Intron RNA Research Articles

Isolation and Identification of Gene-Specific MicroRNAs.

Computer programming has identified hundreds of genomic hairpin sequences, many with functions yet to be determined. Because transfection of hairpin-like microRNA precursors (pre-miRNAs) into mammalian cells is not always sufficient to trigger RNA-induced gene silencing complex (RISC) assembly, a key step for inducingRNA interference (RNAi)-related gene silencing, we havedeveloped an intronic miRNA expressionsystem to overcome this problem by inserting a hairpin-like pre-miRNA structure into the intron region of a gene, and hencesuccessfully increase the efficiency and effectiveness of miRNA-associated RNAi induction in vitro and in vivo. This intronic miRNA biogenesis mechanismhas been found to depend on a coupled interaction of nascent messenger RNA transcription and intron excision within a specific nuclear region proximal to genomic perichromatin fibrils. The intronic miRNAso obtained is transcribed by type-II RNApolymerases, coexpressed within a primary gene transcript, and thenexcised out of the gene transcript by intracellular RNA splicing and processing machineries. After that, ribonuclease III (RNaseIII)endonucleases further process the spliced introns into mature miRNAs. Using this intronicmiRNA expression system, we have shown for the first time that the intron-derived miRNAs are able to elicit strong RNAi effects in not only human and mouse cells in vitrobut also in zebrafishes, chicken embryos, and adult micein vivo. We have also developed a miRNA isolation protocol, based on the complementarity between the designed miRNA and its targeted gene sequence, to purify and identify the mature miRNAs generated. As a result, several intronic miRNA identities and structures have been confirmed. According to this proof-of-principle methodology, we now have full knowledge to design variousintronicpre-miRNA inserts that are more efficient and effective for inducing specific gene silencing effects in vitro and in vivo.

Structure of a Thermostable Group II Intron Reverse Transcriptase with Template-Primer and Its Functional and Evolutionary Implications

Bacterial group II intron reverse transcriptases (RTs) function in both intron mobility and RNA splicing and are evolutionary predecessors of retrotransposon, telomerase, and retroviral RTs as well as the spliceosomal protein Prp8 in eukaryotes. Here we determined a crystal structure of a full-length thermostable group II intron RT in complex with an RNA template-DNA primer duplex and incoming deoxynucleotide triphosphate (dNTP) at 3.0-Å resolution. We find that the binding of template-primer and key aspects of the RT active site are surprisingly different from retroviral RTs but remarkably similar to viral RNA-dependent RNA polymerases. The structure reveals a host of features not seen previously in RTs that may contribute to distinctive biochemical properties of group II intron RTs, and it provides a prototype for many related bacterial and eukaryotic non-LTR retroelement RTs. It also reveals how protein structural features used for reverse transcription evolved to promote the splicing of both group II and spliceosomal introns.

DIP1 modulates stem cell homeostasis in Drosophila through regulation of sisR-1

Stable intronic sequence RNAs (sisRNAs) are by-products of splicing and regulate gene expression. How sisRNAs are regulated is unclear. Here we report that a double-stranded RNA binding protein, Disco-interacting protein 1 (DIP1) regulates sisRNAs in Drosophila. DIP1 negatively regulates the abundance of sisR-1 and INE-1 sisRNAs. Fine-tuning of sisR-1 by DIP1 is important to maintain female germline stem cell homeostasis by modulating germline stem cell differentiation and niche adhesion. Drosophila DIP1 localizes to a nuclear body (satellite body) and associates with the fourth chromosome, which contains a very high density of INE-1 transposable element sequences that are processed into sisRNAs. DIP1 presumably acts outside the satellite bodies to regulate sisR-1, which is not on the fourth chromosome. Thus, our study identifies DIP1 as a sisRNA regulatory protein that controls germline stem cell self-renewal in Drosophila.

Circular RNA profiling reveals an abundant circLMO7 that regulates myoblasts differentiation and survival by sponging miR-378a-3p

Circular RNAs (circRNAs) have been identified from various tissues and species, but their regulatory functions during developmental processes are not well understood. We examined circRNA expression profiles of two developmental stages of bovine skeletal muscle (embryonic and adult musculus longissimus) to provide first insights into their potential involvement in bovine myogenesis. We identified 12 981 circRNAs and annotated them to the Bos taurus reference genome, including 530 circular intronic RNAs (ciRNAs). One parental gene could generate multiple circRNA isoforms, with only one or two isoforms being expressed at higher expression levels. Also, several host genes produced different isoforms when comparing development stages. Most circRNA candidates contained two to seven exons, and genomic distances to back-splicing sites were usually less than 50 kb. The length of upstream or downstream flanking introns was usually less than 105 nt (mean≈11 000 nt). Several circRNAs differed in abundance between developmental stages, and real-time quantitative PCR (qPCR) analysis largely confirmed differential expression of the 17 circRNAs included in this analysis. The second part of our study characterized the role of circLMO7—one of the most down-regulated circRNAs when comparing adult to embryonic muscle tissue—in bovine muscle development. Overexpression of circLMO7 inhibited the differentiation of primary bovine myoblasts, and it appears to function as a competing endogenous RNA for miR-378a-3p, whose involvement in bovine muscle development has been characterized beforehand. Congruent with our interpretation, circLMO7 increased the number of myoblasts in the S-phase of the cell cycle and decreased the proportion of cells in the G0/G1 phase. Moreover, it promoted the proliferation of myoblasts and protected them from apoptosis. Our study provides novel insights into the regulatory mechanisms underlying skeletal muscle development and identifies a number of circRNAs whose regulatory potential will need to be explored in the future.

Therapeutic radiation for childhood cancer drives structural aberrations of NF2 in meningiomas

Cranial radiotherapy improves survival of the most common childhood cancers, including brain tumors and leukemia. Unfortunately, long-term survivors are faced with consequences of secondary neoplasia, including radiation-induced meningiomas (RIMs). We characterized 31 RIMs with exome/NF2 intronic sequencing, RNA sequencing and methylation profiling, and found NF2 gene rearrangements in 12/31 of RIMs, an observation previously unreported in sporadic meningioma (SM). Additionally, known recurrent mutations characteristic of SM, including AKT1, KLF4, TRAF7 and SMO, were not observed in RIMs. Combined losses of chromosomes 1p and 22q were common in RIMs (16/18 cases) and overall, chromosomal aberrations were more complex than that observed in SM. Patterns of DNA methylation profiling supported similar cell of origin between RIMs and SMs. The findings indicate that the mutational landscape of RIMs is distinct from SMs, and have significant therapeutic implications for survivors of childhood cranial radiation and the elucidation of the molecular pathogenesis of meningiomas.

Genome-wide Discovery of Circular RNAs in the Leaf and Seedling Tissues of Arabidopsis Thaliana

Background: Recently, identification and functional studies of circular RNAs, a type of non-coding RNAs arising from a ligation of 3’ and 5’ ends of a linear RNA molecule, were conducted in mammalian cells with the development of RNA-seq technology.Method: Since compared with animals, studies on circular RNAs in plants are less thorough, a genome-wide identification of circular RNA candidates in Arabidopsis was conducted with our own developed bioinformatics tool to several existing RNA-seq datasets specifically for non-coding RNAs.Results: A total of 164 circular RNA candidates were identified from RNA-seq data, and 4 circular RNA transcripts, including both exonic and intronic circular RNAs, were experimentally validated. Interestingly, our results show that circular RNA transcripts are enriched in the photosynthesis system for the leaf tissue and correlated to the higher expression levels of their parent genes. Sixteen out of all 40 genes that have circular RNA candidates are related to the photosynthesis system, and out of the total 146 exonic circular RNA candidates, 63 are found in chloroplast.

Molecular identification of species and production origins of edible bird's nest using FINS and SYBR green I based real-time PCR

The increasing demand and consumption of edible bird's nest (EBN) by people worldwide has contributed to the food fraud issue. To ensure the authenticity of EBN in regard to their origin, rapid and accurate analytical methods are very much needed. In this study, forensically informative nucleotide sequencing (FINS) technique based on mitochondrial and nuclear DNA sequences, and phylogenetic analysis was performed to identify the species and production origins of raw and commercial EBNs. The cytochrome b (Cyt b), NADH dehydrogenase subunit 2 (ND2), 12S ribosomal RNA and beta-fibrinogen intron 7 gene markers used were able to identify and classify EBN produced by Aerodramus fuciphagus and Aerodramus maximus. It was newly discovered that EBN from man-made houses and natural caves were genetically differentiable using the mitochondrial Cyt b and ND2 genes. The phylogenetic results revealed that all EBN samples were well-separated into two groups following their species origin and production origin. A rapid and cost-effective identification alternative of SYBR green I based real-time PCR assay targeting a 177 bp of the mitochondrial Cyt b gene was developed and it efficiently differentiated genuine EBN from counterfeits. This FINS and SYBR green I based real-time PCR are highly sensitive, specific and reliable methods for identification of EBN origins and could be useful for preventing fraud substitution and mislabelling of EBN to ensure food safety.

Seed weight differences between wild and domesticated soybeans are associated with specific changes in gene expression

Our study systematically explored potential genes and molecular pathways as candidates for differences in seed weight resulting from soybean domestication. In addition, potential contributions of lncRNAs to seed weight were also investigated. Soybeans have a long history of domestication in China, and there are several significant phenotypic differences between cultivated and wild soybeans, for example, seeds of cultivars are generally larger and heavier than those from wild accessions. We analyzed seed transcriptomes from thirteen soybean samples, including six landraces and seven wild accessions using strand-specific RNA sequencing. Differentially expressed genes related to seed weight were identified, and some of their homologs were associated with seed development in Arabidopsis. We also identified 1251 long intergenic noncoding RNAs (lincRNAs), 243 intronic RNAs and 81 antisense lncRNAs de novo from these soybean transcriptomes. We then profiled the expression patterns of lncRNAs in cultivated and wild soybean seeds, and found that transcript levels of a number of lncRNAs were sample-specific. Moreover, gene transcript and lincRNA co-expression network analysis showed that some soybean lincRNAs might have functional roles as they were hubs of co-expression modules. In conclusion, this study systematically explored potential genes and molecular pathways as candidates for differences in seed weight resulting from soybean domestication, and will provide a useful future resource for molecular breeding of soybeans.

Coordinated circRNA Biogenesis and Function with NF90/NF110 in Viral Infection

Circular RNAs (circRNAs) generated via back-splicing are enhanced by flanking complementary sequences. Expression levels of circRNAs vary under different conditions, suggesting participation of protein factorsin their biogenesis. Using genome-wide siRNA screening that targets all human unique genes and an efficient circRNA expression reporter, we identify double-stranded RNA-binding domain containing immune factors NF90/NF110 as key regulators in circRNA biogenesis. NF90/NF110 promote circRNA production in the nucleus by associating with intronic RNA pairs juxtaposing the circRNA-forming exon(s); they also interact with mature circRNAs in the cytoplasm. Upon viral infection, circRNA expression is decreased, in part owing to the nuclear export of NF90/NF110 to the cytoplasm. Meanwhile, NF90/NF110 released from circRNP complexes bind to viral mRNAs as part of their functions in antiviral immune response. Our results therefore implicate a coordinated regulation of circRNA biogenesis and function by NF90/NF110 in viral infection.

The effects of structure on pre-mRNA processing and stability.

Pre-mRNA molecules can form a variety of structures, and both secondary and tertiary structures have important effects on processing, function and stability of these molecules. The prediction of RNA secondary structure is a challenging problem and various algorithms that use minimum free energy, maximum expected accuracy and comparative evolutionary based methods have been developed to predict secondary structures. However, these tools are not perfect, and this remains an active area of research. The secondary structure of pre-mRNA molecules can have an enhancing or inhibitory effect on pre-mRNA splicing. An example of enhancing structure can be found in a novel class of introns in zebrafish. About 10% of zebrafish genes contain a structured intron that forms a bridging hairpin that enforces correct splice site pairing. Negative examples of splicing include local structures around splice sites that decrease splicing efficiency and potentially cause mis-splicing leading to disease. Splicing mutations are a frequent cause of hereditary disease. The transcripts of disease genes are significantly more structured around the splice sites, and point mutations that increase the local structure often cause splicing disruptions. Post-splicing, RNA secondary structure can also affect the stability of the spliced intron and regulatory RNA interference pathway intermediates, such as pre-microRNAs. Additionally, RNA secondary structure has important roles in the innate immune defense against viruses. Finally, tertiary structure can also play a large role in pre-mRNA splicing. One example is the G-quadruplex structure, which, similar to secondary structure, can either enhance or inhibit splicing through mechanisms such as creating or obscuring RNA binding protein sites.

Maternally inherited stable intronic sequence RNA triggers a self-reinforcing feedback loop during development

Summary Maternally inherited noncoding RNAs (ncRNAs) can regulate zygotic gene expression across generations [1–4]. Recently, many stable intronic sequence RNAs (sisRNAs), which are byproducts of pre-mRNA splicing, were found to be maternally deposited and persist till zygotic transcription in Xenopus and Drosophila [5–7]. In various organisms, sisRNAs can be in linear or circular conformations, and they have been suggested to regulate host gene expression [5–10]. It is unknown whether maternally deposited sisRNAs can regulate zygotic gene expression in the embryos. Here, we show that a maternally inherited sisRNA ( sisR-4 ) from the deadpan locus is important for embryonic development in Drosophila . Mothers, but not fathers, mutant for sisR-4 produce embryos that fail to hatch. During embryogenesis, sisR-4 promotes transcription of its host gene ( deadpan ), which is essential for development. Interestingly, sisR-4 functions by activating an enhancer present in the intron where sisR-4 is encoded. We propose that a maternal sisRNA triggers expression of its host gene via a positive feedback loop during embryogenesis.

Chloroplast- or Mitochondria-Targeted DEAD-Box RNA Helicases Play Essential Roles in Organellar RNA Metabolism and Abiotic Stress Responses.

The yields and productivity of crops are greatly diminished by various abiotic stresses, including drought, cold, heat, and high salinity. Chloroplasts and mitochondria are cellular organelles that can sense diverse environmental stimuli and alter gene expression to cope with adverse environmental stresses. Organellar gene expression is mainly regulated at posttranscriptional levels, including RNA processing, intron splicing, RNA editing, RNA turnover, and translational control, during which a variety of nucleus-encoded RNA-binding proteins (RBPs) are targeted to chloroplasts or mitochondria where they play essential roles in organellar RNA metabolism. DEAD-box RNA helicases (RHs) are enzymes that can alter RNA structures and affect RNA metabolism in all living organisms. Although a number of DEAD-box RHs have been found to play important roles in RNA metabolism in the nucleus and cytoplasm, our understanding on the roles of DEAD-box RHs in the regulation of RNA metabolism in chloroplasts and mitochondria is only at the beginning. Considering that organellar RNA metabolism and gene expression are tightly regulated by anterograde signaling from the nucleus, it is imperative to determine the functions of nucleus-encoded organellar RBPs. In this review, we summarize the emerging roles of nucleus-encoded chloroplast- or mitochondria-targeted DEAD-box RHs in organellar RNA metabolism and plant response to diverse abiotic stresses.

High-purity circular RNA isolation method (RPAD) reveals vast collection of intronic circRNAs.

High-throughput RNA sequencing methods coupled with specialized bioinformatic analyses have recently uncovered tens of thousands of unique circular (circ)RNAs, but their complete sequences, genes of origin and functions are largely unknown. Given that circRNAs lack free ends and are thus relatively stable, their association with microRNAs (miRNAs) and RNA-binding proteins (RBPs) can influence gene expression programs. While exoribonuclease treatment is widely used to degrade linear RNAs and enrich circRNAs in RNA samples, it does not efficiently eliminate all linear RNAs. Here, we describe a novel method for the isolation of highly pure circRNA populations involving RNase R treatment followed by Polyadenylation and poly(A)+ RNA Depletion (RPAD), which removes linear RNA to near completion. High-throughput sequencing of RNA prepared using RPAD from human cervical carcinoma HeLa cells and mouse C2C12 myoblasts led to two surprising discoveries: (i) many exonic circRNA (EcircRNA) isoforms share an identical backsplice sequence but have different body sizes and sequences, and (ii) thousands of novel intronic circular RNAs (IcircRNAs) are expressed in cells. In sum, isolating high-purity circRNAs using the RPAD method can enable quantitative and qualitative analyses of circRNA types and sequence composition, paving the way for the elucidation of circRNA functions.

Maternally Inherited Stable Intronic Sequence RNA Triggers a Self-Reinforcing Feedback Loop during Development

Maternally inherited noncoding RNAs (ncRNAs) can regulate zygotic gene expression across generations [1-4]. Recently, many stable intronic sequence RNAs (sisRNAs), which are byproducts of pre-mRNA splicing, were found to be maternally deposited and persist till zygotic transcription in Xenopus and Drosophila [5-7]. In various organisms, sisRNAs can be in linear or circular conformations, and they have been suggested to regulate host gene expression [5-10]. It is unknown whether maternally deposited sisRNAs can regulate zygotic gene expression in the embryos. Here, we show that a maternally inherited sisRNA (sisR-4) from the deadpan locus is important for embryonic development in Drosophila. Mothers, but not fathers, mutant for sisR-4 produce embryos that fail to hatch. During embryogenesis, sisR-4 promotes transcription of its host gene (deadpan), which is essential for development. Interestingly, sisR-4 functions by activating an enhancer present in the intron where sisR-4 is encoded. We propose that a maternal sisRNA triggers expression of its host gene via a positive feedback loop during embryogenesis.

5′ End Nicotinamide Adenine Dinucleotide Cap in Human Cells Promotes RNA Decay through DXO-Mediated deNADding

Eukaryotic mRNAs generally possess a 5' end N7methyl guanosine (m7G) cap that promotes theirtranslation and stability. However, mammalian mRNAs can also carry a 5' end nicotinamide adenine dinucleotide (NAD+) cap that, in contrast to the m7G cap, does not support translation but instead promotes mRNA decay. The mammalian and fungal noncanonical DXO/Rai1 decapping enzymes efficiently remove NAD+ caps, and cocrystal structures of DXO/Rai1 with 3'-NADP+ illuminate the molecular mechanism for how the "deNADding" reaction produces NAD+ and 5' phosphate RNA. Removal of DXO from cells increases NAD+-capped mRNA levels and enables detection of NAD+-capped intronic small nucleolar RNAs (snoRNAs), suggesting NAD+ caps can be added to 5'-processed termini. Our findings establish NAD+ as an alternative mammalian RNA cap and DXO as a deNADding enzyme modulating cellular levels of NAD+-capped RNAs. Collectively, these data reveal that mammalian RNAs can harbor a 5' end modification distinct from the classical m7G cap that promotes rather than inhibits RNA decay.

Non-coding RNA may be associated with cytoplasmic male sterility in Silene vulgaris.

Cytoplasmic male sterility (CMS) is a widespread phenomenon in flowering plants caused by mitochondrial (mt) genes. CMS genes typically encode novel proteins that interfere with mt functions and can be silenced by nuclear fertility-restorer genes. Although the molecular basis of CMS is well established in a number of crop systems, our understanding of it in natural populations is far more limited. To identify CMS genes in a gynodioecious plant, Silene vulgaris, we constructed mt transcriptomes and compared transcript levels and RNA editing patterns in floral bud tissue from female and hermaphrodite full siblings. The transcriptomes from female and hermaphrodite individuals were very similar overall with respect to variation in levels of transcript abundance across the genome, the extent of RNA editing, and the order in which RNA editing and intron splicing events occurred. We found only a single genomic region that was highly overexpressed and differentially edited in females relative to hermaphrodites. This region is not located near any other transcribed elements and lacks an open-reading frame (ORF) of even moderate size. To our knowledge, this transcript would represent the first non-coding mt RNA associated with CMS in plants and is, therefore, an important target for future functional validation studies.

Multistep Folding Kinetics of Group I Intron RNA Studied by Mg2+-Concentration Jump Simulations

RNA molecules can function as enzymes forming specific tertiary structures. Since they are highly charged molecules, mono- and divalent cations are essential for the folding reaction. Mg2+ ions are also often important at specific positions for their catalytic activities. Here, we study multistage folding kinetics of Azoarcus group I intron, which is mediated by Mg2+ cations, by Brownian dynamics simulations using a coarse-grained RNA model with explicit ions [1].

C9orf72 Disease-Related Foci Are Each Composed of One Mutant Expanded Repeat RNA

The chromosome 9 open reading frame 72 (c9orf72) gene contains a hexanucleotide (GGGGCC) repeat expansion responsible for many cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The mutant intronic RNA forms "foci" within nuclei, but the connection between transcript expression, foci, and biochemical disease mechanisms is unclear. Knowing the absolute numbers of cellular RNAs, in any system, is important for understanding the molecular mechanisms of natural physiology, disease, and drug action. Absolute numbers, however, are rarely determined, and this absence is a major impediment to understanding complex systems. Using quantitative methods, we demonstrate that foci are single RNA molecules. Most cells have no foci while 1%-2% have more than ten. Knowing the number of disease-causing molecules may contribute to understanding ALS and FTD pathology and successful drug discovery. More broadly, our data suggest that small numbers of RNA molecules may have a sizable impact on disease.

Comparative Analysis of Intronic Noncoding RNA Genes among Organisms

Development of sequencing techniques allowed us to determine genomic sequences in many organisms. Such a determined genome consists of not only protein-coding genes but also noncoding RNA (ncRNA) genes. We should analyze evolutional histories of such genes to estimate evolutional directions in future. Meanwhile, recent studies showed that some ncRNA genes are located in intragenic regions in protein-coding genes, which are called host genes. We considered that such information can help us to discuss gene evolutions. In this study, we constructed a database to analyze evolutions of protein-coding and noncoding genes based on gene locations in genomic sequences. We found that 547 out of 2,691 human host genes are orthologous to 546 out of 1,633 mouse host genes. Such orthologous host genes are involved in similar biological functions but some non-orthologous host genes have different functions. For example, non-orthologous host genes in human are annotated as neuron-related terms but such genes in mouse are not. Meanwhile, similarity searches for intronic microRNA (miRNA) genes between human and mouse showed that 85 out of the orthologous host genes have retained miRNA genes in the intronic regions. 64 out of such genes have retained intronic miRNA genes among human, mouse and rat. These results suggest that some orthologous genes have retained ncRNA genes in the intronic regions in the evolutionary process.

Control of Secreted Protein Gene Expression and the Mammalian Secretome by the Metabolic Regulator PGC-1α

Secreted proteins serve pivotal roles in the development of multicellular organisms, acting as structural matrix, extracellular enzymes, and signal molecules. However, how the secretome is regulated remains incompletely understood. Here we demonstrate, unexpectedly, that peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α), a critical transcriptional co-activator of metabolic gene expression, functions to down-regulate the expression of diverse genes encoding secreted molecules and extracellular matrix components to modulate the secretome. Using cell lines, primary cells, and mice, we show that both endogenous and exogenous PGC-1α down-regulate the expression of numerous genes encoding secreted molecules. Mechanistically, results obtained using mRNA stability measurements as well as intronic RNA expression analysis are consistent with a transcriptional effect of PGC-1α on the expression of genes encoding secreted proteins. Interestingly, PGC-1α requires the central heat shock response regulator heat shock factor protein 1 (HSF1) to affect some of its targets, and both factors co-reside on several target genes encoding secreted molecules in cells. Finally, using a mass spectrometric analysis of secreted proteins, we demonstrate that PGC-1α modulates the secretome of mouse embryonic fibroblasts. Our results define a link between a key pathway controlling metabolic regulation and the regulation of the mammalian secretome.