Rapid preparation of 6S RNA-free B. subtilis σA-RNA polymerase and σA.

The regulatory 6S-1 and 6S-2 RNAs of B. subtilis bind to the housekeeping RNA polymerase holoenzyme (σA-RNAP) with submicromolar affinity. We observed copurification of endogenous 6S RNAs from a published B. subtilis strain expressing a His-tagged RNAP. Such 6S RNA contaminations in σA-RNAP preparations reduce the fraction of enzymes that are accessible for binding to DNA promoters. In addition, this leads to background RNA synthesis by σA-RNAP utilizing copurified 6S RNA as template for the synthesis of short abortive transcripts termed product RNAs (pRNAs). To avoid this problem we constructed a B. subtilis strain expressing His-tagged RNAP but carrying deletions of the two 6S RNA genes. The His-tagged, 6S RNA-free σA-RNAP holoenzyme can be prepared with sufficient purity and activity by a single affinity step. We also report expression and separate purification of B. subtilis σA that can be added to the His-tagged RNAP to maximize the amount of holoenzyme and, by inference, in vitro transcription activity.

Expression and Purification of tRNA/ pre-miRNA-Based Recombinant Noncoding RNAs.

Research on RNA function and therapeutic potential is dominated by the use of chemoengineered RNA mimics. Recent efforts have led to the establishment of novel technologies for the production of recombinant or bioengineered RNA molecules, which should better recapitulate the structures, functions and safety profiles of natural RNAs because both are produced and folded in living cells. Herein, we describe a robust approach for reproducible fermentation production of bioengineered RNA agents (BERAs) carrying warhead miRNAs, siRNAs, aptamers, or other forms of small RNAs, based upon an optimal hybrid tRNA/pre-miRNA carrier. Target BERA/sRNAs are readily purified by fast protein liquid chromatography (FPLC) to a high degree of homogeneity (>97%). This approach offers a consistent high-level expression (>30% of total bacterial RNAs) and large-scale production of ready-to-use BERAs (multiple to tens milligrams from 1 L bacterial culture).

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.

RNA-Seq analysis reveals functionally relevant coding and non-coding RNAs in crossbred bull spermatozoa.

Sperm, which are believed to be transcriptionally and translationally inactive, synthesize RNA and proteins before there is gradual disappearance of the ribosome during chromatin compaction. Sperm transfer several functionally relevant transcripts to the oocyte, controlling maternal-zygotic transition and embryonic development. The present study was undertaken to profile and analyze sperm transcripts comprehensively using Next Generation Ribonucleic acid sequencing technology in Holstein Friesian x Tharparkar crossbred bulls. The results from global transcriptomic profiling revealed transcripts for 13,814 genes; of which 431 transcripts were expressed with >1 FPKM and 13,383 transcripts were expressed with >0 or <1 FPKM. The abundant mRNA transcripts of crossbred bull sperm were PRM1 and HMGB4. Gene ontology of transcripts with>1 FPKM revealed there was a major involvement in the structural constituent of ribosomes and translation. Results from pathway enrichment indicated the connection between ribosome, oxidative phosphorylation and spliceosome pathways and the transcripts of crossbred bull spermatozoa. The transcriptional abundance of selected genes, validated using RT-qPCR, indicated significant variations between bulls. Collectively, it may be inferred that the transcripts in crossbred bull sperm were heavily implicated in functions such as the structural constituent of ribosomes and translation, and pathways such as ribosome, oxidative phosphorylation and spliceosome. Further studies using larger sample sizes are required to understand the possible implications of transcriptomic variations on semen quality and fertility.

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.

Comparative analysis of the down syndrome hippocampal non-coding RNA transcriptomes using a mouse model.

BACKGROUND: Down syndrome (DS), caused by trisomy 21, is the most common human chromosomal disorder. Hippocampal abnormalities have been believed to be responsible for the DS developmental cognitive deficits. Cumulative evidences indicated that non-coding RNAs (ncRNAs) participated in brain development and function. Currently, few was known whether dysregulated ncRNAs existed in DS whether the dysregulated ncRNAs played important pathology roles in DS. OBJECTIVE: The purpose of this study was generating an overview map of the dysregulated ncRNAs in DS, including the microRNA (miRNA), long ncRNA (lncRNA) and circular RNA (circRNAs). DS mouse models are invaluable tools for further mechanism and therapy studies. METHODS: The well-studied DS mouse model Dp(16)1/Yey was used in this study as it contains the trisomy of the whole human chromosome 21 syntenic region on mouse chromosomes 16. Hippocampi were isolated from pups of seven-days-old. Libraries for miRNA, lncRNA and circRNAs were constructed separately, and the next generation sequencing method was utilized. RESULTS: Differentially expressed (DE) miRNAs, lncRNAs and circRNAs were reported. Relative few regulating relationship were found between the DE miRNAs and DE mRNAs. LncRNAs originated from the trisomic regions expressed in clusters, but not all of them were 1.5-fold increased expressed. Dramatic DE circular RNAs were found in the DS hippocampus. The host genes of the DE circRNAs were enriched on functions which were well-known impaired in DS, e.g. long-term-potentiation, glutamatergic synapse, and GABAergic synapse. CONCLUSIONS: We generated the first DS developmental hippocampal ncRNA transcriptome map. This work laid foundations for further investigations on role of ncRNAs in hippocampal functions.

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.

Studying RNA-DNA interactome by Red-C identifies noncoding RNAs associated with various chromatin types and reveals transcription dynamics.

Non-coding RNAs (ncRNAs) participate in various biological processes, including regulating transcription and sustaining genome 3D organization. Here, we present a method termed Red-C that exploits proximity ligation to identify contacts with the genome for all RNA molecules present in the nucleus. Using Red-C, we uncovered the RNA-DNA interactome of human K562 cells and identified hundreds of ncRNAs enriched in active or repressed chromatin, including previously undescribed RNAs. Analysis of the RNA-DNA interactome also allowed us to trace the kinetics of messenger RNA production. Our data support the model of co-transcriptional intron splicing, but not the hypothesis of the circularization of actively transcribed genes.

Isolation and initial structure-functional characterization of endogenous tRNA-derived stress-induced RNAs.

Recent transcriptome-wide studies have identified a diverse pool of transfer RNA (tRNA)-derived RNAs or tRNA-derived fragments (tRFs). Some of these RNAs have been demonstrated to be functional and involved in multiple biological processes ranging from the regulation of gene expression to transgenerational epigenetic inheritance. Post-transcriptional maturation of tRNAs includes various processing events including extensive decoration by various RNA modifications, which are required for correct tRNA folding and stability. Moreover, tRNA modifications determine the pattern and specificity of tRNA cleavage. The major drawbacks of many studies in the field of tRFs are that most of them used synthetic RNAs that closely mimic endogenous tRFs in their sequence, yet lack RNA modification that is found in vivo. Here, we developed a simple method to isolate tRNA-derived stress-induced RNAs (tiRNAs), a specific subset of tRFs. Our approach is scalable, cost-effective and relies on the purification of individual tiRNAs based on a sequence-specific RNA/DNA isolation technique using DNA probes. Our method facilitates functional studies of tiRNAs by addressing how physiological RNA modifications within tRNA fragments affect their biological activities. Here, we report pilot functional studies on selected endogenous tiRNAs, namely tiRNAAla and tiRNAGly. We show that natural 5'-tiRNAAla molecules assemble into G-quadruplex structures, and endogenous 5'-tiRNAGly possesses translation inhibition activity.

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.

Identification and comparative profiling of gonadal microRNAs in the adult pigeon (Columba livia).

1. MicroRNAs are small noncoding RNA molecules that play crucial roles in gene expression. However, the comparative profiling of testicular and ovarian microRNAs in birds are rarely reported, particularly in pigeon.2. In this study, Illumina next-generation sequencing technology was used to sequence miRNA libraries of the gonads from six healthy adult utility pigeons. A total of 344 conserved known miRNAs and 32 novel putative miRNAs candidates were detected. Compared with those of ovaries, 130 differentially expressed (DE) miRNAs were identified in the testes. Among them, 70 miRNAs showed down-regulation in the ovaries, while another 60 miRNAs were up-regulated.3. Combining the results of the expression of target gene measurements and pathway enrichment analyses, it was revealed that some DEmiRNAs from the gonad samples involved in sexual differentiation and development (such as cli-miR-210-3p and cli-miR-214-3p) could down-regulate AR (androgen receptor). Cli-miR-181b-5p, cli-miR-9622-3p and cli-miR-145-5p were highly expressed in both the ovaries and testes, which could co-target HOXC9, and were related to regulation of primary metabolic processes. KEGG enrichment analysis showed that DEmiRNAs may play biological and sex-related roles in pigeon gonads.4. The expression profiles of testicular and ovarian miRNA in adult pigeon gonads are presented for the first time, and the findings may contribute to a better understanding of gonadal expression in poultry.

A review on native and denaturing purification methods for non-coding RNA (ncRNA).

Recently, non-coding RNA (ncRNA) became the centerpiece of human genome research. Modern ncRNA-based research has revolutionized disease diagnosis and therapeutics. However, decoding structural/functional information of ncRNA requires large amount of pure RNA, and hence effective RNA preparation and purification protocols. This review focuses on purification schemes of synthetic oligonucleotides, particularly liquid chromatographic (LC) techniques as improved alternatives to urea-polyacrylamide gel electrophoresis (urea-PAGE) purification. Moreover, the review summarizes the shortcomings of urea-PAGE purification method and details the chromatographic purification such as affinity, ion-exchange (IE) or size-exclusion (SE) chromatography. Specifically, we discuss denaturing and native RNA purification schemes with newest developments. In short, the review evaluates nucleic acid purification schemes required for various structural analyses.

Workflow Development for the Functional Characterization of ncRNAs.

During the last decade, ncRNAs have been investigated intensively and revealed their regulatory role in various biological processes. Worldwide research efforts have identified numerous ncRNAs and multiple RNA subtypes, which are attributed to diverse functionalities known to interact with different functional layers, from DNA and RNA to proteins. This makes the prediction of functions for newly identified ncRNAs challenging. Current bioinformatics and systems biology approaches show promising results to facilitate an identification of these diverse ncRNA functionalities. Here, we review (a) current experimental protocols, i.e., for Next Generation Sequencing, for a successful identification of ncRNAs; (b) sequencing data analysis workflows as well as available computational environments; and (c) state-of-the-art approaches to functionally characterize ncRNAs, e.g., by means of transcriptome-wide association studies, molecular network analyses, or artificial intelligence guided prediction. In addition, we present a strategy to cover the identification and functional characterization of unknown transcripts by using connective workflows.

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.

A novel non-coding RNA within an intron of CDH2 and association of its SNP with non-syndromic cleft lip and palate.

BACKGROUND: Genome-wide linkage analysis and whole genome sequencing in a Van der Woude syndrome (VWS) family revealed that the SNP, rs539075, within intron 2 of the cadherin 2 gene (CDH2) co-segregated with the disease phenotype. RESULTS: A study with nonsyndromic cleft lip with or without cleft palate (NSCL ±â€¯P) cases (N = 292) and controls (N = 287) established association of this SNP with NSCL ±â€¯P as a risk factor. RT-PCR based expression analysis of the SNP-harbouring region of intron 2 of CDH2 in the clefted lip and/or palate tissues of 16 patients revealed that the mutant allele expressed in all those individuals having it (hetero-/homozygous), whereas the wild type allele expressed in <50% of the samples in which it was present. The intronic transcript was also present in the prospective lip and palate region of 13.5 dpc mouse embryo, detected by RNA in situ hybridization and RT-PCR. CONCLUSIONS: These results including the in silico, characterization of the ~200 nt-intronic transcript showed that conformationally it fits best with noncoding small RNA, possibly a precursor of miRNA. Its function in the orofacial organogenesis remains to be elucidated which will enable us to define the role of this mutant ncRNA in the clefting of lip and palate.

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.

Evaluating the Stability of mRNAs and Noncoding RNAs.

Changes in RNA stability have an important impact in the gene expression regulation. Different methods based on the transcription blockage with RNA polymerase inhibitors or metabolic labeling of newly synthesized RNAs have been developed to evaluate RNA decay rates in cultured cell. Combined with techniques to measure transcript abundance genome-wide, these methods have been used to reveal novel features of the eukaryotic transcriptome. The stability of protein-coding mRNAs is in general closely associated to the physiological function of their encoded proteins, with short-lived mRNAs being significantly enriched among regulatory genes whereas genes associated with housekeeping functions are predominantly stable. Likewise, the stability of noncoding RNAs (ncRNAs) seems to reflect their functional role in the cell. Thus, investigating RNA stability can provide insights regarding the function of yet uncharacterized regulatory ncRNAs. In this chapter, we discuss the methodologies currently used to estimate RNA decay and outline an experimental protocol for genome-wide estimation of RNA stability of protein-coding and lncRNAs. This protocol details the transcriptional blockage of cultured cells with actinomycin D, followed by RNA isolation at different time points, the determination of transcript abundance by qPCR/DNA oligoarray hybridization, and the calculation of individual transcript half-lives.

Analysis RNA-seq and Noncoding RNA.

RNA-Seq is an approach to transcriptome profiling that uses deep-sequencing technologies to detect and accurately quantify RNA molecules originating from a genome at a given moment in time. In recent years, the advent of RNA-Seq has facilitated genome-wide expression profiling, including the identification of novel and rare transcripts like noncoding RNAs and novel alternative splicing isoforms.Here, we describe the analytical steps required for the identification and characterization of noncoding RNAs starting from RNA-Seq raw samples, with a particular emphasis on long noncoding RNAs (lncRNAs).