriched for cell adhesion, cellular senescence and TGF-β receptor signaling pathway. We also found that aged ASCs exhibited loss of eccDNAs, including CAMK2G (chr10: 75577899-75578176), TRABD2B (chr1: 48305638-48307008) and TRABD2B (chr1: 48305425-48307091).ConclusionIn this study, we elucidated the first eccDNA profile relating to ASCs and demonstrated that three eccDNAs are lost in aged ASCs, which may be potential biomarkers of stem cell aging and valuable targets for stem cell rejuvenation.

Targeted long-read sequencing identifies and characterizes structural variants in cases of inherited platelet disorders

BackgroundGenetic diagnosis of inherited platelet disorders (IPDs) is mainly performed by high-throughput sequencing (HTS). These short-read–based sequencing methods sometimes fail to characterize the genetics of the disease. ObjectivesTo evaluate nanopore long-read DNA sequencing for characterization of structural variants (SVs) in patients with IPDs. MethodsFour patients with a clinical and laboratory diagnosis of Glanzmann thrombasthenia (GT) (P1 and P2) and Hermansky–Pudlak syndrome (HPS) (P3 and P4) in whom HTS missed the underlying molecular cause were included. DNA was analyzed by both standard HTS and nanopore sequencing on a MinION device (Oxford Nanopore Technologies) after enrichment of DNA spanning regions covering GT and HPS genes. ResultsIn patients with GT, HTS identified only 1 heterozygous ITGB3 splice variant c.2301+1G>C in P2. In patients with HPS, a homozygous deletion in HPS5 was suspected in P3, and 2 heterozygous HPS3 variants, c.2464C>T (p.Arg822∗) and a deletion affecting 2 exons, were reported in P4. Nanopore sequencing revealed a complex SV affecting exons 2 to 6 in ITGB3 (deletion-inversion-duplication) in homozygosity in P1 and compound heterozygosity with the splice variant in P2. In the 2 patients with HPS, nanopore defined the length of the SVs, which were characterized at nucleotide resolution. This allowed the identification of repetitive Alu elements at the breakpoints and the design of specific polymerase chain reactions for family screening. ConclusionThe nanopore technology overcomes the limitations of standard short-read sequencing techniques in SV characterization. Using nanopore, we characterized novel defects in ITGB3, HPS5, and HPS3, highlighting the utility of long-read sequencing as an additional diagnostic tool in IPDs.

Oligonucleotide skews enable comprehensive and insightful characterization of GC- and TA-skew properties observed throughout the human genome with support of unsupervised AI with reference to gene- and Alu-polarity skews

GC and TA skew are fundamental genomic features observed widely from bacteria to humans. They have been described as replication- and transcription-coupled skews because they are mainly caused by differences in mutation and repair biases between complementary DNA strands in replication and transcription processes. The skews are clearly observed in bacteria, plants and birds, but are more difficult to accurately identify in mammals, including humans. The main reason for this difficulty is that the correlation between GC and TA skew is not high for their variation in the mammalian genomes. In this study, we focused not only on these mononucleotide skews but also on di- and trinucleotide skews, first in 100-kb sequences of two short human chromosomes (chr21 and 22), and by using the conventional distribution map analysis, we clearly observed these skews over these chromosomes, except for centromeric heterochromatin regions where diverse repetitive sequences are densely clustered. Then, by using a batch learning self-organizing map (BLSOM), which can display large amounts of sequence data at once, we detected the oligonucleotide skews across nearly the entire human genome. Next, to understand skews within centromeric heterochromatin regions, we focused on Alu sequences ubiquitously distributed in the human genome and analyzed mono-, di-, and trinucleotide skews by referring to the polarity of Alu sequences. Since this polarity is unevenly distributed in the human genome, a clear TA skew was observed by reflecting the evident A-richness to T of Alu elements. However, due to some degree of their G-richness to C, TA and GC skews showed inversely correlated variations, when analyzing Alu sequences alone. Notably, when we analyzed genome sequences after excluding Alu sequences, the concordant correlation between GC- and TA-skew variations increased, indicating that a large amount of Alu sequences has made the detection of these mononucleotide skews in the human genome more difficult than in other organisms. Analysis of oligonucleotide skews using BLSOM is thought to be suitable for characterizing sequence features in skew transition regions.

Adenosine Deaminase Acting on RNA (ADAR) Enzymes: A Journey from Weird to Wondrous.

The adenosine deaminase acting on RNA (ADAR) enzymes that catalyze the conversion of adenosine to inosine in double-stranded (ds)RNA are evolutionarily conserved and are essential for many biological functions including nervous system function, hematopoiesis, and innate immunity. Initially it was assumed that the wide-ranging biological roles of ADARs are due to inosine in mRNA being read as guanosine by the translational machinery, allowing incomplete RNA editing in a target codon to generate two different proteins from the same primary transcript. In humans, there are approximately seventy-six positions that undergo site-specific editing in tissues at greater than 20% efficiency that result in recoding. Many of these transcripts are expressed in the central nervous system (CNS) and edited by ADAR2. Exploiting mouse genetic models revealed that transgenic mice lacking the gene encoding Adar2 die within 3 weeks of birth. Therefore, the role of ADAR2 in generating protein diversity in the nervous system is clear, but why is ADAR RNA editing activity essential in other biological processes, particularly editing mainly involving ADAR1? ADAR1 edits human transcripts having embedded Alu element inverted repeats (AluIRs), but the link from this activity to innate immunity activation was elusive. Mice lacking the gene encoding Adar1 are embryonically lethal, and a major breakthrough was the discovery that the role of Adar1 in innate immunity is due to its ability to edit such repetitive element inverted repeats which have the ability to form dsRNA in transcripts. The presence of inosine prevents activation of the dsRNA sensor melanoma differentiation-associated protein 5 (Mda5). Thus, inosine helps the cell discriminate self from non-self RNA, acting like a barcode on mRNA. As innate immunity is key to many different biological processes, the basis for this widespread biological role of the ADAR1 enzyme became evident.Our group has been studying ADARs from the outset of research on these enzymes. In this Account, we give a historical perspective, moving from the initial purification of ADAR1 and ADAR2 and cloning of their encoding genes up to the current research focus in the field and what questions still remain to be addressed. We discuss the characterizations of the proteins, their localizations, posttranslational modifications, and dimerization, and how all of these affect their biological activities. Another aspect we explore is the use of mouse and Drosophila genetic models to study ADAR functions and how these were crucial in determining the biological functions of the ADAR proteins. Finally, we describe the severe consequences of rare mutations found in the human genes encoding ADAR1 and ADAR2.

Emerging Opportunities to Study Mobile Element Insertions and Their Source Elements in an Expanding Universe of Sequenced Human Genomes.

Three mobile element classes, namely Alu, LINE-1 (L1), and SVA elements, remain actively mobile in human genomes and continue to produce new mobile element insertions (MEIs). Historically, MEIs have been discovered and studied using several methods, including: (1) Southern blots, (2) PCR (including PCR display), and (3) the detection of MEI copies from young subfamilies. We are now entering a new phase of MEI discovery where these methods are being replaced by whole genome sequencing and bioinformatics analysis to discover novel MEIs. We expect that the universe of sequenced human genomes will continue to expand rapidly over the next several years, both with short-read and long-read technologies. These resources will provide unprecedented opportunities to discover MEIs and study their impact on human traits and diseases. They also will allow the MEI community to discover and study the source elements that produce these new MEIs, which will facilitate our ability to study source element regulation in various tissue contexts and disease states. This, in turn, will allow us to better understand MEI mutagenesis in humans and the impact of this mutagenesis on human biology.

Proximity Proteomics Revealed Aberrant mRNA Splicing Elicited by ALS-Linked Profilin-1 Mutants.

Profilin 1 (PFN1) is a cytoskeleton protein that modulates actin dynamics through binding to monomeric actin and polyproline-containing proteins. Mutations in PFN1 have been linked to the pathogenesis of familial amyotrophic lateral sclerosis (ALS). Here, we employed an unbiased proximity labeling strategy in combination with proteomic analysis for proteome-wide profiling of proteins that differentially interact with mutant and wild-type (WT) PFN1 proteins in human cells. We uncovered 11 mRNA splicing proteins that are preferentially enriched in the proximity proteomes of the two ALS-linked PFN1 variants, C71G and M114T, over that of wild-type PFN1. We validated the preferential interactions of the ALS-linked PFN1 variants with two mRNA splicing factors, hnRNPC and U2AF2, by immunoprecipitation, followed with immunoblotting. We also found that the two ALS-linked PFN1 variants promoted the exonization of Alu elements in the mRNAs of MTO1, TCFL5, WRN and POLE genes in human cells. Together, we showed that the two ALS-linked PFN1 variants interacted preferentially with mRNA splicing proteins, which elicited aberrant exonization of the Alu elements in mRNAs. Thus, our work provided pivotal insights into the perturbations of ALS-linked PFN1 variants in RNA biology and their potential contributions to ALS pathology.

Genome-wide perturbations of A-to-I RNA editing dysregulated circular RNAs promoting the development of cervical cancer

Cervical cancer, the second most common female malignant tumor, seriously threatens women's health and lives. Despite the availability of the HPV vaccine, effective treatment options for cervical cancer are still lacking. New research perspectives now clarify that RNA editing dysregulation and changes in circRNA expression are jointly involved in disease pathogenesis, so molecular changes associated with circRNA and RNA editing may provide clues for the development of new therapeutic strategies for cervical cancer. In this study, we designed a series of pipelines to identify and analyze dysregulated RNA editing events in circRNAs. Our findings indicate a decrease in A-to-I RNA editing levels in cervical cancer compared to normal tissues, and editing may influence the back-splicing process of circRNAs through structural modifications of Alu elements. Moreover, our research reveals that RNA editing could modulate circRNA biogenesis by influencing RNA binding protein (RBP) binding on a transcriptome-wide scale, as well as influence the expression and coding potential of circRNAs. Importantly, we identified three RNA editing sites that could serve as potential biomarkers. In summary, our study presents a comprehensive landscape of RNA editing perturbations in circRNAs, providing new insights into the complex relationship between RNA editing and circRNA dysregulation in cervical cancer.

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.

CapTEs enables locus-specific dissection of transcriptional outputs from reference and nonreference transposable elements

Transposable elements (TEs) serve as both insertional mutagens and regulatory elements in cells, and their aberrant activity is increasingly being revealed to contribute to diseases and cancers. However, measuring the transcriptional consequences of nonreference and young TEs at individual loci remains challenging with current methods, primarily due to technical limitations, including short read lengths generated and insufficient coverage in target regions. Here, we introduce a long-read targeted RNA sequencing method, Cas9-assisted profiling TE expression sequencing (capTEs), for quantitative analysis of transcriptional outputs for individual TEs, including transcribed nonreference insertions, noncanonical transcripts from various transcription patterns and their correlations with expression changes in related genes. This method selectively identified TE-containing transcripts and outputted data with up to 90% TE reads, maintaining a comparable data yield to whole-transcriptome sequencing. We applied capTEs to human cancer cells and found that internal and inserted Alu elements may employ distinct regulatory mechanisms to upregulate gene expression. We expect that capTEs will be a critical tool for advancing our understanding of the biological functions of individual TEs at the locus level, revealing their roles as both mutagens and regulators in biological and pathogenic processes.

Epigenomic signature of accelerated ageing in progeroid Cockayne syndrome.

Cockayne syndrome (CS) and UV-sensitive syndrome (UVSS) are rare genetic disorders caused by mutation of the DNA repair and multifunctional CSA or CSB protein, but only CS patients display a progeroid and neurodegenerative phenotype, providing a unique conceptual and experimental paradigm. As DNA methylation (DNAm) remodelling is a major ageing marker, we performed genome-wide analysis of DNAm of fibroblasts from healthy, UVSS and CS individuals. Differential analysis highlighted a CS-specific epigenomic signature (progeroid-related; not present in UVSS) enriched in three categories: developmental transcription factors, ion/neurotransmitter membrane transporters and synaptic neuro-developmental genes. A large fraction of CS-specific DNAm changes were associated with expression changes in CS samples, including in previously reported post-mortem cerebella. The progeroid phenotype of CS was further supported by epigenomic hallmarks of ageing: the prediction of DNAm of repetitive elements suggested an hypomethylation of Alu sequences in CS, and the epigenetic clock returned a marked increase in CS biological age respect to healthy and UVSS cells. The epigenomic remodelling of accelerated ageing in CS displayed both commonalities and differences with other progeroid diseases and regular ageing. CS shared DNAm changes with normal ageing more than other progeroid diseases do, and included genes functionally validated for regular ageing. Collectively, our results support the existence of an epigenomic basis of accelerated ageing in CS and unveil new genes and pathways that are potentially associated with the progeroid/degenerative phenotype.

Distribution of the macaques genus-specific Alu repeat AluMacYa3 in the MGMT gene orthologs of old world monkeys

Aim. To follow the distribution and evolution of the macaques genus-specific Alu repeat AluMacYa3 in Cercopithecidae MGMT gene orthologs. Methods. The homology between nucleotide sequences has been determined by the BLAST 2.6.1 program. The results of the search and identification of MGE have been obtained by the CENSOR program. Results. It has been shown on the example of Cercopithecidae MGMT gene orthologs that the genus-specific Alu repeat AluMacYa3 is present not only in the intron sequences of macaques, but also in other representatives of the Old World monkeys and in its evolutionary history not a deletion degradation of nucleotide sequences, as it is in the case of species-specific Alu repeats, but a single nucleotide polymorphism is dominated. Conclusions. Thus, the macaque genus-specific Alu repeat AluMacYa3 has been identified in different representatives of the Old World monkeys and its evolutionary history combines nucleotide polymorphism and deletion degradation of nucleotide sequences.