Analytical ultracentrifuge: an ideal tool for characterization of non-coding RNAs.

Analytical ultracentrifugation (AUC) has emerged as a robust and reliable technique for biomolecular characterization with extraordinary sensitivity. AUC is widely used to study purity, conformational changes, biomolecular interactions, and stoichiometry. Furthermore, AUC is used to determine the molecular weight of biomolecules such as proteins, carbohydrates, and DNA and RNA. Due to the multifaceted role(s) of non-coding RNAs from viruses, prokaryotes, and eukaryotes, research aimed at understanding the structure-function relationships of non-coding RNAs is rapidly increasing. However, due to their large size, flexibility, complicated secondary structures, and conformations, structural studies of non-coding RNAs are challenging. In this review, we are summarizing the application of AUC to evaluate the homogeneity, interactions, and conformational changes of non-coding RNAs from adenovirus as well as from Murray Valley, Powassan, and West Nile viruses. We also discuss the application of AUC to characterize eukaryotic long non-coding RNAs, Xist, and HOTAIR. These examples highlight the significant role AUC can play in facilitating the structural determination of non-coding RNAs and their complexes.

Principles and innovative technologies for decrypting noncoding RNAs: from discovery and functional prediction to clinical application.

Noncoding RNAs (ncRNAs) are a large segment of the transcriptome that do not have apparent protein-coding roles, but they have been verified to play important roles in diverse biological processes, including disease pathogenesis. With the development of innovative technologies, an increasing number of novel ncRNAs have been uncovered; information about their prominent tissue-specific expression patterns, various interaction networks, and subcellular locations will undoubtedly enhance our understanding of their potential functions. Here, we summarized the principles and innovative methods for identifications of novel ncRNAs that have potential functional roles in cancer biology. Moreover, this review also provides alternative ncRNA databases based on high-throughput sequencing or experimental validation, and it briefly describes the current strategy for the clinical translation of cancer-associated ncRNAs to be used in diagnosis.

Synthesis, purification and crystallization of a putative critical bulge of HAR1 RNA.

Non-coding RNAs have raised a lot of interest because of their capabilities to perform enzymatic reactions and regulate gene expression in various ways. Human Accelerated Region 1 (HAR1) has been identified during the search for highly conserved regions in mammalian genomes, over one hundred base pairs long, and with high rates of substitution in the human genome. Its potential for coding for a protein is very minimal. However, the HAR1 transcript has been computationally predicted to have a stable secondary structure. Previous structure-probing experiments have suggested that the majority of differences between human and chimp constructs are in helices, designated C and D. For this reason, a 47nt construct consisting of the C and D helices along with two additional C-G pairs was synthesized, purified, and crystallized, and its x-ray structure is reported in this study. The final structure is an artificial dimer, with a bulge that forms different conformations on each monomer. This bulge has been observed in predicted secondary structures, footprinting assays, enzymatic degradation assays, NMR studies, in silico studies, and in this crystalized dimer structure. It is proposed that the HAR1 transcript is a non-coding RNA that interacts with an unknown binding partner responsible for brain development through this inherent structural motif of bulged adenosines.

Biosensors for epigenetic biomarkers detection: A review.

Epigenetic inheritance is a heritable change in gene function independent of alterations in nucleotide sequence. It regulates the normal cellular activities of the organisms by affecting gene expression and transcription, and its abnormal expression may lead to the developmental disorder, senile dementia, and carcinogenesis progression. Thus, epigenetic inheritance is recognized as an important biomarker, and the accurate quantification of epigenetic inheritance is crucial to clinical diagnosis, drug development and cancer treatment. Noncoding RNA, DNA methylation and histone modification are the most common epigenetic biomarkers. The conventional biosensors (e.g., northern blotting, radiometric, mass spectrometry and immunosorbent biosensors) for epigenetic biomarkers assay usually suffer from hazardous radiation, complicated manipulation, and time-consuming procedures. To facilitate the practical applications, some new biosensors including colorimetric, luminescent, Raman scattering spectroscopy, electrochemical and fluorescent biosensors have been developed for the detection of epigenetic biomarkers with simplicity, rapidity, high throughput and high sensitivity. In this review, we summarize the recent advances in epigenetic biomarkers assay. We classify the biosensors into the direct amplification-free and the nucleotide amplification-assisted ones, and describe the principles of various biosensors, and further compare their performance for epigenetic biomarkers detection. Moreover, we discuss the emerging trends and challenges in the future development of epigenetic biomarkers biosensors.

Concerted regulation of mitochondrial and nuclear non-coding RNAs by a dual-targeted RNase Z.

The molecular roles of the dually targeted ElaC domain protein 2 (ELAC2) during nuclear and mitochondrial RNA processing in vivo have not been distinguished. We generated conditional knockout mice of ELAC2 to identify that it is essential for life and its activity is non-redundant. Heart and skeletal muscle-specific loss of ELAC2 causes dilated cardiomyopathy and premature death at 4 weeks. Transcriptome-wide analyses of total RNAs, small RNAs, mitochondrial RNAs, and miRNAs identified the molecular targets of ELAC2 in vivo We show that ELAC2 is required for processing of tRNAs and for the balanced maintenance of C/D box snoRNAs, miRNAs, and a new class of tRNA fragments. We identify that correct biogenesis of regulatory non-coding RNAs is essential for both cytoplasmic and mitochondrial protein synthesis and the assembly of mitochondrial ribosomes and cytoplasmic polysomes. We show that nuclear tRNA processing is required for the balanced production of snoRNAs and miRNAs for gene expression and that 3' tRNA processing is an essential step in the production of all mature mitochondrial RNAs and the majority of nuclear tRNAs.

Isolation of Extracellular RNA from Bile.

The study of extracellular RNA has been recently reported as a tool for biomarker discovery. Extracellular vesicles can be isolated from different types of body fluids which contain protein, mRNA, and noncoding RNA. Extracellular RNA isolated from bile could be a useful tool for analyzing biliary tract diseases or cancer. Herein, we describe protocols based on modifications of commercially available kits for the collection, processing, and isolation of extracellular RNA from bile.

Mining Exosomal Genes for Pancreatic Cancer Targets.

BACKGROUND: Exosomes, cell-derived vesicles encompassing lipids, DNA, proteins coding genes and noncoding RNAs (ncRNAs) are present in diverse body fluids. They offer novel biomarker and drug therapy potential for diverse diseases, including cancer. MATERIALS AND METHODS: Using gene ontology, exosomal genes database and GeneCards metadata analysis tools, a database of cancer-associated protein coding genes and ncRNAs (n=2,777) was established. Variant analysis, expression profiling and pathway mapping were used to identify putative pancreatic cancer exosomal gene candidates. RESULTS: Five hundred and seventy-five protein-coding genes, 26 RNA genes and one pseudogene directly associated with pancreatic cancer were identified in the study. Nine open reading frames (ORFs) encompassing enzymes, apoptosis and transcriptional regulators, and secreted factors and five cDNAs (enzymes) emerged from the analysis. Among the ncRNA class, 26 microRNAs (miRs), one pseudogene, one long noncoding RNA (LNC) and one antisense gene were identified. Furthermore, 22 exosome-associated protein-coding targets (a cytokine, enzymes, membrane glycoproteins, receptors, and a transporter) emerged as putative leads for pancreatic cancer therapy. Seven of these protein-coding targets are FDA-approved and the drugs-based on these could provide repurposing opportunities for pancreatic cancer. CONCLUSION: The database of exosomal genes established in this study provides a framework for developing novel biomarkers and drug therapy targets for pancreatic cancer.

Characterization of novel transcripts of human papillomavirus type 16 using cap analysis gene expression technology.

We have performed cap-analysis gene expression (CAGE) sequencing to identify the regulatory networks that orchestrate genome-wide transcription in human papillomavirus type 16 (HPV16)-positive cervical cell lines of different grades: W12E, SiHa, and CaSki. Additionally, a cervical intraepithelial neoplasia grade 1 (CIN1) lesion was assessed for identifying the transcriptome expression profile. Here we have precisely identified a novel antisense noncoding viral transcript in HPV16. In conclusion, CAGE sequencing should pave the way for understanding a diversity of viral transcript expression.

Isolation of small noncoding RNAs from human serum.

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 µl of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/µl from serum but can also be adapted for other biological tissues.

RNA networks in ovarian cancer.

Development of ovarian cancer is known to be associated with alterations in the expression of cellular RNAs. Most of the clinical and biological characteristics of ovarian cancer have been correlated with significant changes in the expression of a subset of genes. Over the last few years, considerable resources have been focused on understanding the complex structure and function of noncoding RNAs, and this paradigm is also applicable to ovarian cancer research. The chapter provides a brief review of methodological approaches used to study alterations in protein coding and noncoding RNAs in the context of ovarian cancer.

The single-nucleotide resolution transcriptome of Pseudomonas aeruginosa grown in body temperature.

One of the hallmarks of opportunistic pathogens is their ability to adjust and respond to a wide range of environmental and host-associated conditions. The human pathogen Pseudomonas aeruginosa has an ability to thrive in a variety of hosts and cause a range of acute and chronic infections in individuals with impaired host defenses or cystic fibrosis. Here we report an in-depth transcriptional profiling of this organism when grown at host-related temperatures. Using RNA-seq of samples from P. aeruginosa grown at 28°C and 37°C we detected genes preferentially expressed at the body temperature of mammalian hosts, suggesting that they play a role during infection. These temperature-induced genes included the type III secretion system (T3SS) genes and effectors, as well as the genes responsible for phenazines biosynthesis. Using genome-wide transcription start site (TSS) mapping by RNA-seq we were able to accurately define the promoters and cis-acting RNA elements of many genes, and uncovered new genes and previously unrecognized non-coding RNAs directly controlled by the LasR quorum sensing regulator. Overall we identified 165 small RNAs and over 380 cis-antisense RNAs, some of which predicted to perform regulatory functions, and found that non-coding RNAs are preferentially localized in pathogenicity islands and horizontally transferred regions. Our work identifies regulatory features of P. aeruginosa genes whose products play a role in environmental adaption during infection and provides a reference transcriptional landscape for this pathogen.

Isolation and characterization of a novel noncoding RNA from nickel-induced lung cancer.

Noncoding RNAs have drawn significant attention in carcinogenesis. In this study, we identified a novel gene named nickel-related gene1 (NRG1) associated with nickel-induced cancer. By using rapid amplification of cDNA end PCR, we obtained the full length of the cDNA. The sequence was analyzed by using related bioinformatics software and comparative genomics methods. The results showed that NRG1 was located on chromosome 2q12, within intron2 of ADAMTS6, a disintegrin and metalloproteinase with thrombospondin motifs. And, NRG1 had a high level of homology (76 %) to rat LINE1 sequence RL1.3 (long interspersed middle repetitive DNA). What's more, there was no continuous open reading frame present in NRG1 sequence. Taken together, these data demonstrate that NRG1 is a novel noncoding RNA, and we predicted it may be a transposon-like gene. The identification of NRG1 emphasized the potential role of noncoding RNA in nickel carcinogenesis.

Steroid Receptor RNA Activator - A nuclear receptor coregulator with multiple partners: Insights and challenges.

Steroid Receptor RNA Activator (SRA) occupies a unique and enigmatic position within the nuclear receptor (NR) field and more broadly in transcriptional regulation. This is as a result of its transcripts having both coding and non-coding coactivator activities along with its protein product SRAP performing mixed coactivator/repressor functions. Recent publications have provided greater understanding of SRA gene product activities and how they affect not only NR function, but now more broadly, signalling pathways involved in differentiation and metabolism. This review will discuss the isolation of SRA, its gene products, regulation of transcription along with its in vitro and in vivo activities with a particular focus on its actions as an RNA and its binding partners.

Identification and characterisation of a novel antisense non-coding RNA from the RBM5 gene locus.

Previous work from our lab identified a 326 base-pair (bp) cDNA, termed Je2, which mapped to the antisense strand of intron 6 of the putative tumour suppressor gene RBM5/LUCA-15/H37, and functioned as an apoptosis suppressor. The purpose of the work described herein was to determine if Je2 is part of a larger transcript, to clone that transcript and to examine its ability to modulate RBM5 expression. Northern blot analyses in conjunction with strand-specific reverse transcription and PCR revealed two novel transcripts, one antisense and one sense, that included Je2 as well as RBM5 intron 4 sequence. Using rapid amplification of cDNA ends (RACE), a novel 1.4 kb product including Je2 and intron 4 was cloned. In vitro transcription/translation did not result in the production of any protein product, from either strand. Genomic DNA analysis revealed the presence of a putative promoter region 5' to Je2, suggesting that the cloned 1.4 kb RACE product represents an antisense transcript that initiates within intron 6 and terminates within intron 4 of the RBM5 gene. This novel antisense, non-coding RNA was termed LUST, for LUCA-15-specific transcript. Ectopic overexpression of LUST coincided with elevated expression of the full-length RBM5+5+6 alternative RBM5 RNA splice variant, and reduced expression of the truncated, cytotoxic RBM5+5+6t/Clone 26 alternative RBM5 RNA splice variant. A model is proposed whereby LUST functions co-transcriptionally to mask a sense-strand regulatory sequence, common to both RBM5+5+6 and RBM5+5+6t/Clone 26 transcripts, that when unmasked results in premature termination of RBM5+5+6, thereby generating the cytotoxic truncated product, RBM5+5+6t/Clone 26. These results suggest that LUST is a novel, functional, non-coding RNA that plays a role in determining the apoptotic fate of a cell by regulating the expression of RBM5 splice variants.

Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5' terminal nucleotide.

Argonaute (AGO) proteins recruit small RNAs to form the core of RNAi effector complexes. Arabidopsis encodes ten AGO proteins and a large network of small RNAs. How these small RNAs are sorted into specific AGO complexes remains largely unknown. We have cataloged small RNAs resident in four AGO complexes. We found that AGO2 and AGO4 preferentially recruit small RNAs with a 5' terminal adenosine, whereas AGO1 harbors microRNAs (miRNAs) that favor a 5' terminal uridine. AGO5 predominantly binds small RNAs that initiate with cytosine. Changing the 5' terminal nucleotide of an miRNA predictably redirected it into a different AGO complex and alters its biological activity. These results reveal a role for small RNA sequences in assorting among AGO complexes. This suggests that specialization of AGO complexes might involve remodeling the 5' end-binding pocket to accept certain small RNA sequences, perhaps explaining the evolutionary drive for miRNAs to initiate with uridine.

Identification of two small RNAs within the first 1.5-kb of the herpes simplex virus type 1-encoded latency-associated transcript.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is abundantly expressed in latently infected neurons. In the rabbit or mouse ocular models of infection, expression of the first 1.5 kb of LAT coding sequences is sufficient for and necessary for wild-type levels of spontaneous reactivation from latency. The antiapoptosis functions of LAT, which maps to the same 1.5 kb of LAT, are important for the latency-reactivation cycle because replacement of LAT with other antiapoptosis genes (the baculovirus IAP gene or the bovine herpesvirus type 1 latency-related gene) restores wild-type levels of reactivation to a LAT null mutant. A recent study identified a micro-RNA within LAT that can inhibit apoptosis (Gupta et al, Nature 442: 82-85). In this study, the authors analyzed the first 1.5 kb of LAT for additional small RNAs that may have regulatory functions. Two LAT-specific small RNAs were detected in productively infected human neuroblastoma cells within the first 1.5 kb of LAT, in a region that is important for inhibiting apoptosis. Although these small RNAs possess extensive secondary structure and a stem-loop structure, bands migrating near 23 bases were not detected suggesting these small RNAs are not true micro-RNAs. Both of the small LAT-specific RNAs have the potential to base pair with the ICP4 mRNA. These two small LAT RNAs may play a role in the latency-reactivation cycle by reducing apoptosis and/or by reducing ICP4 RNA expression.

Use of tiling array data and RNA secondary structure predictions to identify noncoding RNA genes.

BACKGROUND: Within the last decade a large number of noncoding RNA genes have been identified, but this may only be the tip of the iceberg. Using comparative genomics a large number of sequences that have signals concordant with conserved RNA secondary structures have been discovered in the human genome. Moreover, genome wide transcription profiling with tiling arrays indicate that the majority of the genome is transcribed. RESULTS: We have combined tiling array data with genome wide structural RNA predictions to search for novel noncoding and structural RNA genes that are expressed in the human neuroblastoma cell line SK-N-AS. Using this strategy, we identify thousands of human candidate RNA genes. To further verify the expression of these genes, we focused on candidate genes that had a stable hairpin structures or a high level of covariance. Using northern blotting, we verify the expression of 2 out of 3 of the hairpin structures and 3 out of 9 high covariance structures in SK-N-AS cells. CONCLUSION: Our results demonstrate that many human noncoding, structured and conserved RNA genes remain to be discovered and that tissue specific tiling array data can be used in combination with computational predictions of sequences encoding structural RNAs to improve the search for such genes.

Characterization of the piRNA complex from rat testes.

Small noncoding RNAs regulate processes essential for cell growth and development, including mRNA degradation, translational repression, and transcriptional gene silencing (TGS). During a search for candidate mammalian factors for TGS, we purified a complex that contains small RNAs and Riwi, the rat homolog to human Piwi. The RNAs, frequently 29 to 30 nucleotides in length, are called Piwi-interacting RNAs (piRNAs), 94% of which map to 100 defined (< or = 101 kb) genomic regions. Within these regions, the piRNAs generally distribute across only one genomic strand or distribute on two strands but in a divergent, nonoverlapping manner. Preparations of piRNA complex (piRC) contain rRecQ1, which is homologous to qde-3 from Neurospora, a gene implicated in silencing pathways. Piwi has been genetically linked to TGS in flies, and slicer activity cofractionates with the purified complex. These results are consistent with a gene-silencing role for piRC in mammals.

Identification of new small non-coding RNAs from tobacco and Arabidopsis.

Small non-coding RNAs (ncRNAs) have typically been searched in fully sequenced genomes using one of two approaches-experimental or computational. We developed a mixed method, using both types of information, which has the advantage of applying bio-computing methods to actually expressed sequences. Our method allowed the identification of new small ncRNAs in Arabidopsis thaliana and in the unfinished genome of Nicotiana tabacum. We constructed a N. tabacum cDNA library from small RNAs ranging from 20 to 30 nucleotides (nt). The sequences from 73 unique clones were compared to the A. thaliana genome and to all plant sequences using a pattern-matching approach (program Patbank). Thus, we selected 15 clones from the library corresponding mostly to A. thaliana or N. tabacum non-coding sequences. By Northern blot analyses, we confirmed the presence of most RNA candidates in Arabidopsis and in Nicotiana sylvestris with a size range of 21-100 nt. To gain more insight into the possible genesis of 21-24 nt sequences, stable folding of sRNAs with their flanking regions were predicted with the software MIRFOLD dedicated to the folding of microRNAs (miRNA). Stable hairpins structures were observed for some putative miRNAs.