B2 and ALU retrotransposons are self-cleaving ribozymes whose activity is enhanced by EZH2.

Transposable elements make up half of the mammalian genome. One of the most abundant is the short interspersed nuclear element (SINE). Among their million copies, B2 accounts for ∼350,000 in the mouse genome and has garnered special interest because of emerging roles in epigenetic regulation. Our recent work demonstrated that B2 RNA binds stress genes to retard transcription elongation. Although epigenetically silenced, B2s become massively up-regulated during thermal and other types of stress. Specifically, an interaction between B2 RNA and the Polycomb protein, EZH2, results in cleavage of B2 RNA, release of B2 RNA from chromatin, and activation of thermal stress genes. Although an established RNA-binding protein and histone methyltransferase, EZH2 is not known to be a nuclease. Here, we provide evidence for the surprising conclusion that B2 is a self-cleaving ribozyme. Ribozyme activity depends on Mg+2 and monovalent cations but is resistant to protease treatment. However, contact with EZH2 accelerates cleavage rate by >100-fold, suggesting that EZH2 promotes a cleavage-competent RNA conformation. B2 modification-interference analysis demonstrates that phosphorothioate changes at A and C nucleotides can substitute for EZH2. B2 nucleotides 45 to 55 and 100 to 101 are essential for activity. Finally, another family of SINEs, the human ALU element, also produces a self-cleaving RNA and is cleaved during T-cell activation as well as thermal and endoplasmic reticulum (ER) stress. Thus, B2/ALU SINEs may be classified as "epigenetic ribozymes" that function as transcriptional switches during stress. Given their high copy numbers, B2 and ALU may represent the predominant ribozyme activity in mammalian cells.

TFIIIC Binding to Alu Elements Controls Gene Expression via Chromatin Looping and Histone Acetylation.

How repetitive elements, epigenetic modifications, and architectural proteins interact ensuring proper genome expression remains poorly understood. Here, we report regulatory mechanisms unveiling a central role of Alu elements (AEs) and RNA polymerase III transcription factor C (TFIIIC) in structurally and functionally modulating the genome via chromatin looping and histone acetylation. Upon serum deprivation, a subset of AEs pre-marked by the activity-dependent neuroprotector homeobox Protein (ADNP) and located near cell-cycle genes recruits TFIIIC, which alters their chromatin accessibility by direct acetylation of histone H3 lysine-18 (H3K18). This facilitates the contacts of AEs with distant CTCF sites near promoter of other cell-cycle genes, which also become hyperacetylated at H3K18. These changes ensure basal transcription of cell-cycle genes and are critical for their re-activation upon serum re-exposure. Our study reveals how direct manipulation of the epigenetic state of AEs by a general transcription factor regulates 3D genome folding and expression.

Warning SINEs: Alu elements, evolution of the human brain, and the spectrum of neurological disease.

Alu elements are a highly successful family of primate-specific retrotransposons that have fundamentally shaped primate evolution, including the evolution of our own species. Alus play critical roles in the formation of neurological networks and the epigenetic regulation of biochemical processes throughout the central nervous system (CNS), and thus are hypothesized to have contributed to the origin of human cognition. Despite the benefits that Alus provide, deleterious Alu activity is associated with a number of neurological and neurodegenerative disorders. In particular, neurological networks are potentially vulnerable to the epigenetic dysregulation of Alu elements operating across the suite of nuclear-encoded mitochondrial genes that are critical for both mitochondrial and CNS function. Here, we highlight the beneficial neurological aspects of Alu elements as well as their potential to cause disease by disrupting key cellular processes across the CNS. We identify at least 37 neurological and neurodegenerative disorders wherein deleterious Alu activity has been implicated as a contributing factor for the manifestation of disease, and for many of these disorders, this activity is operating on genes that are essential for proper mitochondrial function. We conclude that the epigenetic dysregulation of Alu elements can ultimately disrupt mitochondrial homeostasis within the CNS. This mechanism is a plausible source for the incipient neuronal stress that is consistently observed across a spectrum of sporadic neurological and neurodegenerative disorders.

Analysis of chromatin accessibility in decidualizing human endometrial stromal cells.

Spontaneous decidualization of the endometrium in response to progesterone signaling is confined to menstruating species, including humans and other higher primates. During this process, endometrial stromal cells (EnSCs) differentiate into specialized decidual cells that control embryo implantation. We subjected undifferentiated and decidualizing human EnSCs to an assay for transposase accessible chromatin with sequencing (ATAC-seq) to map the underlying chromatin changes. A total of 185,084 open DNA loci were mapped accurately in EnSCs. Altered chromatin accessibility upon decidualization was strongly associated with differential gene expression. Analysis of 1533 opening and closing chromatin regions revealed over-representation of DNA binding motifs for known decidual transcription factors (TFs) and identified putative new regulators. ATAC-seq footprint analysis provided evidence of TF binding at specific motifs. One of the largest footprints involved the most enriched motif-basic leucine zipper-as part of a triple motif that also comprised the estrogen receptor and Pax domain binding sites. Without exception, triple motifs were located within Alu elements, which suggests a role for this primate-specific transposable element (TE) in the evolution of decidual genes. Although other TEs were generally under-represented in open chromatin of undifferentiated EnSCs, several classes contributed to the regulatory DNA landscape that underpins decidual gene expression.-Vrljicak, P., Lucas, E. S., Lansdowne, L., Lucciola, R., Muter, J., Dyer, N. P., Brosens, J. J., Ott, S. Analysis of chromatin accessibility in decidualizing human endometrial stromal cells.

The role of Alu elements in the cis-regulation of RNA processing.

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.

Correlations in global DNA methylation measures in peripheral blood mononuclear cells and granulocytes.

Alterations in global DNA methylation levels have been associated with chronic diseases. Despite the increase in the number of studies measuring markers of global methylation, few have adequately examined within-individual differences by source of DNA and whether within-individual differences by source of DNA differ by age, race and other lifestyle factors. We examined correlations between peripheral mononuclear cell (PBMC) and granulocyte DNA methylation levels measured by the luminometric methylation assay (LUMA), and in LINE-1, Sat2, and Alu by MethyLight and pyrosequencing, in the same individual in 112 women participating in The New York City Multiethnic Breast Cancer Project. Levels of DNA methylation of Sat2 by MethyLight (r = 0.57; P < 0.01) and LINE-1 by pyrosequencing (r = 0.30; P < 0.01) were correlated between PBMC and granulocyte DNA of the same individuals, but LUMA and Alu levels were not. The magnitude of the correlations for Sat2 and LINE-1 varied when stratified by selected demographic and lifestyle factors, although the study sample size limited our comparisons across subgroups. These results lend further support to the importance of considering the source of DNA in epidemiologic studies of white blood cell DNA methylation. Results from studies that combine individuals with different available DNA sources need to be interpreted with caution.

Dynamic Alu methylation during normal development, aging, and tumorigenesis.

DNA methylation primarily occurs on CpG dinucleotides and plays an important role in transcriptional regulations during tissue development and cell differentiation. Over 25% of CpG dinucleotides in the human genome reside within Alu elements, the most abundant human repeats. The methylation of Alu elements is an important mechanism to suppress Alu transcription and subsequent retrotransposition. Decades of studies revealed that Alu methylation is highly dynamic during early development and aging. Recently, many environmental factors were shown to have a great impact on Alu methylation. In addition, aberrant Alu methylation has been documented to be an early event in many tumors and Alu methylation levels have been associated with tumor aggressiveness. The assessment of the Alu methylation has become an important approach for early diagnosis and/or prognosis of cancer. This review focuses on the dynamic Alu methylation during development, aging, and tumor genesis. The cause and consequence of Alu methylation changes will be discussed.

PKR is activated by cellular dsRNAs during mitosis and acts as a mitotic regulator.

dsRNA-dependent protein kinase R (PKR) is a ubiquitously expressed enzyme well known for its roles in immune response. Upon binding to viral dsRNA, PKR undergoes autophosphorylation, and the phosphorylated PKR (pPKR) regulates translation and multiple signaling pathways in infected cells. Here, we found that PKR is activated in uninfected cells, specifically during mitosis, by binding to dsRNAs formed by inverted Alu repeats (IRAlus). While PKR and IRAlu-containing RNAs are segregated in the cytosol and nucleus of interphase cells, respectively, they interact during mitosis when nuclear structure is disrupted. Once phosphorylated, PKR suppresses global translation by phosphorylating the α subunit of eukaryotic initiation factor 2 (eIF2α). In addition, pPKR acts as an upstream kinase for c-Jun N-terminal kinase and regulates the levels of multiple mitotic factors such as cyclins A and B and Polo-like kinase 1 and phosphorylation of histone H3. Disruption of PKR activation via RNAi or expression of a transdominant-negative mutant leads to misregulation of the mitotic factors, delay in mitotic progression, and defects in cytokinesis. Our study unveils a novel function of PKR and endogenous dsRNAs as signaling molecules during the mitosis of uninfected cells.

[Genetic diversity and relationships of northern eurasia populations for polymorphic Alu-insertions].

We for the first time have examined the autosomal gene pool of the Siberia, Central Asian and the Far East populations (27 populations of 12 ethnic groups) using a set of polymorphic Alu insertions in the human genome. The results of the analysis testify (i) to a significant level of genetic diversity in the Northern Eurasian populations and (ii) to a considerable differentiation of gene pool in the population of this region. It has been shown that at the CD4 locus, the frequency of Alu (-) is inversely related to the Mongoloid component of the population, the lowest and highest frequencies of the Alu deletion at locus CD4 were recorded respectively in Eskimo (0.012) and Russian and Ukrainian (0.35). The analysis of gene flow proved Caucasoid populations (Russian, Tajik and Uzbek), as well as those of Turkic ethnic groups from the Southern Siberia (Altaians and Tuvinians) and Khanty and Mansy populations to be the recipients of a considerable gene flow from the outside of the concerned population system, as compared with the East Siberian and the Far East ethnic groups. The results of the correlation analysis received with use polymorphic Alu insertion testify to the greatest correlation of genetic distances with anthropological characteristics of populations.

Alu-mediated recombination defect in IGF1R: haploinsufficiency in a patient with short stature.

BACKGROUND: The insulin-like growth factor (IGF) receptor (IGF1R) is essential for normal development and growth. IGF1R mutations cause IGF-1 resistance resulting in intrauterine and postnatal growth failure. The phenotypic spectrum related to IGF1R mutations remains to be fully understood. METHODS: Auxological and endocrinological data of a patient identified previously were assessed. The patient's fibroblasts were studied to characterize the IGF1R deletion, mRNA fate, protein expression and signalling capabilities. RESULTS: The boy, who carries a heterozygous IGF1R exon 6 deletion caused by Alu element-mediated recombination and a heterozygous SHOX variant (p.Met240Ile), was born appropriate for gestational age but developed proportionate short stature postnatally. IGF-1 levels were low-normal. None of the stigmata associated with SHOX deficiency or sporadically observed in IGF1R mutation carriers were present. Nonsense-mediated mRNA decay led to a substantial decline of IGF1R dosage and IGF-1-dependent receptor autophosphorylation but not impaired downstream signalling. CONCLUSION: We present the first detailed report of an intragenic IGF1R deletion identified in a patient who, apart from short stature, deviates from all established markers that qualify a growth-retarded child for IGF1R analysis. Although such children will usually escape routine clinical mutation screenings, they can contribute to the understanding of factors and mechanisms that cooperate with the IGF1R.

The intertwining of transposable elements and non-coding RNAs.

Growing evidence shows a close association of transposable elements (TE) with non-coding RNAs (ncRNA), and a significant number of small ncRNAs originate from TEs. Further, ncRNAs linked with TE sequences participate in a wide-range of regulatory functions. Alu elements in particular are critical players in gene regulation and molecular pathways. Alu sequences embedded in both long non-coding RNAs (lncRNA) and mRNAs form the basis of targeted mRNA decay via short imperfect base-pairing. Imperfect pairing is prominent in most ncRNA/target RNA interactions and found throughout all biological kingdoms. The piRNA-Piwi complex is multifunctional, but plays a major role in protection against invasion by transposons. This is an RNA-based genetic immune system similar to the one found in prokaryotes, the CRISPR system. Thousands of long intergenic non-coding RNAs (lincRNAs) are associated with endogenous retrovirus LTR transposable elements in human cells. These TEs can provide regulatory signals for lincRNA genes. A surprisingly large number of long circular ncRNAs have been discovered in human fibroblasts. These serve as "sponges" for miRNAs. Alu sequences, encoded in introns that flank exons are proposed to participate in RNA circularization via Alu/Alu base-pairing. Diseases are increasingly found to have a TE/ncRNA etiology. A single point mutation in a SINE/Alu sequence in a human long non-coding RNA leads to brainstem atrophy and death. On the other hand, genomic clusters of repeat sequences as well as lncRNAs function in epigenetic regulation. Some clusters are unstable, which can lead to formation of diseases such as facioscapulohumeral muscular dystrophy. The future may hold more surprises regarding diseases associated with ncRNAs andTEs.

Functional regulation of Alu element of human angiotensin-converting enzyme gene in neuron cells.

The angiotensin-converting enzyme (ACE) insertion/deletion (I/D) genotype and its protein activity have been widely implicated to be associated with Alzheimer's disease (AD). However, whether the insertion sequence, Alu element, in intron 16 of the human ACE gene plays a functional role remains uncertain. To investigate the influence of the I/D polymorphism on ACE promoter, we recombined the I and D form fragments with the human ACE promoter sequence before the reporter gene in pSEAP-Basic2 vector. The effect of the Alu element on regulating the transcriptional activity of ACE promoter was examined using transient transfection in SH-SY5Y cells. We found that the I form fragment upregulated the transcriptional activity of ACE promoter by approximately 70% but that the D form fragment did not. Our study first reveals that Alu sequence in human ACE gene possesses a regulatory function on the ACE promoter activity in neuron. This novel finding bridges the gap between the association of ACE I/D genotype with AD, and suggests that Alu sequence is not merely a "junk" DNA in human ACE gene.

DICER- and AGO3-dependent generation of retinoic acid-induced DR2 Alu RNAs regulates human stem cell proliferation.

Although liganded nuclear receptors have been established to regulate RNA polymerase II (Pol II)-dependent transcription units, their role in regulating Pol III-transcribed DNA repeats remains largely unknown. Here we report that ~2-3% of the ~100,000-200,000 total human DR2 Alu repeats located in proximity to activated Pol II transcription units are activated by the retinoic acid receptor (RAR) in human embryonic stem cells to generate Pol III-dependent RNAs. These transcripts are processed, initially in a DICER-dependent fashion, into small RNAs (~28-65 nt) referred to as repeat-induced RNAs that cause the degradation of a subset of crucial stem-cell mRNAs, including Nanog mRNA, which modulate exit from the proliferative stem-cell state. This regulation requires AGO3-dependent accumulation of processed DR2 Alu transcripts and the subsequent recruitment of AGO3-associated decapping complexes to the target mRNA. In this way, the RAR-dependent and Pol III-dependent DR2 Alu transcriptional events in stem cells functionally complement the Pol II-dependent neuronal transcriptional program.

Human intron-encoded Alu RNAs are processed and packaged into Wdr79-associated nucleoplasmic box H/ACA RNPs.

Alu repetitive sequences are the most abundant short interspersed DNA elements in the human genome. Full-length Alu elements are composed of two tandem sequence monomers, the left and right Alu arms, both derived from the 7SL signal recognition particle RNA. Since Alu elements are common in protein-coding genes, they are frequently transcribed into pre-mRNAs. Here, we demonstrate that the right arms of nascent Alu transcripts synthesized within pre-mRNA introns are processed into metabolically stable small RNAs. The intron-encoded Alu RNAs, termed AluACA RNAs, are structurally highly reminiscent of box H/ACA small Cajal body (CB) RNAs (scaRNAs). They are composed of two hairpin units followed by the essential H (AnAnnA) and ACA box motifs. The mature AluACA RNAs associate with the four H/ACA core proteins: dyskerin, Nop10, Nhp2, and Gar1. Moreover, the 3' hairpin of AluACA RNAs carries two closely spaced CB localization motifs, CAB boxes (UGAG), which bind Wdr79 in a cumulative fashion. In contrast to canonical H/ACA scaRNPs, which concentrate in CBs, the AluACA RNPs accumulate in the nucleoplasm. Identification of 348 human AluACA RNAs demonstrates that intron-encoded AluACA RNAs represent a novel, large subgroup of H/ACA RNAs, which are apparently confined to human or primate cells.

Widespread occurrence of power-law distributions in inter-repeat distances shaped by genome dynamics.

Repetitive DNA sequences derived from transposable elements (TE) are distributed in a non-random way, co-clustering with other classes of repeat elements, genes and other genomic components. In a previous work we reported power-law-like size distributions (linearity in log-log scale) in the spatial arrangement of Alu and LINE1 elements in the human genome. Here we investigate the large-scale features of the spatial arrangement of all principal classes of TEs in 14 genomes from phylogenetically distant organisms by studying the size distribution of inter-repeat distances. Power-law-like size distributions are found to be widespread, extending up to several orders of magnitude. In order to understand the emergence of this distributional pattern, we introduce an evolutionary scenario, which includes (i) Insertions of DNA segments (e.g., more recent repeats) into the considered sequence and (ii) Eliminations of members of the studied TE family. In the proposed model we also incorporate the potential for transposition events (characteristic of the DNA transposons' life-cycle) and segmental duplications. Simulations reproduce the main features of the observed size distributions. Furthermore, we investigate the effects of various genomic features on the presence and extent of power-law size distributions including TE class and age, mode of parental TE transmission, GC content, deletion and recombination rates in the studied genomic region, etc. Our observations corroborate the hypothesis that insertions of genomic material and eliminations of repeats are at the basis of power-laws in inter-repeat distances. The existence of these power-laws could facilitate the formation of the recently proposed "fractal globule" for the confined chromatin organization.

Misfolded human tRNA isodecoder binds and neutralizes a 3' UTR-embedded Alu element.

Several classes of small noncoding RNAs are key players in cellular metabolism including mRNA decoding, RNA processing, and mRNA stability. Here we show that a tRNA(Asp) isodecoder, corresponding to a human tRNA-derived sequence, binds to an embedded Alu RNA element contained in the 3' UTR of the human aspartyl-tRNA synthetase mRNA. This interaction between two well-known classes of RNA molecules, tRNA and Alu RNA, is driven by an unexpected structural motif and induces a global rearrangement of the 3' UTR. Besides, this 3' UTR contains two functional polyadenylation signals. We propose a model where the tRNA/Alu interaction would modulate the accessibility of the two alternative polyadenylation sites and regulate the stability of the mRNA. This unique regulation mechanism would link gene expression to RNA polymerase III transcription and may have implications in a primate-specific signal pathway.

Human nucleosomes: special role of CG dinucleotides and Alu-nucleosomes.

BACKGROUND: The periodical occurrence of dinucleotides with a period of 10.4 bases now is undeniably a hallmark of nucleosome positioning. Whereas many eukaryotic genomes contain visible and even strong signals for periodic distribution of dinucleotides, the human genome is rather featureless in this respect. The exact sequence features in the human genome that govern the nucleosome positioning remain largely unknown. RESULTS: When analyzing the human genome sequence with the positional autocorrelation method, we found that only the dinucleotide CG shows the 10.4 base periodicity, which is indicative of the presence of nucleosomes. There is a high occurrence of CG dinucleotides that are either 31 (10.4 × 3) or 62 (10.4 × 6) base pairs apart from one another - a sequence bias known to be characteristic of Alu-sequences. In a similar analysis with repetitive sequences removed, peaks of repeating CG motifs can be seen at positions 10, 21 and 31, the nearest integers of multiples of 10.4. CONCLUSIONS: Although the CG dinucleotides are dominant, other elements of the standard nucleosome positioning pattern are present in the human genome as well.The positional autocorrelation analysis of the human genome demonstrates that the CG dinucleotide is, indeed, one visible element of the human nucleosome positioning pattern, which appears both in Alu sequences and in sequences without repeats. The dominant role that CG dinucleotides play in organizing human chromatin is to indicate the involvement of human nucleosomes in tuning the regulation of gene expression and chromatin structure, which is very likely due to cytosine-methylation/-demethylation in CG dinucleotides contained in the human nucleosomes. This is further confirmed by the positions of CG-periodical nucleosomes on Alu sequences. Alu repeats appear as monomers, dimers and trimers, harboring two to six nucleosomes in a run. Considering the exceptional role CG dinucleotides play in the nucleosome positioning, we hypothesize that Alu-nucleosomes, especially, those that form tightly positioned runs, could serve as "anchors" in organizing the chromatin in human cells.