Direct conversion of fibroblasts to neurons by reprogramming PTB-regulated microRNA circuits.

The induction of pluripotency or trans-differentiation of one cell type to another can be accomplished with cell-lineage-specific transcription factors. Here, we report that repression of a single RNA binding polypyrimidine-tract-binding (PTB) protein, which occurs during normal brain development via the action of miR-124, is sufficient to induce trans-differentiation of fibroblasts into functional neurons. Besides its traditional role in regulated splicing, we show that PTB has a previously undocumented function in the regulation of microRNA functions, suppressing or enhancing microRNA targeting by competitive binding on target mRNA or altering local RNA secondary structure. A key event during neuronal induction is the relief of PTB-mediated blockage of microRNA action on multiple components of the REST complex, thereby derepressing a large array of neuronal genes, including miR-124 and multiple neuronal-specific transcription factors, in nonneuronal cells. This converts a negative feedback loop to a positive one to elicit cellular reprogramming to the neuronal lineage.

Profiling of RNA editing events in plant organellar transcriptomes with high-throughput sequencing.

RNA editing is a crucial post-transcriptional modification process in plant organellar RNA metabolism. rRNA removal-based total RNA-seq is one of the most common methods to study this event. However, the lack of commercial kits to remove rRNAs limits the usage of this method, especially for non-model plant species. DSN-seq is a transcriptome sequencing method utilizing duplex-specific nuclease (DSN) to degrade highly abundant cDNA species especially those from rRNAs while keeping the robustness of transcript levels of the majority of other mRNAs, and has not been applied to study RNA editing in plants before. In this study, we evaluated the capability of DSN-seq to reduce rRNA content and profile organellar RNA editing events in plants, as well we used commercial Ribo-off-seq and standard mRNA-seq as comparisons. Our results demonstrated that DSN-seq efficiently reduced rRNA content and enriched organellar transcriptomes in rice. With high sensitivity to RNA editing events, DSN-seq and Ribo-off-seq provided a more complete and accurate RNA editing profile of rice, which was further validated by Sanger sequencing. Furthermore, DSN-seq also demonstrated efficient organellar transcriptome enrichment and high sensitivity for profiling RNA editing events in Arabidopsis thaliana. Our study highlights the capability of rRNA removal-based total RNA-seq for profiling RNA editing events in plant organellar transcriptomes and also suggests DSN-seq as a widely accessible RNA editing profiling method for various plant species.

Transcriptome analysis reveals downregulation of virulence-associated genes expression in a low virulence Verticillium dahliae strain.

Verticillium dahliae causes wilt diseases and early senescence in numerous plants, including agricultural crops such as cotton. In this study, we studied two closely related V. dahliae strains, and found that V991w showed significantly reduced virulence on cotton than V991b. Comprehensive transcriptome analysis revealed various differentially expressed genes between the two strains, with more genes repressed in V991w. The downregulated genes in V991w were involved in production of hydrophobins, melanin, predicted aflatoxin, and membrane proteins, most of which are related to pathogenesis and multidrug resistance. Consistently, melanin production in V991w in vitro was compromised. We next obtained genomic variations between the two strains, demonstrating that transcription factor genes containing fungi specific transcription factor domain and fungal Zn2-Cys6 binuclear cluster domain were enriched in V991w, which might be related to pathogenicity-related genes downregulation. Thus, this study supports a model in which some virulence factors involved in V. dahliae pathogenicity were pre-expressed during in vitro growth before host interaction.

The dynamics of FTO binding and demethylation from the m6A motifs.

N6-methyladenosine (m6A) is considered as a reversible RNA modification occurring more frequently on the GAC than AAC context in vivo, which regulates post-transcriptional gene expression in mammalian cells. m6A 'writers' METTL3 and METTL14 demonstrate a strong preference for binding AC-containing motifs in living cells. However, this evidence is currently lacking for m6A erasers, leaving the dynamics of the internal m6A modification under debate recently. We analysed three recently published FTO CLIP-seq data sets and two generated in this study, one of the two known m6A 'erasers'. FTO binding peaks from all cell lines contain RRACH motifs. Only those from K562, 3T3-L1and HeLa cells were enriched in AC-containing motifs, while those from HEK293 were not. The exogenously overexpressed FTO effectively binds to m6A motif-containing RNA sites. FTO overexpression specifically removed m6A modification from GGACU and RRACU motifs in a concentration-dependent manner. These findings underline the dynamics of FTO in target selection, which is predicted to contribute to both the m6A dynamics and the FTO plasticity in biological functions and diseases.

Transcriptome analysis reveals the complexity of alternative splicing regulation in the fungus Verticillium dahliae.

BACKGROUND: Alternative splicing (AS) regulation is extensive and shapes the functional complexity of higher organisms. However, the contribution of alternative splicing to fungal biology is not well studied. RESULTS: This study provides sequences of the transcriptomes of the plant wilt pathogen Verticillium dahliae, using two different strains and multiple methods for cDNA library preparations. We identified alternatively spliced mRNA isoforms in over a half of the multi-exonic fungal genes. Over one-thousand isoforms involve TopHat novel splice junction; multiple types of combinatory alternative splicing patterns were identified. We showed that one Verticillium gene could use four different 5' splice sites and two different 3' donor sites to produce up to five mature mRNAs, representing one of the most sophisticated alternative splicing model in eukaryotes other than animals. Hundreds of novel intron types involving a pair of new splice sites were identified in the V. dahliae genome. All the types of AS events were validated by using RT-PCR. Functional enrichment analysis showed that AS genes are involved in most known biological functions and enriched in ATP biosynthesis, sexual/asexual reproduction, morphogenesis, signal transduction etc., predicting that the AS regulation modulates mRNA isoform output and shapes the V. dahliae proteome plasticity of the pathogen in response to the environmental and developmental changes. CONCLUSIONS: These findings demonstrate the comprehensive alternative splicing mechanisms in a fungal plant pathogen, which argues the importance of this fungus in developing complicate genome regulation strategies in eukaryotes.

In situ detection of human glioma based on tissue optical properties using diffuse reflectance spectroscopy.

Safely maximizing brain cancer removal without injuring adjacent healthy tissue is crucial for optimal treatment outcomes. However, it is challenging to distinguish cancer from noncancer intraoperatively. This study aimed to explore the feasibility of diffuse reflectance spectroscopy (DRS) as a label-free and real-time detection technology for discrimination between brain cancer and noncancer tissues. Fifty-five fresh cancer and noncancer specimens from 19 brain surgeries were measured with DRS, and the results were compared with co-registered clinical standard histopathology. Tissue optical properties were quantitatively obtained from the diffuse reflectance spectra and compared among different types of brain tissues. A machine learning-based classifier was trained to differentiate cancerous versus noncancerous tissues. Our method could achieve a sensitivity of 93% and specificity of 95% for discriminating high-grade glioma from normal white matter. Our results showed that DRS has the potential to be used for label-free, real-time in vivo cancer detection during brain surgery.

Deciphering the Biochemical Similarities and Differences Among Human Neuroglial Cells and Glioma Cells Using Fourier Transform Infrared Spectroscopy.

BACKGROUND: The use of Fourier transform infrared spectroscopy to identify the peritumoral tissue of gliomas proves the potential of this technique to distinguish normal brain tissues from glioma tissues. However, due to the heterogeneity of gliomas, it is difficult to characterize the representative spectra of normal brain tissues and glioma tissues. The linear spectra of major cellular components, such as microglia, astrocytes, and glioma cells, were obtained to quantify the biochemical changes between healthy cells and tumor cells, and provide supporting data for the final distinction between tumor and normal brain tissue. METHODS: Fourier transform infrared was used to measure human astrocytes, microglia (HM1900), and glioma cells (U87, BT325), and the cellular components of the 4 types of cells were analyzed by means of average spectra, second-derivative spectra, principal component analysis, hierarchical cluster analysis, and difference spectra. RESULTS: The proteomics, lipidomics, genomics, and metabolic statuses of the cells were different. The amide I, lipid, and nuclear acid regions of the spectra are the most obvious regions to use for distinguishing the 4 types of cells. CONCLUSIONS: We conclude that an improved understanding of both similarities and differences in the cellular components of astrocytes, microglia, and glioma cells can help us better understand the heterogeneity of gliomas. We suggest that targeting cellular metabolism (protein, lipid, and nuclear acids) is helpful to distinguish between normal brain tissue and glioma tissue, which has broad application prospects.

ILF3 represses repeat-derived microRNAs targeting RIG-I mediated type I interferon response.

MicroRNAs (miRNAs) play important roles in regulated gene expression and miRNA biogenesis is also subject to regulation, together constituting critical regulatory circuitries in numerous physiological and pathological processes. As a dsRNA binding protein, interleukin enhancer binding factor 3 (ILF3) has been implicated as a negative regulator in miRNA biogenesis, but the mechanism and specificity have remained undefined. Here, combining small-RNA-seq and CLIP-seq, we showed that ILF3 directly represses many miRNAs or perhaps other types of small RNAs annotated in both miRBase and MirGeneDB. We demonstrated that ILF3 preferentially binds to A/U-enriched motifs, which tend to lengthen and/or stabilize the stem-loop in pri-miRNAs, thereby effectively competing with the Microprocessor to block miRNA biogenesis. Focusing on the biological function of ILF3-suppressed miR-582-3p, we discovered that this LINE-derived miRNA targets a critical interferon-inducible gene RIG-I for repression, thus establishing a novel ILF3/miR-582/RIG-I axis in the antiviral response.

The Welwitschia genome reveals a unique biology underpinning extreme longevity in deserts.

The gymnosperm Welwitschia mirabilis belongs to the ancient, enigmatic gnetophyte lineage. It is a unique desert plant with extreme longevity and two ever-elongating leaves. We present a chromosome-level assembly of its genome (6.8 Gb/1 C) together with methylome and transcriptome data to explore its astonishing biology. We also present a refined, high-quality assembly of Gnetum montanum to enhance our understanding of gnetophyte genome evolution. The Welwitschia genome has been shaped by a lineage-specific ancient, whole genome duplication (~86 million years ago) and more recently (1-2 million years) by bursts of retrotransposon activity. High levels of cytosine methylation (particularly at CHH motifs) are associated with retrotransposons, whilst long-term deamination has resulted in an exceptionally GC-poor genome. Changes in copy number and/or expression of gene families and transcription factors (e.g. R2R3MYB, SAUR) controlling cell growth, differentiation and metabolism underpin the plant's longevity and tolerance to temperature, nutrient and water stress.

GISSD: Group I Intron Sequence and Structure Database.

Group I Intron Sequence and Structure Database (GISSD) is a specialized and comprehensive database for group I introns, focusing on the integration of useful group I intron information from available databases and providing de novo data that is essential for understanding these introns at a systematic level. This database presents 1789 complete intron records, including the nucleotide sequence of each annotated intron plus 15 nt of the upstream and downstream exons, and the pseudoknots-containing secondary structures predicted by integrating comparative sequence analyses and minimal free energy algorithms. These introns represent all 14 subgroups, with their structure-based alignments being separately provided. Both structure predictions and alignments were done manually and iteratively adjusted, which yielded a reliable consensus structure for each subgroup. These consensus structures allowed us to judge the confidence of 20 085 group I introns previously found by the INFERNAL program and to classify them into subgroups automatically. The database provides intron-associated taxonomy information from GenBank, allowing one to view the detailed distribution of all group I introns. CDSs residing in introns and 3D structure information are also integrated if available. About 17 000 group I introns have been validated in this database; approximately 95% of them belong to the IC3 subgroup and reside in the chloroplast tRNA(Leu) gene. The GISSD database can be accessed at http://www.rna.whu.edu.cn/gissd/

Coordination of two sequential ester-transfer reactions: exogenous guanosine binding promotes the subsequent omegaG binding to a group I intron.

Self-splicing of group I introns is accomplished by two sequential ester-transfer reactions mediated by sequential binding of two different guanosine ligands, but it is yet unclear how the binding is coordinated at a single G-binding site. Using a three-piece trans-splicing system derived from the Candida intron, we studied the effect of the prior GTP binding on the later omegaG binding by assaying the ribozyme activity in the second reaction. We showed that adding GTP simultaneously with and prior to the esterified omegaG in a substrate strongly accelerated the second reaction, suggesting that the early binding of GTP facilitates the subsequent binding of omegaG. GTP-mediated facilitation requires C2 amino and C6 carbonyl groups on the Watson-Crick edge of the base but not the phosphate or sugar groups, suggesting that the base triple interactions between GTP and the binding site are important for the subsequent omegaG binding. Strikingly, GTP binding loosens a few local structures of the ribozyme including that adjacent to the base triple, providing structural basis for a rapid exchange of omegaG for bound GTP.

Poly(A)-seq: A method for direct sequencing and analysis of the transcriptomic poly(A)-tails.

Poly(A) tails at the 3' end of eukaryotic messenger RNAs control mRNA stability and translation efficiency. Facilitated by various NGS methods, alternative polyadenylation sites determining the 3'-UTR length of gene transcripts have been extensively studied. However, poly(A) lengths demonstrating dynamic and developmental regulation remain largely unexplored. The recently developed NGS-based methods for genome-wide poly(A) profiling have promoted the study of genom-wide poly(A) dynamics. Here we present a straight forward NGS-method for poly(A) profiling, which applies a direct 3'-end adaptor ligation and the template switching for 5'-end adaptor ligation for cDNA library construction. Poly(A) lengths are directly calculated from base call data using a self-developed pipeline pA-finder. The libraries were directly sequenced from the 3'-UTR regions into the followed poly(A) tails, firstly on NextSeq 500 to produce single-end 300-nt reads, demonstrating the method feasibility and that optimization of the fragmented RNA size for cDNA library construction could detecting longer poly (A) tails. We next applied Poly(A)-seq cDNA libraries containing 40-nt and 120-nt poly(A) tail spike-in RNAs on HiSeq X-ten and NovaSeq 6000 to obtain 150-nt and 250-nt pair-end reads. The sequencing profiles of the spike-in RNAs demonstrated both high accuracy and high quality score in reading poly(A) tails. The poly(A) signal bleeding into the 3' adaptor sequence and a sharp decreased quality score at the junction were observed, allowing the modification of pA-finder to remove homopolymeric signal bleeding. We hope that wide applications of Poly(A)-seq help facilitate the study of the development- and disease-related poly(A) dynamics and regulation, and of the recent emerging mixed tailing regulation.

A phage RNA-binding protein binds to a non-cognate structured RNA and stabilizes its core structure.

Recent studies suggest that some RNA-binding proteins facilitate the folding of non-cognate RNAs. Here, we report that bacteriophage MS2 coat protein (MS2 CP) bound and promoted the catalytic activity of Candida group I ribozyme. Cloning of the MS2-bound RNA segments showed that this protein primarily interacts with the P5ab-P5 structure. Ultraviolet cross-linking and the T1 footprinting assay further showed that MS2 binding stabilized tertiary interactions, including the conserved L9-P5 interaction, and led to a more compact core structure. This mechanism is similar to that of the yeast mitochondrial tyrosyl-tRNA synthetase on other group I introns, suggesting that different RNA-binding proteins may use common mechanisms to support RNA structures.

The structure and function of catalytic RNAs.

Before the discovery of ribozymes, RNA had been proposed to function as a catalyst, based on the discovery that RNA folded into high-ordered structures as protein did. This hypothesis was confirmed in the 1980s, after the discovery of Tetrahymena group I intron and RNase P ribozyme. There have been about ten ribozymes identified during the past thirty years, as well as the fact that ribosomes function as ribozymes. Advances have been made in understanding the structures and functions of ribozymes, with numerous crystal structures resolved in the past years. Here we review the structure-function relationship of both small and large ribozymes, especially the structural basis of their catalysis. ribozyme, structure, catalysis.

Seasonal variations in body melanism and size of the wolf spider Pardosa astrigera (Araneae: Lycosidae).

Variations in species morphology and life-history traits strongly correlate with geographic and climatic characteristics. Most studies on morphological variations in animals focus on ectotherms distributed on a large geographic scale across latitudinal and/or altitudinal gradient. However, the morphological variations of spiders living in the same habitats across different seasons have not been reported. In this study, we used the wolf spider, Pardosa astrigera, as a model to determine seasonal differences in adult body size, melanism, fecundity, and egg diameter both in the overwintering and the first generation for 2010 and 2016. The results showed that in 2010, both females and males of the overwintering generation were significantly darker than the first generation. Moreover, the overwintering females were markedly larger and produced more and bigger eggs than the first generation in both 2010 and 2016. Considering the overwintering P. astrigera experiencing low temperature and/or desiccation stress, these results suggest that substantially darker and larger body of the overwintering generation is adaptive to adverse conditions.

CELF1 preferentially binds to exon-intron boundary and regulates alternative splicing in HeLa cells.

The current RIP-seq approach has been developed for the identification of genome-wide interaction between RNA binding protein (RBP) and the bound RNA transcripts, but still rarely for identifying its binding sites. In this study, we performed RIP-seq experiments in HeLa cells using a monoclonal antibody against CELF1. Mapping of the RIP-seq reads showed a biased distribution at the 3'UTR and intronic regions. A total of 15,285 and 1384 CELF1-specific sense and antisense peaks were identified using the ABLIRC software tool. Our bioinformatics analyses revealed that 5' and 3' splice site motifs and GU-rich motifs were highly enriched in the CELF1-bound peaks. Furthermore, transcriptome analyses revealed that alternative splicing was globally regulated by CELF1 in HeLa cells. For example, the inclusion of exon 16 of LMO7 gene, a marker gene of breast cancer, is positively regulated by CELF1. Taken together, we have shown that RIP-seq data can be used to decipher RBP binding sites and reveal an unexpected landscape of the genome-wide CELF1-RNA interactions in HeLa cells. In addition, we found that CELF1 globally regulates the alternative splicing by binding the exon-intron boundary in HeLa cells, which will deepen our understanding of the regulatory roles of CELF1 in the pre-mRNA splicing process.

NEAT1 scaffolds RNA-binding proteins and the Microprocessor to globally enhance pri-miRNA processing.

MicroRNA (miRNA) biogenesis is known to be modulated by a variety of RNA-binding proteins (RBPs), but in most cases, individual RBPs appear to influence the processing of a small subset of target miRNAs. Here, we report that the RNA-binding NONO-PSF heterodimer binds a large number of expressed pri-miRNAs in HeLa cells to globally enhance pri-miRNA processing by the Drosha-DGCR8 Microprocessor. NONO and PSF are key components of paraspeckles organized by the long noncoding RNA (lncRNA) NEAT1. We further demonstrate that NEAT1 also has a profound effect on global pri-miRNA processing. Mechanistic dissection reveals that NEAT1 broadly interacts with the NONO-PSF heterodimer as well as many other RBPs and that multiple RNA segments in NEAT1, including a 'pseudo pri-miRNA' near its 3' end, help attract the Microprocessor. These findings suggest a 'bird nest' model in which an lncRNA orchestrates efficient processing of potentially an entire class of small noncoding RNAs in the nucleus.

A structured RNA in hepatitis B virus post-transcriptional regulatory element represses alternative splicing in a sequence-independent and position-dependent manner.

Hepatitis B virus (HBV) transcripts are subjected to multiple splicing decisions, but the mechanism of splicing regulation remains poorly understood. In this study, we used a well-investigated alternative splicing reporter to dissect splicing regulatory elements residing in the post-transcriptional regulatory element (PRE) of HBV. A strong intronic splicing silencer (ISS) with a minimal functional element of 105 nucleotides (referred to as PRE-ISS) was identified and, interestingly, both the sense and antisense strands of the element were found to strongly suppress alternative splicing in multiple human cell lines. PRE-ISS folds into a double-hairpin structure, in which substitution mutations disrupting the double-hairpin structure abolish the splicing silencer activity. Although it harbors two previously identified binding sites for polypyrimidine tract binding protein, PRE-ISS represses splicing independent of this protein. The silencing function of PRE-ISS exhibited a strong position dependence, decreasing with the distance from affected splice sites. PRE-ISS does not belong to the intronic region of any HBV splicing variants identified thus far, preventing the testing of this intronic silencer function in the regulation of HBV splicing. These findings, together with the identification of multiple sense-antisense ISSs in the HBV genome, support the hypothesis that a sequence-independent and structure-dependent regulatory mechanism may have evolved to repress cryptic splice sites in HBV transcripts, thereby preventing their aberrant splicing during viral replication in the host.

A maturase that specifically stabilizes and activates its cognate group I intron at high temperatures.

Folding of large structured RNAs into their functional tertiary structures at high temperatures is challenging. Here we show that I-TnaI protein, a small LAGLIDADG homing endonuclease encoded by a group I intron from a hyperthermophilic bacterium, acts as a maturase that is essential for the catalytic activity of this intron at high temperatures and physiological cationic conditions. I-TnaI specifically binds to and induces tertiary packing of the P4-P6 domain of the intron; this RNA-protein complex might serve as a thermostable platform for active folding of the entire intron. Interestingly, the binding affinity of I-TnaI to its cognate intron RNA largely increases with temperature; over 30-fold stronger binding at higher temperatures relative to 37 °C correlates with a switch from an entropy-driven (37 °C) to an enthalpy-driven (55-60 °C) interaction mode. This binding mode may represent a novel strategy how an RNA binding protein can promote the function of its target RNA specifically at high temperatures.