Repeat-containing RNA Research Articles

Telomere functions grounding on TERRA firma.

Long noncoding telomeric repeat-containing RNAs - TERRAs - are transcribed in a regulated manner from telomeres throughout eukaryotes. TERRA molecules consist of chromosome end-specific subtelomeric sequences and telomeric repeats at their 3' ends. Recent work suggests that TERRA sustains several important functions at chromosome ends. TERRA can regulate telomere length through modulation of exonuclease 1 and telomerase, it may promote recruitment of chromatin modifiers to damaged telomeres and thereby enable DNA end-processing, and it may promote telomere protein composition changes during cell cycle progression. Furthermore, telomere transcription regulates chromosome-end mobility within the nucleus. We review how TERRA, by regulated expression and by providing a molecular scaffold for various protein enzymes, can support a large variety of vital functions.

Dipeptide repeat peptides on the attack

Neurodegeneration![Figure][1] Studying neurodegeneration in Drosophila CREDIT: TERESA NICCOLI/UCL INSTITUTE OF AGEING, LONDON, UK Certain neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), are associated with expanded dipeptides translated from RNA transcripts of disease-associated genes (see the Perspective by Paul and Gitler). Kwon et al. show that the peptides encoded by the expanded repeats in the C9orf72 gene interfere with the way cells make RNA and kill cells. These effects may account for how this genetic form of ALS causes disease. Working in Drosophila , Mizielinska et al. aimed to distinguish between the effects of repeat-containing RNAs and the dipeptide repeat peptides that they encode. The findings provide evidence that dipeptide repeat proteins can cause toxicity directly. Science , this issue p. [1139][2] and p. [1192][3]; see also p. [1118][4] [1]: pending:yes [2]: /lookup/doi/10.1126/science.1254917 [3]: /lookup/doi/10.1126/science.1256800 [4]: /lookup/doi/10.1126/science.1259461

Identification of TERRA locus unveils a telomere protection role through association to nearly all chromosomes.

Telomeric RNAs (TERRAs) are UUAGGG repeat-containing RNAs that are transcribed from the subtelomere towards the telomere. The precise genomic origin of TERRA has remained elusive. Using a whole-genome RNA-sequencing approach, we identify novel mouse transcripts arising mainly from the subtelomere of chromosome 18, and to a lesser extend chromosome 9, that resemble TERRA in several key aspects. Those transcripts contain UUAGGG-repeats and are heterogeneous in size, fluctuate in abundance in a TERRA-like manner during the cell cycle, are bound by TERRA RNA-binding proteins and are regulated in a manner similar to TERRA in response to stress and the induction of pluripotency. These transcripts are also found to associate with nearly all chromosome ends and downregulation of the transcripts that originate from chromosome 18 causes a reduction in TERRA abundance. Interestingly, downregulation of either chromosome 18 transcripts or TERRA results in increased number of telomere dysfunction-induced foci, suggesting a protective role at telomeres.

C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins.

An expanded GGGGCC repeat in C9orf72 is the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis. A fundamental question is whether toxicity is driven by the repeat RNA itself and/or by dipeptide repeat proteins generated by repeat-associated, non-ATG translation. To address this question, we developed in vitro and in vivo models to dissect repeat RNA and dipeptide repeat protein toxicity. Expression of pure repeats, but not stop codon-interrupted "RNA-only" repeats in Drosophila caused adult-onset neurodegeneration. Thus, expanded repeats promoted neurodegeneration through dipeptide repeat proteins. Expression of individual dipeptide repeat proteins with a non-GGGGCC RNA sequence revealed that both poly-(glycine-arginine) and poly-(proline-arginine) proteins caused neurodegeneration. These findings are consistent with a dual toxicity mechanism, whereby both arginine-rich proteins and repeat RNA contribute to C9orf72-mediated neurodegeneration.

Construction of a microsatellites-based linkage map for the white grouper (Epinephelus aeneus).

The white grouper (Epinephelus aeneus) is a promising candidate for domestication and aquaculture due to its fast growth, excellent taste, and high market price. A linkage map is an essential framework for mapping quantitative trait loci for economic traits and the study of genome evolution. DNA of a single individual was deep-sequenced, and microsatellite markers were identified in 177 of the largest scaffolds of the sequence assembly. The success rate of developing polymorphic homologous markers was 94.9% compared with 63.1% of heterologous markers from other grouper species. Of the 12 adult mature fish present in the broodstock tank, two males and two females were identified as parents of the assigned offspring by parenthood analysis using 34 heterologous markers. A single full-sib family of 48 individuals was established for the construction of first-generation linkage maps based on genotyping data of 222 microsatellites. The markers were assigned to 24 linkage groups in accordance to the 24 chromosomal pairs. The female and male maps consisting of 203 and 202 markers spanned 1053 and 886 cM, with an average intermarker distance of 5.8 and 5.0 cM, respectively. Mapping of markers to linkage groups ends was enriched by using markers originating from scaffolds harboring telomeric repeat-containing RNA. Comparative mapping showed high synteny relationships among the white grouper, kelp grouper (E. bruneus), orange-spotted grouper (E. coioides), and Nile tilapia (Oreochromis niloticus). Thus, it would be useful to integrate the markers that were developed for different groupers, depending on sharing of sequence data, into a comprehensive consensus map.

Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells.

Many RNA regulatory proteins controlling pre-messenger RNA splicing contain serine:arginine (SR) repeats. Here, we found that these SR domains bound hydrogel droplets composed of fibrous polymers of the low-complexity domain of heterogeneous ribonucleoprotein A2 (hnRNPA2). Hydrogel binding was reversed upon phosphorylation of the SR domain by CDC2-like kinases 1 and 2 (CLK1/2). Mutated variants of the SR domains changing serine to glycine (SR-to-GR variants) also bound to hnRNPA2 hydrogels but were not affected by CLK1/2. When expressed in mammalian cells, these variants bound nucleoli. The translation products of the sense and antisense transcripts of the expansion repeats associated with the C9orf72 gene altered in neurodegenerative disease encode GRn and PRn repeat polypeptides. Both peptides bound to hnRNPA2 hydrogels independent of CLK1/2 activity. When applied to cultured cells, both peptides entered cells, migrated to the nucleus, bound nucleoli, and poisoned RNA biogenesis, which caused cell death.

Telomere homeostasis is compromised in spermatocytes from patients with idiopathic infertility

To study whether the telomere structure of germ cells from idiopathic infertile men is altered and if this impairment is influenced by meiotic recombination and telomere length. We performed a detailed analysis of both telomeric repeat-containing RNA (TERRA) and telomerase distribution in testis cell spreads by combining immunofluorescence and RNA fluorescent in situ hybridization. In addition we analyzed meiotic recombination between homologous chromosomes by immunofluorescence and telomere length by quantitative fluorescent in situ hybridization. University. Men consulting for fertility problems. Unilateral testicular biopsies. We observed that TERRA levels and its nuclear distribution were compromised in infertile patients. In addition, the presence of the protein component of telomerase at telomeres decreased in the affected patients. However, neither telomerase-TERRA association nor telomere length was altered in spermatocytes I of infertile samples compared with control individuals. In addition, we observed that meiotic recombination was reduced in infertile individuals. Telomere homeostasis is impaired in infertile patients, and this was translated into a decrease in TERRA levels together with an alteration of the TERRA-protein component of telomerase telomeric association in primary spermatocytes. This study demonstrates for the first time that telomere structure and homeostasis in germ cells is compromised in infertile individuals. In the light of our results we propose that the analysis of telomeric structure (i.e., TERRA levels and telomere association with TERRA and telomerase) would provide new tools for our understanding of the origin of human infertility.

Finding a human telomere DNA–RNA hybrid G-quadruplex formed by human telomeric 6-mer RNA and 16-mer DNA using click chemistry: A protective structure for telomere end

Telomeric repeat-containing RNA is a non-coding RNA molecule newly found in mammalian cells. The telomere RNA has been found to localize to the telomere DNA, but how the newly discovered RNA molecule interacts with telomere DNA is less known. In this study, using the click chemistry we successfully found that a 6-mer human telomere RNA and 16-mer human telomere DNA sequence can form a DNA–RNA hybrid type G-quadruplex structure. Detection of the click-reaction products directly probes DNA–RNA G-quadruplex structures in a complicated solution, whereas traditional methods such as NMR and crystallography may not be suitable. Importantly, we found that formation of DNA–RNA G-quadruplex induced an exonuclease resistance for telomere DNA, indicating that such structures might be important for protecting telomeric DNA from enzyme digestion to avoid telomere DNA shortening. These results provide the direct evidence for formation of DNA–RNA hybrid G-quadruplex structure by human telomere DNA and RNA sequence, suggesting DNA–RNA hybrid G-quadruplex structure associated between telomere DNA and RNA may respond to chromosome end protection and/or present a valuable target for drug design.

Discovery of selective ligands for telomeric RNA G-quadruplexes (TERRA) through 19F-NMR based fragment screening.

Telomeric repeat-containing RNA (TERRA) is a novel and very attractive antitumoral target. Here, we report the first successful application of (19)F-NMR fragment-based screening to identify chemically diverse compounds that bind to an RNA molecule such as TERRA. We have built a library of 355 fluorinated fragments, and checked their interaction with a long telomeric RNA as a target molecule. The screening resulted in the identification of 20 hits (hit rate of 5.6%). For a number of binders, their interaction with TERRA was confirmed by (19)F- and (1)H NMR as well as by CD melting experiments. We have also explored the selectivity of the ligands for RNA G-quadruplexes and found that some of the hits do not interact with other nucleic acids such as tRNA and duplex DNA and, most importantly, favor the propeller-like parallel conformation in telomeric DNA G-quadruplexes. This suggests a selective recognition of this particular quadruplex topology and that different ligands may recognize specific sites in propeller-like parallel G-quadruplexes. Such features make some of the resulting binders promising lead compounds for fragment based drug discovery.

TERRA enhances production of senescence-associated inflammatory cytokines IL-6 and IL-8 (IRC5P.452)

Abstract Aged or senescent cells develop a complex senescence-associated secretory phenotype (SASP). However, the mechanisms underlying the induction of the SASP are unclear. Telomeres are critical for genome stability and aging. Telomeres are transcribed into long non-coding telomeric repeat-containing RNA (TERRA) molecules. We demonstrate that TERRA and inflammatory cytokines were significantly elevated during senescence. Over expression of TERRA significantly upregulated IL-6 and IL-8, but IL-6 and IL-8 did not modulate TERRA expression. The results show that TERRA expression is related to senescence and increases production of SASP. Our findings expand the biological function of TERRA in senscence-assosciated inflammatory responses.

Telomeric Repeat-Containing RNA (TERRA) and Telomerase Are Components of Telomeres During Mammalian Gametogenesis1

Telomeres are ribonucleoprotein structures at the end of chromosomes composed of telomeric DNA, specific-binding proteins, and noncoding RNA (TERRA). Despite their importance in preventing chromosome instability, little is known about the cross talk between these three elements during the formation of the germ line. Here, we provide evidence that both TERRA and the telomerase enzymatic subunit (TERT) are components of telomeres in mammalian germ cells. We found that TERRA colocalizes with telomeres during mammalian meiosis and that its expression progressively increases during spermatogenesis until the beginning of spermiogenesis. While both TERRA levels and distribution would be regulated in a gender-specific manner, telomere-TERT colocalization appears to be regulated based on species-specific characteristics of the telomeric structure. Moreover, we found that TERT localization at telomeres is maintained throughout spermatogenesis as a structural component without affecting telomere elongation. Our results represent the first evidence of colocalization between telomerase and telomeres during mammalian gametogenesis.

A Human Artificial Chromosome Recapitulates the Metabolism of Native Telomeres in Mammalian Cells

Telomeric and subtelomeric regions of human chromosomes largely consist of highly repetitive and redundant DNA sequences, resulting in a paucity of unique DNA sequences specific to individual telomeres. Accordingly, it is difficult to analyze telomere metabolism on a single-telomere basis. To circumvent this problem, we have exploited a human artificial chromosome (HAC#21) derived from human chromosome 21 (hChr21). HAC#21 was generated through truncation of the long arm of native hChr21 by the targeted telomere seeding technique. The newly established telomere of HAC#21 lacks canonical subtelomere structures but possesses unique sequences derived from the target vector backbone and the internal region of hChr21 used for telomere targeting, which enabled us to molecularly characterize the single HAC telomere. We established HeLa and NIH-3T3 sub-lines containing a single copy of HAC#21, where it was robustly maintained. The seeded telomere is associated with telomeric proteins over a length similar to that reported in native telomeres, and is faithfully replicated in mid-S phase in HeLa cells. We found that the seeded telomere on HAC#21 is transcribed from the newly juxtaposed site. The transcript, HAC-telRNA, shares several features with TERRA (telomeric repeat-containing RNA): it is a short-lived RNA polymerase II transcript, rarely contains a poly(A) tail, and associates with chromatin. Interestingly, HAC-telRNA undergoes splicing. These results suggest that transcription into TERRA is locally influenced by the subtelomeric context. Taken together, we have established human and mouse cell lines that will be useful for analyzing the behavior of a uniquely identifiable, functional telomere.

Telomeric transcripts stimulate telomere recombination to suppress senescence in cells lacking telomerase

In human somatic cells or yeast cells lacking telomerase, telomeres are shortened upon each cell division. This gradual shortening of telomeres eventually leads to senescence. However, a small population of telomerase-deficient cells can survive by bypassing senescence through the activation of alternative recombination pathways to maintain their telomeres. Although genes involved in telomere recombination have been identified, mechanisms that trigger telomere recombination are less known. The THO (suppressor of the transcriptional defects of Hpr1 mutants by overexpression) complex is involved in transcription elongation and mRNA export. Here we demonstrate that mutations in THO complex components can stimulate early senescence and type II telomere recombination in cells lacking telomerase. The accumulation of telomere-associated noncoding telomere repeat-containing RNA (TERRA) is required for the observed telomere effects in THO complex mutants; reduced transcriptional efficiency, or overexpression of RNase H or C(1-3)A RNA can severely impair the type II telomere recombination. The results highlight a unique function for telomere-associated TERRA, in the formation of type II survivors. Moreover, because TERRA is a long noncoding RNA, these results reveal a function for long noncoding RNA in regulating recombination.

Telomeric noncoding RNA: telomeric repeat‐containing RNA in telomere biology

Telomeres are nucleoprotein structures that cap the ends of eukaryotic chromosomes, protecting them from degradation and activation of DNA damage response. For this reason, functional telomeres are vital to genome stability. For years, telomeres were assumed to be transcriptionally silent, because of their heterochromatic state. It was only recently shown that, in several organisms, telomeres are transcribed, giving rise to a long noncoding RNA (lncRNA) called telomeric repeat-containing RNA (TERRA). Several lines of evidence now indicate that TERRA molecules play crucial roles in telomere homeostasis and telomere functions. Recent studies have shown that the expression and regulation of TERRA are dynamically controlled by each chromosome end. TERRA has been involved in the regulation of telomere length, telomerase activity, and heterochromatin formation at telomeres. The correct regulation of the telomeric transcripts may be essential to genome stability, and altered TERRA levels associate with tumorigenesis and cellular senescence. Thus, the study of the molecular mechanisms of TERRA biogenesis and function may advance the understanding of telomere-related diseases, including cancer and aging.

Probe development for detection of TERRA 1 intramolecular G-quadruplex formation using a fluorescent adenosine derivative

We developed a probing system to detect the intramolecular G-quadruplex of telomeric repeat-containing RNA (TERRA 1). We used a fluorescent adenosine derivative rApy as a fluorophore and incorporated it into the dangling position of the parallel-type G-quadruplex sequence of TERRA 1. The rApy-modified G-quadruplex structure exhibited a strong fluorescence emission signal, while the emission signals of the single-strand and duplex structures were much lower.

Mechanisms of toxicity in C9FTLD/ALS

A hexanucleotide repeat expansion within a non-coding region of the C9ORF72 gene is the most common mutation causative of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Elucidating how this bidirectionally transcribed G4C2·C4G2 expanded repeat causes "C9FTLD/ALS" has since become an important goal of the field. Likely pathogenic mechanisms include toxicity induced by repeat-containing RNAs, and loss of C9orf72 function due to epigenetic changes resulting in decreased C9ORF72 mRNA expression. With regards to the former, sense and antisense transcripts of the expanded repeat aberrantly interact with various RNA-binding proteins and form discrete nuclear structures, termed RNA foci. These foci have the capacity to sequester select RNA-binding proteins, thereby impairing their function. (G4C2)exp and (C4G2)exp transcripts also succumb to an alternative fate: repeat-associated non-ATG (RAN) translation. This unconventional mode of translation, which occurs in the absence of an initiating codon, results in the abnormal production of poly(GA), poly(GP), poly(GR), poly(PR) and poly(PA) peptides, collectively referred to as C9RAN proteins. C9RAN proteins form neuronal inclusions throughout the central nervous system of C9FTLD/ALS patients and may contribute to disease pathogenesis. This review aims to summarize the important findings from studies examining mechanisms of disease in C9FTLD/ALS, and will also highlight some of the many questions in need of further investigation.

Chapter One - Telomere and Telomerase Biology

Telomeres are the physical ends of eukaryotic linear chromosomes. Telomeres form special structures that cap chromosome ends to prevent degradation by nucleolytic attack and to distinguish chromosome termini from DNA double-strand breaks. With few exceptions, telomeres are composed primarily of repetitive DNA associated with proteins that interact specifically with double- or single-stranded telomeric DNA or with each other, forming highly ordered and dynamic complexes involved in telomere maintenance and length regulation. In proliferative cells and unicellular organisms, telomeric DNA is replicated by the actions of telomerase, a specialized reverse transcriptase. In the absence of telomerase, some cells employ a recombination-based DNA replication pathway known as alternative lengthening of telomeres. However, mammalian somatic cells that naturally lack telomerase activity show telomere shortening with increasing age leading to cell cycle arrest and senescence. In another way, mutations or deletions of telomerase components can lead to inherited genetic disorders, and the depletion of telomeric proteins can elicit the action of distinct kinases-dependent DNA damage response, culminating in chromosomal abnormalities that are incompatible with life. In addition to the intricate network formed by the interrelationships among telomeric proteins, long noncoding RNAs that arise from subtelomeric regions, named telomeric repeat-containing RNA, are also implicated in telomerase regulation and telomere maintenance. The goal for the next years is to increase our knowledge about the mechanisms that regulate telomere homeostasis and the means by which their absence or defect can elicit telomere dysfunction, which generally results in gross genomic instability and genetic diseases.

Specific Binding of Modified RGG Domain in TLS/FUS to G-Quadruplex RNA: Tyrosines in RGG Domain Recognize 2′-OH of the Riboses of Loops in G-Quadruplex

Telomeric repeat-containing RNA (TERRA), which contains tandem arrays of short RNA repeats, r(UUAGGG), is an integral component of the telomere and contributes to telomeric heterochromatin formation and telomere-length regulation. TERRA forms a G-quadruplex, but the biologic significance of its G-quadruplex formation is unknown. Compounds that selectively bind to G-quadruplex RNA are useful for understanding G-quadruplex TERRA. Here we report that an engineered RGG domain translocated in liposarcoma (TLS) specifically binds to G-quadruplex TERRA. The Arg-Gly-Gly repeat (RGG) TLS binds to G-quadruplex human telomere DNA and TERRA simultaneously, but we show that substitution of Tyr for Phe in the RGG domain of TLS (TLSRGG3Y) converts its binding specificity solely toward G-quadruplex TERRA. TLSRGG3Y binds to dG tetrads with abasic RNA loops, but fails to bind to rG tetrads without loops or dG tetrads with abasic DNA loops. These findings suggest that TLSRGG3Y binds to loops within the G-quadruplexes of TERRA by recognizing the 2'-OH of the riboses. To our knowledge, TLSRGG3Y is the first known molecule that specifically recognizes the 2'-OH of the riboses of loops in the G-quadruplex. TLSRGG3Y will be useful for investigating the role of the G-quadruplex form of TERRA without affecting G-quadruplex telomere DNA functions.

Targeted degradation of sense and antisense C9orf72 RNA foci as therapy for ALS and frontotemporal degeneration

Expanded hexanucleotide repeats in the chromosome 9 open reading frame 72 (C9orf72) gene are the most common genetic cause of ALS and frontotemporal degeneration (FTD). Here, we identify nuclear RNA foci containing the hexanucleotide expansion (GGGGCC) in patient cells, including white blood cells, fibroblasts, glia, and multiple neuronal cell types (spinal motor, cortical, hippocampal, and cerebellar neurons). RNA foci are not present in sporadic ALS, familial ALS/FTD caused by other mutations (SOD1, TDP-43, or tau), Parkinson disease, or nonneurological controls. Antisense oligonucleotides (ASOs) are identified that reduce GGGGCC-containing nuclear foci without altering overall C9orf72 RNA levels. By contrast, siRNAs fail to reduce nuclear RNA foci despite marked reduction in overall C9orf72 RNAs. Sustained ASO-mediated lowering of C9orf72 RNAs throughout the CNS of mice is demonstrated to be well tolerated, producing no behavioral or pathological features characteristic of ALS/FTD and only limited RNA expression alterations. Genome-wide RNA profiling identifies an RNA signature in fibroblasts from patients with C9orf72 expansion. ASOs targeting sense strand repeat-containing RNAs do not correct this signature, a failure that may be explained, at least in part, by discovery of abundant RNA foci with C9orf72 repeats transcribed in the antisense (GGCCCC) direction, which are not affected by sense strand-targeting ASOs. Taken together, these findings support a therapeutic approach by ASO administration to reduce hexanucleotide repeat-containing RNAs and raise the potential importance of targeting expanded RNAs transcribed in both directions.

Telomeric RNA-DNA hybrids affect telomere-length dynamics and senescence

Although telomeres are heterochromatic, they are transcribed into noncoding telomeric repeat-containing RNA (TERRA). Here we show that RNA-DNA hybrids form at telomeres and are removed by RNase H enzymes in the budding yeast, Saccharomyces cerevisiae. In recombination-competent telomerase mutants, telomeric RNA-DNA hybrids promote recombination-mediated elongation events that delay the onset of cellular senescence. Reduction of TERRA and telomeric RNA-DNA-hybrid levels diminishes rates of recombination-mediated telomere elongation in cis. Overexpression of RNase H decreases telomere recombination rates and accelerates senescence in recombination-competent but not recombination-deficient cells. In contrast, in the absence of both telomerase and homologous recombination, accumulation of telomeric RNA-DNA hybrids leads to telomere loss and accelerated rates of cellular senescence. Therefore, the regulation of TERRA transcription and telomeric RNA-DNA-hybrid formation are important determinants of both telomere-length dynamics and proliferative potential after the inactivation of telomerase.