Interphase chromatin LINEd with RNA.

RNA has been proposed to be a component of an underlying nuclear matrix. Hall et al. show that noncoding, repetitive RNAs, some derived from LINE1 elements, stably associate with interphase chromosomes and copurify with nuclear scaffold, indicating that RNAs might impact interphase chromosome architecture.

Stable C0T-1 repeat RNA is abundant and is associated with euchromatic interphase chromosomes.

Recent studies recognize a vast diversity of noncoding RNAs with largely unknown functions, but few have examined interspersed repeat sequences, which constitute almost half our genome. RNA hybridization in situ using C0T-1 (highly repeated) DNA probes detects surprisingly abundant euchromatin-associated RNA comprised predominantly of repeat sequences (C0T-1 RNA), including LINE-1. C0T-1-hybridizing RNA strictly localizes to the interphase chromosome territory in cis and remains stably associated with the chromosome territory following prolonged transcriptional inhibition. The C0T-1 RNA territory resists mechanical disruption and fractionates with the nonchromatin scaffold but can be experimentally released. Loss of repeat-rich, stable nuclear RNAs from euchromatin corresponds to aberrant chromatin distribution and condensation. C0T-1 RNA has several properties similar to XIST chromosomal RNA but is excluded from chromatin condensed by XIST. These findings impact two "black boxes" of genome science: the poorly understood diversity of noncoding RNA and the unexplained abundance of repetitive elements.

Ro60 and Y RNAs: structure, functions, and roles in autoimmunity.

Ro60, also known as SS-A or TROVE2, is an evolutionarily conserved RNA-binding protein that is found in most animal cells, approximately 5% of sequenced prokaryotic genomes and some archaea. Ro60 is present in cells as both a free protein and as a component of a ribonucleoprotein complex, where its best-known partners are members of a class of noncoding RNAs called Y RNAs. Structural and biochemical analyses have revealed that Ro60 is a ring-shaped protein that binds Y RNAs on its outer surface. In addition to Y RNAs, Ro60 binds misfolded and aberrant noncoding RNAs in some animal cell nuclei. Although the fate of these defective Ro60-bound noncoding RNAs in animal cells is not well-defined, a bacterial Ro60 ortholog functions with 3' to 5' exoribonucleases to assist structured RNA degradation. Studies of Y RNAs have revealed that these RNAs regulate the subcellular localization of Ro60, tether Ro60 to effector proteins and regulate the access of other RNAs to its central cavity. As both mammalian cells and bacteria lacking Ro60 are sensitized to ultraviolet irradiation, Ro60 function may be important during exposure to some environmental stressors. Here we summarize the current knowledge regarding the functions of Ro60 and Y RNAs in animal cells and bacteria. Because the Ro60 RNP is a clinically important target of autoantibodies in patients with rheumatic diseases such as Sjogren's syndrome, systemic lupus erythematosus, and neonatal lupus, we also discuss potential roles for Ro60 RNPs in the initiation and pathogenesis of systemic autoimmune rheumatic disease.

Comprehensive discovery of novel structured noncoding RNAs in 26 bacterial genomes.

Comparative sequence analysis methods are highly effective for uncovering novel classes of structured noncoding RNAs (ncRNAs) from bacterial genomic DNA sequence datasets. Previously, we developed a computational pipeline to more comprehensively identify structured ncRNA representatives from individual bacterial genomes. This search process exploits the fact that genomic regions serving as templates for the transcription of structured RNAs tend to be present in longer than average noncoding 'intergenic regions' (IGRs) that are enriched in G and C nucleotides compared to the remainder of the genome. In the present study, we apply this computational pipeline to identify structured ncRNA candidates from 26 diverse bacterial species. Numerous novel structured ncRNA motifs were discovered, including several riboswitch candidates, one whose ligand has been identified and others that have yet to be experimentally validated. Our findings support recent predictions that hundreds of novel ribo-switch classes and other ncRNAs remain undiscovered among the limited number of bacterial species whose genomes have been completely sequenced.

Assessing the involvement of the placental microbiome and virome in preeclampsia using non coding RNA sequencing.

OBJECTIVES: Preeclampsia is a dangerous pregnancy complication. The source of preeclampsia is unknown, though the placenta is believed to have a central role in its pathogenesis. An association between maternal infection and preeclampsia has been demonstrated, yet the involvement of the placental microbiome in the etiology of preeclampsia has not been determined. In this study, we examined whether preeclampsia is associated with an imbalanced microorganism composition in the placenta. METHODS: To this end, we developed a novel method for the identification of bacteria/viruses based on sequencing of small non-coding RNA, which increases the microorganism-to-host ratio, this being a major challenge in microbiome methods. We validated the method on various infected tissues and demonstrated its efficiency in detecting microorganisms in samples with extremely low bacterial/viral biomass. We then applied the method to placenta specimens from preeclamptic and healthy pregnancies. Since the placenta is a remarkably large and heterogeneous organ, we explored the bacterial and viral RNA at each of 15 distinct locations. RESULTS: Bacterial RNA was detected at all locations and was consistent with previous studies of the placental microbiome, though without significant differences between the preeclampsia and control groups. Nevertheless, the bacterial RNA composition differed significantly between various areas of the placenta. Viral RNA was detected in extremely low quantities, below the threshold of significance, thus viral abundance could not be determined. CONCLUSIONS: Our results suggest that the bacterial and viral abundance in the placenta may have only limited involvement in the pathogenesis of preeclampsia. The evidence of a heterogenic bacterial RNA composition in the various placental locations warrants further investigation to capture the true nature of the placental microbiome.

Are Non-Coding RNAs Useful Biomarkers in Parathyroid Tumorigenesis?

Tumors of the parathyroid glands are common endocrine diseases almost always characterized by parathyroid hormone hypersecretion that determines the clinical manifestations of primary hyperparathyroidism, such as fatigue, kidney problems, weakness, brittle bones, and other symptoms. Most parathyroid neoplasia are benign adenomas, although rare malignant forms have been described. They are heterogeneous in terms of clinical presentation and the associated signs and symptoms overlap with those of disease and aging. Furthermore, most patients with hypercalcemia are discovered during routine blood tests for other reasons. Surgical removal is considered the main therapeutic option to cure these endocrine tumors and, therefore, innovative therapeutic approaches are actively required. Recently, a growing number of studies have suggested that alterations to the epigenetic mechanisms could play a pivotal role in parathyroid tumorigenesis. Most of the attention has been focused on non-coding RNAs (ncRNAs) (i.e., miRNAs, lncRNAs, and circRNAs) whose expression profile has been found to be deregulated in parathyroid tumors. The aim of the present paper is to give an insight into the ncRNAs involved in parathyroid tumorigenesis, which could be used in the future either as innovative diagnostic biomarkers or as therapeutic targets for the treatment of this endocrine neoplasia.

Quantitative analysis of noncoding RNA from paired fresh and formalin-fixed paraffin-embedded brain tissues.

Formalin-fixed paraffin-embedded (FFPE) tissues are commonly used both clinically and in forensic pathology. Recently, noncoding RNA (ncRNA) has attracted interest among molecular medical researchers. However, it remains unclear whether newly identified ncRNAs, such as long noncoding RNA (lncRNA) and circular RNA (circRNA), remain stable for downstream molecular analysis in FFPE tissues. Here, we assessed the feasibility of using autoptic FFPE brain tissues from eight individuals to perform quantitative molecular analyses. Selected RNA targets (9 mRNAs and 15 ncRNAs) with different amplicon lengths were studied by RT-qPCR in paired fresh and FFPE specimens. For RNA quality assessment, RNA purity and yield were comparable between the two sample cohorts; however, the RNA integrity number decreased significantly during FFPE sampling. Amplification efficiency also displayed certain variability related with amplicon length and RNA species. We found molecular evidence that short amplicons of mRNA, lncRNA, and circRNA were amplified more efficiently than long amplicons. With the assistance of RefFinder, 5S, SNORD48, miR-103a, and miR-125b were selected as reference genes given their high stability. After normalization, we found that short amplicon markers (e.g., ACTB mRNA and MALAT1 lncRNA) exhibited high consistency of quantification in paired fresh/FFPE samples. In particular, circRNAs (XPO1, HIPK3, and TMEM56) presented relatively consistent and stable expression profiles in FFPE tissues compared with their corresponding linear transcripts. Additionally, we evaluated the influence of prolonged storage time on the amplification of gene transcripts and found that short amplicons still work effectively in archived FFPE biospecimens. In conclusion, our findings demonstrate the possibility of performing accurate quantitative analysis of ncRNAs using short amplicons and standardized RT-qPCR assays in autopsy-derived FFPE samples.

Evolving methods for rational de novo design of functional RNA molecules.

Artificial RNA molecules with novel functionality have many applications in synthetic biology, pharmacy and white biotechnology. The de novo design of such devices using computational methods and prediction tools is a resource-efficient alternative to experimental screening and selection pipelines. In this review, we describe methods common to many such computational approaches, thoroughly dissect these methods and highlight open questions for the individual steps. Initially, it is essential to investigate the biological target system, the regulatory mechanism that will be exploited, as well as the desired components in order to define design objectives. Subsequent computational design is needed to combine the selected components and to obtain novel functionality. This process can usually be split into constrained sequence sampling, the formulation of an optimization problem and an in silico analysis to narrow down the number of candidates with respect to secondary goals. Finally, experimental analysis is important to check whether the defined design objectives are indeed met in the target environment and detailed characterization experiments should be performed to improve the mechanistic models and detect missing design requirements.

Immobilization of proteins in the nucleolus by ribosomal intergenic spacer noncoding RNA.

Cellular pathways are established and maintained by stochastic interactions of highly mobile molecules. The nucleolus plays a central role in the regulation of these molecular networks by capturing and immobilizing proteins. Here, we report a function for noncoding RNA (ncRNA) in the regulation of protein dynamics of key cellular factors, including VHL, Hsp70 and MDM2/PML. Stimuli-specific loci of the nucleolar intergenic spacer produce ncRNA capable of capturing and immobilizing proteins that encode a discrete peptidic code referred to as the nucleolar detention sequence (NoDS). Disruption of the NoDS/intergenic RNA interaction enables proteins to evade nucleolar sequestration and retain their dynamic profiles. Mislocalization of intergenic ncRNA triggers protein immobilization outside of the nucleolus, demonstrating that these ncRNA species can operate independently from the nucleolar architecture. We propose a model whereby protein immobilization by ncRNA is a posttranslational regulatory mechanism.

Non-Coding RNAs as New Therapeutic Targets in the Context of Renal Fibrosis.

Fibrosis, or tissue scarring, is defined as the excessive, persistent and destructive accumulation of extracellular matrix components in response to chronic tissue injury. Renal fibrosis represents the final stage of most chronic kidney diseases and contributes to the progressive and irreversible decline in kidney function. Limited therapeutic options are available and the molecular mechanisms governing the renal fibrosis process are complex and remain poorly understood. Recently, the role of non-coding RNAs, and in particular microRNAs (miRNAs), has been described in kidney fibrosis. Seminal studies have highlighted their potential importance as new therapeutic targets and innovative diagnostic and/or prognostic biomarkers. This review will summarize recent scientific advances and will discuss potential clinical applications as well as future research directions.

Modular regulatory principles of large non-coding RNAs.

It is clear that RNA has a diverse set of functions and is more than just a messenger between gene and protein. The mammalian genome is extensively transcribed, giving rise to thousands of non-coding transcripts. Whether all of these transcripts are functional is debated, but it is evident that there are many functional large non-coding RNAs (ncRNAs). Recent studies have begun to explore the functional diversity and mechanistic role of these large ncRNAs. Here we synthesize these studies to provide an emerging model whereby large ncRNAs might achieve regulatory specificity through modularity, assembling diverse combinations of proteins and possibly RNA and DNA interactions.

The non-coding RNA composition of the mitotic chromosome by 5'-tag sequencing.

Mitotic chromosomes are one of the most commonly recognized sub-cellular structures in eukaryotic cells. Yet basic information necessary to understand their structure and assembly, such as their composition, is still lacking. Recent proteomic studies have begun to fill this void, identifying hundreds of RNA-binding proteins bound to mitotic chromosomes. However, by contrast, there are only two RNA species (U3 snRNA and rRNA) that are known to be associated with the mitotic chromosome, suggesting that there are many mitotic chromosome-associated RNAs (mCARs) not yet identified. Here, using a targeted protocol based on 5'-tag sequencing to profile the mammalian mCAR population, we report the identification of 1279 mCARs, the majority of which are ncRNAs, including lncRNAs that exhibit greater conservation across 60 vertebrate species than the entire population of lncRNAs. There is also a significant enrichment of snoRNAs and specific SINE RNAs. Finally, ∼40% of the mCARs are presently unannotated, many of which are as abundant as the annotated mCARs, suggesting that there are also many novel ncRNAs in the mCARs. Overall, the mCARs identified here, together with the previous proteomic and genomic data, constitute the first comprehensive catalogue of the molecular composition of the eukaryotic mitotic chromosomes.

Long noncoding RNA papillary thyroid carcinoma susceptibility candidate 3 (PTCSC3) inhibits proliferation and invasion of glioma cells by suppressing the Wnt/ß-catenin signaling pathway.

BACKGROUND: The dysregulation of long noncoding RNAs (lncRNAs) has been identified in a variety of cancers. An increasing number of studies have found the critical role of lncRNAs in the regulation of cellular processes, such as proliferation, invasion and differentiation. Long noncoding RNA papillary thyroid carcinoma susceptibility candidate 3 (PTCSC3) is a novel lncRNA that was primarily detected in papillary thyroid carcinoma. However, the biological function and molecular mechanism of lncRNA PTCSC3 in glioma are still unknown. METHODS: The expression level of lncRNA PTCSC3 in human microglia and glioma cell lines was examined using quantitative real-time polymerase chain reaction (qRT-PCR). The influence of lncRNA PTCSC3 on cell proliferation were studied using the cell counting kit-8, and cell cycle and apoptosis were analyzed by flow cytometry assays. The migration and invasion abilities were investigated by transwell and wound healing assays. The target genes of lncRNA PTCSC3 were explored by qRT-PCR, immunofluorescence and western blot. RESULTS: LncRNA PTCSC3 was significantly downregulated in glioma cell lines. The overexpression of lncRNA PTCSC3 suppressed proliferation and induced apoptosis in U87 and U251 cells. Additionally, the overexpression of lncRNA PTCSC3 inhibited the migration and invasion of U87 and U251 cells. Moreover, lncRNA PTCSC3 inhibited the epithelial-mesenchymal transition of U87 cells. The study also demonstrated that LRP6, as a receptor of the Wnt/ß-catenin pathway, was a target of lncRNA PTCSC3. By evaluating the expression levels of Axin1, active ß-catenin, c-myc, and cyclin D1, the study indicated that lncRNA PTCSC3 inhibited the activation of the Wnt/ß-cateninpathway through targeting LRP6. CONCLUSIONS: LncRNA PTCSC3 inhibits the proliferation and migration of glioma cells and suppresses Wnt/ß-catenin signaling pathway by targeting LRP6. LncRNA PTCSC3 is a potential therapeutic target for treatment of glioma.

Combining DGE and RNA-sequencing data to identify new polyA+ non-coding transcripts in the human genome.

Recent sequencing technologies that allow massive parallel production of short reads are the method of choice for transcriptome analysis. Particularly, digital gene expression (DGE) technologies produce a large dynamic range of expression data by generating short tag signatures for each cell transcript. These tags can be mapped back to a reference genome to identify new transcribed regions that can be further covered by RNA-sequencing (RNA-Seq) reads. Here, we applied an integrated bioinformatics approach that combines DGE tags, RNA-Seq, tiling array expression data and species-comparison to explore new transcriptional regions and their specific biological features, particularly tissue expression or conservation. We analysed tags from a large DGE data set (designated as 'TranscriRef'). We then annotated 750,000 tags that were uniquely mapped to the human genome according to Ensembl. We retained transcripts originating from both DNA strands and categorized tags corresponding to protein-coding genes, antisense, intronic- or intergenic-transcribed regions and computed their overlap with annotated non-coding transcripts. Using this bioinformatics approach, we identified ∼34,000 novel transcribed regions located outside the boundaries of known protein-coding genes. As demonstrated using sequencing data from human pluripotent stem cells for biological validation, the method could be easily applied for the selection of tissue-specific candidate transcripts. DigitagCT is available at http://cractools.gforge.inria.fr/softwares/digitagct.

AGO2 and SETDB1 cooperate in promoter-targeted transcriptional silencing of the androgen receptor gene.

In mammals, RNA interference is primarily a post-transcriptional mechanism. Evidence has accumulated for additional role in transcriptional gene silencing (TGS) but the question for a good paradigm for small interfering antigene RNA (agRNA)-induced chromatin modification remains unanswered. Here, we show that SETDB1, a histone H3-lysine 9 (H3K9)-specific methyltransferase, cooperates with Argonaute-2 (AGO2) and plays an essential role in agRNA-induced TGS. The androgen receptor (AR) gene was transcriptionally silenced by agRNA targeted to its promoter, and we show that this repression was mitigated by knockdown of SETDB1 or AGO2. Chromatin immunoprecipitation demonstrated that agRNA-driven AGO2 was first targeted to the AR promoter, followed by SETDB1. SIN3A and HDAC1/2, the components of the SIN3-HDAC complex, immunoprecipitated with SETDB1, and localized at the agRNA-targeted promoter. Agreeing with the presence of SETDB1, trimethyl-H3K9 was enriched in the AR promoter. Both EZH2 and trimethyl-H3K27 were also present in the targeted locus; accordingly, EZH2 immunoprecipitated with SETDB1. DNA methylation level was not significantly changed, suggesting the absence of de novo methylating activity in agRNA-induced AR promoter. Our results demonstrate that SETDB1, together with AGO2, plays an essential role in TGS through recruiting chromatin remodeler and/or other modifiers, consequently creating a repressive chromatin milieu at the targeted promoter.

Non-coding RNAs as Biomarkers for Colorectal Cancer Screening and Early Detection.

Early detection of colorectal cancer (CRC) is the key for prevention and the ability to impact long-term survival of CRC patients. Current CRC screening modalities are inadequate for global application because of low sensitivity and specificity in case of conventional stool-based screening tests, and high costs and a low participation compliance in colonoscopy. An accurate stool- or blood-based screening test with use of innovative biomarkers is an appealing alternative as it is non-invasive and poses minimal risk to patients. It is easy to perform, can be repeated at shorter intervals, and therefore would likely lead to a much higher compliance rates. Non-coding RNAs (ncRNAs) have recently gained attention because of their involvement in different biological processes, such as proliferation, differentiation, migration, angiogenesis and apoptosis. An increasing number of studies have demonstrated that mutations or abnormal expression of ncRNAs are closely associated with various cancers, including CRC. The discovery that ncRNAs (mainly microRNAs) are stable in stool and in blood plasma and serum presents the opportunity to develop novel strategies taking advantage of circulating ncRNAs as early diagnostic biomarkers of CRC. This chapter is a comprehensive examination of aberrant ncRNAs expression levels in tumor tissue, stool and blood of CRC patients and a summary of the current findings on ncRNAs, including microRNAs, small nucleolar RNAs, small nuclear RNAs, Piwi-interacting RNAs, circular RNAs and long ncRNAs in regards to their potential usage for screening or early detection of CRC.

The identification and characterization of non-coding and coding RNAs and their modified nucleosides by mass spectrometry.

The analysis of ribonucleic acids (RNA) by mass spectrometry has been a valuable analytical approach for more than 25 years. In fact, mass spectrometry has become a method of choice for the analysis of modified nucleosides from RNA isolated out of biological samples. This review summarizes recent progress that has been made in both nucleoside and oligonucleotide mass spectral analysis. Applications of mass spectrometry in the identification, characterization and quantification of modified nucleosides are discussed. At the oligonucleotide level, advances in modern mass spectrometry approaches combined with the standard RNA modification mapping protocol enable the characterization of RNAs of varying lengths ranging from low molecular weight short interfering RNAs (siRNAs) to the extremely large 23 S rRNAs. New variations and improvements to this protocol are reviewed, including top-down strategies, as these developments now enable qualitative and quantitative measurements of RNA modification patterns in a variety of biological systems.

4D Non-uniformly sampled C,C-NOESY experiment for sequential assignment of 13C, 15N-labeled RNAs.

A 4D (13)C(aromatic),(13)C(ribose)-edited NOESY experiment is introduced to improve sequential assignment of non-coding RNA, often hampered by a limited dispersion of (1)H and (13)C chemical shifts. The (13)C-labeling of RNA is fully utilized by inclusion of two (13)C evolution periods. These dimensions provide enhanced dispersion of resonances in the 4D spectrum. High spectral resolution is obtained using random non-uniform sampling in three indirect dimensions. The autocorrelation peaks are efficiently suppressed using band-selective pulses. Since the dynamic range of observed resonances is significantly decreased, the reconstruction of the 4D spectrum is greatly simplified. The experiment can replace two conventionally sampled 3D NOESY spectra (either ribose-(13)C- or aromatic-(13)C-separated), and remove most ambiguities encountered during sequential walks. The assignment strategy based on a homonuclear and 4D C,C-edited NOESY experiments is proposed and verified on a 34-nt RNA showing typical structure elements.

A single-base resolution map of an archaeal transcriptome.

Organisms of the third domain of life, the Archaea, share molecular characteristics both with Bacteria and Eukarya. These organisms attract scientific attention as research models for regulation and evolution of processes such as transcription, translation, and RNA processing. We have reconstructed the primary transcriptome of Sulfolobus solfataricus P2, one of the most widely studied model archaeal organisms. Analysis of 625 million bases of sequenced cDNAs yielded a single-base-pair resolution map of transcription start sites and operon structures for more than 1000 transcriptional units. The analysis led to the discovery of 310 expressed noncoding RNAs, with an extensive expression of overlapping cis-antisense transcripts to a level unprecedented in any bacteria or archaea but resembling that of eukaryotes. As opposed to bacterial transcripts, most Sulfolobus transcripts completely lack 5'-UTR sequences, suggesting that mRNA/ncRNA interactions differ between Bacteria and Archaea. The data also reveal internal hotspots for transcript cleavage linked to RNA degradation and predict sequence motifs that promote RNA destabilization. This study highlights transcriptome sequencing as a key tool for understanding the mechanisms and extent of RNA-based regulation in Bacteria and Archaea.

RNAspace.org: An integrated environment for the prediction, annotation, and analysis of ncRNA.

The annotation of noncoding RNA genes remains a major bottleneck in genome sequencing projects. Most genome sequences released today still come with sets of tRNAs and rRNAs as the only annotated RNA elements, ignoring hundreds of other RNA families. We have developed a web environment that is dedicated to noncoding RNA (ncRNA) prediction, annotation, and analysis and allows users to run a variety of tools in an integrated and flexible manner. This environment offers complementary ncRNA gene finders and a set of tools for the comparison, visualization, editing, and export of ncRNA candidates. Predictions can be filtered according to a large set of characteristics. Based on this environment, we created a public website located at http://RNAspace.org. It accepts genomic sequences up to 5 Mb, which permits for an online annotation of a complete bacterial genome or a small eukaryotic chromosome. The project is hosted as a Source Forge project (http://rnaspace.sourceforge.net/).