End Of Transcript Research Articles

Recurrent tumor-specific regulation of alternative polyadenylation of cancer-related genes

BackgroundAlternative polyadenylation (APA) results in messenger RNA molecules with different 3′ untranslated regions (3’ UTRs), affecting the molecules’ stability, localization, and translation. APA is pervasive and implicated in cancer. Earlier reports on APA focused on 3’ UTR length modifications and commonly characterized APA events as 3’ UTR shortening or lengthening. However, such characterization oversimplifies the processing of 3′ ends of transcripts and fails to adequately describe the various scenarios we observe.ResultsWe built a cloud-based targeted de novo transcript assembly and analysis pipeline that incorporates our previously developed cleavage site prediction tool, KLEAT. We applied this pipeline to elucidate the APA profiles of 114 genes in 9939 tumor and 729 tissue normal samples from The Cancer Genome Atlas (TCGA). The full set of 10,668 RNA-Seq samples from 33 cancer types has not been utilized by previous APA studies. By comparing the frequencies of predicted cleavage sites between normal and tumor sample groups, we identified 77 events (i.e. gene-cancer type pairs) of tumor-specific APA regulation in 13 cancer types; for 15 genes, such regulation is recurrent across multiple cancers. Our results also support a previous report showing the 3’ UTR shortening of FGF2 in multiple cancers. However, over half of the events we identified display complex changes to 3’ UTR length that resist simple classification like shortening or lengthening.ConclusionsRecurrent tumor-specific regulation of APA is widespread in cancer. However, the regulation pattern that we observed in TCGA RNA-seq data cannot be described as straightforward 3’ UTR shortening or lengthening. Continued investigation into this complex, nuanced regulatory landscape will provide further insight into its role in tumor formation and development.

Characterization of Chlamydial Rho and the Role of Rho-Mediated Transcriptional Polarity during Interferon Gamma-Mediated Tryptophan Limitation.

As an obligate intracellular, developmentally regulated bacterium, Chlamydia is sensitive to amino acid fluctuations within its host cell. When human epithelial cells are treated with the cytokine interferon gamma (IFN-γ), the tryptophan (Trp)-degrading enzyme, indoleamine-2,3-dioxygenase, is induced. Chlamydiae within such cells are starved for Trp and enter a state of so-called persistence. Chlamydia lacks the stringent response used by many eubacteria to respond to this stress. Unusually, chlamydial transcription is globally elevated during Trp starvation with transcripts for Trp codon-containing genes disproportionately increased. Yet, the presence of Trp codons destabilized 3' ends of transcripts in operons or large genes. We initially hypothesized that ribosome stalling on Trp codons rendered the 3' ends sensitive to RNase activity. The half-life of chlamydial transcripts containing different numbers of Trp codons was thus measured in untreated and IFN-γ-treated infected cells to determine whether Trp codons influenced the stability of transcripts. However, no effect of Trp codon content was detected. Therefore, we investigated whether Rho-dependent transcription termination could play a role in mediating transcript instability. Rho is expressed as a midcycle gene product, interacts with itself as predicted, and is present in all chlamydial species. Inhibition of Rho via the Rho-specific antibiotic, bicyclomycin, and overexpression of Rho are detrimental to chlamydiae. Finally, when we measured transcript abundance 3' to Trp codons in the presence of bicyclomycin, we observed that transcript abundance increased. These data are the first to demonstrate the importance of Rho in Chlamydia and the role of Rho-dependent transcription polarity during persistence.

PolyA tracks, polybasic peptides, poly-translational hurdles.

The abundance of messenger RNA (mRNA) is one of the major determinants of protein synthesis. As such, factors that influence mRNA stability often contribute to gene regulation. Polyadenylation of the 3' end of mRNA transcripts, the poly(A) tail, has long been recognized as one of these regulatory elements given its influence on translation efficiency and mRNA stability. Unwanted translation of the poly(A) tail signals to the cell an aberrant polyadenylation event or the lack of stop codons, which makes this sequence an important element in translation fidelity and mRNA surveillance response. Consequently, investigations into the effects of the poly(A) tail lead to the discoveries that poly-lysine as well as other polybasic peptide sequences and, to a much greater extent, polyA mRNA sequences within the open reading frame influence mRNA stability and translational efficiency. Conservation and evolutionary selection of codon usage in polyA track sequences across multiple organisms suggests a biological significance for coding polyA tracks in the regulation of gene expression. Here, we discuss the cellular responses and consequences of coding polyA track translation and synthesis of polybasic peptides. This article is categorized under: Translation > Translation Mechanisms Translation > Translation Regulation RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms.

Comprehensive comparative analysis of 5'-end RNA-sequencing methods.

RNA-Seq is an effective method to study the transcriptome, but specialized methods are required to identify 5’ ends of transcripts. Several published strategies exist for this specific purpose, but their relative merits have not been systematically analyzed. Here, we directly compare the performance of six such methods – testing five with cellular RNA as well as a novel spike-in RNA assay that helps address interpretation challenges that arise from uncertainties in annotation or RNA processing. Using a single human RNA sample, we constructed and sequenced 18 libraries with these methods and one standard, control RNA-Seq library. We find that the CAGE method performed best for mRNA and that most of its unannotated peaks are supported by evidence from other genomic methods. We then applied CAGE to eight brain-related samples and revealed sample-specific transcription start site (TSS) usage as well as a transcriptome-wide shift in TSS usage between fetal and adult brain.

High-resolution RNA 3'-ends mapping of bacterial Rho-dependent transcripts.

Transcription termination in bacteria can occur either via Rho-dependent or independent (intrinsic) mechanisms. Intrinsic terminators are composed of a stem-loop RNA structure followed by a uridine stretch and are known to terminate in a precise manner. In contrast, Rho-dependent terminators have more loosely defined characteristics and are thought to terminate in a diffuse manner. While transcripts ending in an intrinsic terminator are protected from 3′-5′ exonuclease digestion due to the stem-loop structure of the terminator, it remains unclear what protects Rho-dependent transcripts from being degraded. In this study, we mapped the exact steady-state RNA 3′ ends of hundreds of Escherichia coli genes terminated either by Rho-dependent or independent mechanisms. We found that transcripts generated from Rho-dependent termination have precise 3′-ends at steady state. These termini were localized immediately downstream of energetically stable stem-loop structures, which were not followed by uridine rich sequences. We provide evidence that these structures protect Rho-dependent transcripts from 3′-5′ exonucleases such as PNPase and RNase II, and present data localizing the Rho-utilization (rut) sites immediately downstream of these protective structures. This study represents the first extensive in-vivo map of exact RNA 3′-ends of Rho-dependent transcripts in E. coli.

SMRT‐Cappable‐seq reveals complex operon variants in bacteria

Gene expression in bacteria is organized into operons, functional units of genomic DNA containing clusters of genes under the control of single promoters. The definition of an operon relies on the precise identification of the start of transcription (TSS), the end of transcription and (TTS) and the relation between the TSS and the TTS for each transcript molecule. Yet current experimental methods for sequencing RNA in bacteria rely on the necessary fragmentation of transcripts for short read sequencing. Consequently the larger context by which genes are expressed and the phasing between the start and the end of primary transcripts are lost. As a result, and despite overwhelming mass of transcriptome information, accurate operon structure have so far been solved only for a handful of cases. Here we describe SMRT‐cappable‐seq as the first experimental methodology to identify operons genome‐wide in prokaryotes. It combines the isolation of full‐length primary transcripts with long read SMRT sequencing. Applied to E.coli, SMRT‐cappable‐seq identifies a total of 2300 operons from which around 900 are novel. Importantly, our result reveals a pervasive read‐through of previous experimentally validated transcription termination sites. Termination read‐through represents a powerful strategy to control gene expression and redefines operons with thousand of new operons containing more genes than previously thought. Taken together this data provides a first glance at the complexity of the ‘operome’ in bacteria and presents an invaluable resource for understanding gene regulation and function in bacteria.Support or Funding InformationNew England Biolabs Inc. and Pacific Biosciences of California, Inc supported this research.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Multiple Mechanisms Driving Alternative Polyadenylation of Cyclin D1 (CCND1) pre‐mRNA Processing

Eukaryotic 3′end formation involves binding of 3′end processing factors to the pre‐mRNA, cleavage downstream of a polyadenylation signal (PAS), followed by polyadenylation. The existence of multiple PASs in ~70% of human genes has led to the increased recognition of alternative polyadenylation (APA) as a new paradigm of global gene regulation involving the 3′end. Transcripts resulting from APA of the same terminal exon share the same open reading frame but have 3′UTRs of different length which affect the stability, localization and translatability of the mRNA transcript. One of the genes that undergoes APA is cyclin D1 (CCND1), which is rate limiting for G1‐S phase cell cycle transition. Cyclin D1 expression is tightly regulated and is normally transiently expressed in response to mitogenic stimuli. Both the normal protein and full length CCND1 mRNA transcript have a short half–life (~30 minutes). It is therefore not surprising that cyclin D1 expression is deregulated in several pathological states including cancer. In the B‐cell malignancy mantle cell lymphoma (MCL), the initiating lesion is a t(11; 14) (q13; q32) chromosomal translocation event that juxtaposes the CCND1 gene downstream of the IgH locus, resulting in constitutive expression of cyclin D1. Some MCL patients express CCND1 transcripts with truncated 3′UTRs and this is associated with a decrease in survival of almost two years. The truncated CCND1 transcript eliminates ~ 3kb from the 3′UTR leaving a 3′UTR <0.5kb in length. Our goal is to determine the mechanisms involved in APA as well as develop tools to identify and detect changes in the 3′UTR of genes. We developed different qRT‐PCR primers that can detect CCND1 transcripts with long/short 3′UTRs as well as nested primers for 3′RACE to help map the 3′ends of different CCND1 transcripts. In addition, used Illumina HiSeq and Pacific Biosciences (PacBio) next generation sequencing platforms to detect alternative transcripts. Reporter plasmids containing 3′UTRs were used to determine effects of RNAi. We found that highly proliferative cell lines were associated with higher levels of truncated CCND1 3′UTRs. One cell line had a mutation that resulted in the generation of a canonical PAS from a non‐optimal PAS proximal to the stop codon causing a switch from using a more distal PAS to the mutated PAS proximal to the stop codon. Illumina transcriptome sequencing after knockdown of a 3′end processing factor, CFIm25, resulted in increased shortening of the 3′UTR of CCND1 and over 1,400 other genes. Hence APA of CCND1 may be regulated by altered expression/function of genes involved in 3′end formation. When we mapped the 3′ends of CCND1 transcripts from multiple cell lines using 3′RACE, we identified a novel CCND1 transcript with a chimeric 3′UTR consisting of sequences from CCND1 fused to another gene, MCRK. This transcript containing the chimeric CCND1‐MRCK sequence was only detected in some MCL cell lines and patient DNA. Full length CCND1 3′UTR has over 60 potential miRNA target sites and reporter assays showed that the CCND1‐MRCK 3′UTR was able to evade miRNA regulation. Preliminary full length transcript PacBio sequencing resulted in the de novo identification of the CCND1‐MRCK fusion transcript. In conclusion, there are multiple ways in which CCND1 optimizes its expression in cells at the mRNA level and these mechanisms may be used by other genes under different physiological states.Support or Funding InformationDoD W81XWH‐12‐1‐0218This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cap‐Independent Translation Initiation Driven by a 13‐nucleotide motif

In eukaryotes, production of proteins is an essential biological process that typically occurs by a mechanism dependent on the 7‐methylguanosine cap at the 5′ end of an mRNA transcript. During normal cellular processes, it is this cap‐dependent (CD) mechanism that synthesizes proteins in three phases: initiation, elongation, and termination. The initiation phase is dependent upon recognition of the cap structure and attachment of the ribosomal pre‐initiation complex (PIC) to facilitate scanning of the mRNA 5′ ‐ untranslated region (UTR) in search for the initiation codon. Translation then proceeds with elongation after the initiation codon is identified. Current research indicates an alternative to the CD translation initiation mechanism, referred to as cap‐independent (CI) translation. In CI translation, recognition of the 5′ cap structure is not required for initiation of translation to occur. Several studies have demonstrated the CI mechanism to function during circumstances including stress conditions of apoptosis, hypoxia, mitosis, or inhibition of the CD mechanism. The literature suggests an internal ribosome entry site (IRES) is one possible mode CI translation can occur. An IRES directly recruits the ribosome to that specific site, regardless of its location within the RNA strand. In efforts to address the uncertainty of IRES occurrences within RNA leader sequences in human transcripts, a recent study revealed a library of human translation enhancing elements (TEEs) that can stimulate the CI mechanism. Detailed comparative functional sequence alignment analysis of the TEEs revealed a common 13‐nucleotide (13mer) motif capable of stimulating translation initiation. In order to evaluate the potential of the 13mer to function as an IRES, a series of RNA leader sequences were constructed in a reporter plasmid system containing a stable hairpin loop in the 5′‐UTR. In this system, the hairpin loop blocks ribosomal scanning due to CD initiation, resulting in reporter expression as evidence of the 13mer functioning as an IRES downstream of the hairpin loop. A series of TEEs were evaluated in this system with and without the 13mer motif. The presence of the 13mer up‐regulated translational activity, however only in certain sequence contexts. The IRES activity of the 13mer was then confirmed in a dual luciferase reporter plasmid (dlp), with the RNA leader sequences located internally between the renilla luciferase and firefly luciferase genes. In addition, translational activity was observed when CD translation was specifically inhibited using a eIF4E/eIF4G‐1 protein interaction inhibitor. Results of this study elucidate the capacity for the CI mode of translation from the human genome by demonstrating IRES activity. The identification of the 13mer as an IRES provides an opportunity to explore the mechanistic details of human IRESs and the contributions to protein production from the human genome.Support or Funding InformationIntramural Midwestern University funds awarded to BPW, and by an NIH grant awarded to JCC (Eureka Award; GM085530)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High-throughput detection of RNA processing in bacteria

BackgroundUnderstanding the RNA processing of an organism’s transcriptome is an essential but challenging step in understanding its biology. Here we investigate with unprecedented detail the transcriptome of Pseudomonas aeruginosa PAO1, a medically important and innately multi-drug resistant bacterium. We systematically mapped RNA cleavage and dephosphorylation sites that result in 5′-monophosphate terminated RNA (pRNA) using monophosphate RNA-Seq (pRNA-Seq). Transcriptional start sites (TSS) were also mapped using differential RNA-Seq (dRNA-Seq) and both datasets were compared to conventional RNA-Seq performed in a variety of growth conditions.ResultsThe pRNA-Seq library revealed known tRNA, rRNA and transfer-messenger RNA (tmRNA) processing sites, together with previously uncharacterized RNA cleavage events that were found disproportionately near the 5′ ends of transcripts associated with basic bacterial functions such as oxidative phosphorylation and purine metabolism. The majority (97%) of the processed mRNAs were cleaved at precise codon positions within defined sequence motifs indicative of distinct endonucleolytic activities. The most abundant of these motifs corresponded closely to an E. coli RNase E site previously established in vitro. Using the dRNA-Seq library, we performed an operon analysis and predicted 3159 potential TSS. A correlation analysis uncovered 105 antiparallel pairs of TSS that were separated by 18 bp from each other and were centered on single palindromic TAT(A/T)ATA motifs (likely − 10 promoter elements), suggesting that, consistent with previous in vitro experimentation, these sites can initiate transcription bi-directionally and may thus provide a novel form of transcriptional regulation. TSS and RNA-Seq analysis allowed us to confirm expression of small non-coding RNAs (ncRNAs), many of which are differentially expressed in swarming and biofilm formation conditions.ConclusionsThis study uses pRNA-Seq, a method that provides a genome-wide survey of RNA processing, to study the bacterium Pseudomonas aeruginosa and discover extensive transcript processing not previously appreciated. We have also gained novel insight into RNA maturation and turnover as well as a potential novel form of transcription regulation.NOTE: All sequence data has been submitted to the NCBI sequence read archive. Accession numbers are as follows: [NCBI sequence read archive: SRX156386, SRX157659, SRX157660, SRX157661, SRX157683 and SRX158075]. The sequence data is viewable using Jbrowse on www.pseudomonas.com.

Tn5Prime, a Tn5 based 5' capture method for single cell RNA-seq.

RNA-sequencing (RNA-seq) is a powerful technique to investigate and quantify entire transcriptomes. Recent advances in the field have made it possible to explore the transcriptomes of single cells. However, most widely used RNA-seq protocols fail to provide crucial information regarding transcription start sites. Here we present a protocol, Tn5Prime, that takes advantage of the Tn5 transposase-based Smart-seq2 protocol to create RNA-seq libraries that capture the 5′ end of transcripts. The Tn5Prime method dramatically streamlines the 5′ capture process and is both cost effective and reliable. By applying Tn5Prime to bulk RNA and single cell samples, we were able to define transcription start sites as well as quantify transcriptomes at high accuracy and reproducibility. Additionally, similar to 3′ end-based high-throughput methods like Drop-seq and 10× Genomics Chromium, the 5′ capture Tn5Prime method allows the introduction of cellular identifiers during reverse transcription, simplifying the analysis of large numbers of single cells. In contrast to 3′ end-based methods, Tn5Prime also enables the assembly of the variable 5′ ends of the antibody sequences present in single B-cell data. Therefore, Tn5Prime presents a robust tool for both basic and applied research into the adaptive immune system and beyond.

SEASTAR: systematic evaluation of alternative transcription start sites in RNA.

Alternative first exons diversify the transcriptomes of eukaryotes by producing variants of the 5′ Untranslated Regions (5′UTRs) and N-terminal coding sequences. Accurate transcriptome-wide detection of alternative first exons typically requires specialized experimental approaches that are designed to identify the 5′ ends of transcripts. We developed a computational pipeline SEASTAR that identifies first exons from RNA-seq data alone then quantifies and compares alternative first exon usage across multiple biological conditions. The exons inferred by SEASTAR coincide with transcription start sites identified directly by CAGE experiments and bear epigenetic hallmarks of active promoters. To determine if differential usage of alternative first exons can yield insights into the mechanism controlling gene expression, we applied SEASTAR to an RNA-seq dataset that tracked the reprogramming of mouse fibroblasts into induced pluripotent stem cells. We observed dynamic temporal changes in the usage of alternative first exons, along with correlated changes in transcription factor expression. Using a combined sequence motif and gene set enrichment analysis we identified N-Myc as a regulator of alternative first exon usage in the pluripotent state. Our results demonstrate that SEASTAR can leverage the available RNA-seq data to gain insights into the control of gene expression and alternative transcript variation in eukaryotic transcriptomes.

RNA secondary structure profiling in zebrafish reveals unique regulatory features

BackgroundRNA is known to play diverse roles in gene regulation. The clues for this regulatory function of RNA are embedded in its ability to fold into intricate secondary and tertiary structure.ResultsWe report the transcriptome-wide RNA secondary structure in zebrafish at single nucleotide resolution using Parallel Analysis of RNA Structure (PARS). This study provides the secondary structure map of zebrafish coding and non-coding RNAs. The single nucleotide pairing probabilities of 54,083 distinct transcripts in the zebrafish genome were documented. We identified RNA secondary structural features embedded in functional units of zebrafish mRNAs. Translation start and stop sites were demarcated by weak structural signals. The coding regions were characterized by the three-nucleotide periodicity of secondary structure and display a codon base specific structural constrain. The splice sites of transcripts were also delineated by distinct signature signals. Relatively higher structural signals were observed at 3’ Untranslated Regions (UTRs) compared to Coding DNA Sequence (CDS) and 5’ UTRs. The 3′ ends of transcripts were also marked by unique structure signals. Secondary structural signals in long non-coding RNAs were also explored to better understand their molecular function.ConclusionsOur study presents the first PARS-enabled transcriptome-wide secondary structure map of zebrafish, which documents pairing probability of RNA at single nucleotide precision. Our findings open avenues for exploring structural features in zebrafish RNAs and their influence on gene expression.

Transcriptome sequencing of the human pathogen Corynebacterium diphtheriae NCTC 13129 provides detailed insights into its transcriptional landscape and into DtxR-mediated transcriptional regulation

BackgroundThe human pathogen Corynebacterium diphtheriae is the causative agent of diphtheria. In the 1990s a large diphtheria outbreak in Eastern Europe was caused by the strain C. diphtheriae NCTC 13129. Although the genome was sequenced more than a decade ago, not much is known about its transcriptome. Our aim was to use transcriptome sequencing (RNA-Seq) to close this knowledge gap and gain insights into the transcriptional landscape of a C. diphtheriae tox+ strain.ResultsWe applied two different RNA-Seq techniques, one to retrieve 5′-ends of primary transcripts and the other to characterize the whole transcriptional landscape in order to gain insights into various features of the C. diphtheriae NCTC 13129 transcriptome. By examining the data we identified 1656 transcription start sites (TSS), of which 1202 were assigned to genes and 454 to putative novel transcripts. By using the TSS data promoter regions recognized by the housekeeping sigma factor σA and its motifs were analyzed in detail, revealing a well conserved −10 but an only weakly conserved −35 motif, respectively. Furthermore, with the TSS data 5’-UTR lengths were explored. The observed 5’-UTRs range from zero length (leaderless transcripts), which make up 20% of all genes, up to over 450 nt long leaders, which may harbor regulatory functions. The C. diphtheriae transcriptome consists of 471 operons which are further divided into 167 sub-operon structures. In a differential expression analysis approach, we discovered that genetic disruption of the iron-sensing transcription regulator DtxR, which controls expression of diphtheria toxin (DT), causes a strong influence on general gene expression. Nearly 15% of the genome is differentially transcribed, indicating that DtxR might have other regulatory functions in addition to regulation of iron metabolism and DT. Furthermore, our findings shed light on the transcriptional landscape of the DT encoding gene tox and present evidence for two tox antisense RNAs, which point to a new way of transcriptional regulation of toxin production.ConclusionsThis study presents extensive insights into the transcriptome of C. diphtheriae and provides a basis for future studies regarding gene characterization, transcriptional regulatory networks, and regulation of the tox gene in particular.

Multi-Platform Sequencing Approach Reveals a Novel Transcriptome Profile in Pseudorabies Virus.

Third-generation sequencing is an emerging technology that is capable of solving several problems that earlier approaches were not able to, including the identification of transcripts isoforms and overlapping transcripts. In this study, we used long-read sequencing for the analysis of pseudorabies virus (PRV) transcriptome, including Oxford Nanopore Technologies MinION, PacBio RS-II, and Illumina HiScanSQ platforms. We also used data from our previous short-read and long-read sequencing studies for the comparison of the results and in order to confirm the obtained data. Our investigations identified 19 formerly unknown putative protein-coding genes, all of which are 5′ truncated forms of earlier annotated longer PRV genes. Additionally, we detected 19 non-coding RNAs, including 5′ and 3′ truncated transcripts without in-frame ORFs, antisense RNAs, as well as RNA molecules encoded by those parts of the viral genome where no transcription had been detected before. This study has also led to the identification of three complex transcripts and 50 distinct length isoforms, including transcription start and end variants. We also detected 121 novel transcript overlaps, and two transcripts that overlap the replication origins of PRV. Furthermore, in silico analysis revealed 145 upstream ORFs, many of which are located on the longer 5′ isoforms of the transcripts.

High-throughput sequencing of the chloroplast and mitochondrion of Chlamydomonas reinhardtii to generate improved de novo assemblies, analyze expression patterns and transcript speciation, and evaluate diversity among laboratory strains and wild isolates.

Chlamydomonas reinhardtii is a unicellular chlorophyte alga that is widely studied as a reference organism for understanding photosynthesis, sensory and motile cilia, and for development of an algal-based platform for producing biofuels and bio-products. Its highly repetitive, ~205-kbp circular chloroplast genome and ~15.8-kbp linear mitochondrial genome were sequenced prior to the advent of high-throughput sequencing technologies. Here, high coverage shotgun sequencing was used to assemble both organellar genomes de novo. These new genomes correct dozens of errors in the prior genome sequences and annotations. Genome sequencing coverage indicates that each cell contains on average 83 copies of the chloroplast genome and 130 copies of the mitochondrial genome. Using protocols and analyses optimized for organellar transcripts, RNA-Seq was used to quantify their relative abundances across 12 different growth conditions. Forty-six percent of total cellular mRNA is attributable to high expression from a few dozen chloroplast genes. RNA-Seq data were used to guide gene annotation, to demonstrate polycistronic gene expression, and to quantify splicing of psaA and psbA introns. In contrast to a conclusion from a recent study, we found that chloroplast transcripts are not edited. Unexpectedly, cytosine-rich polynucleotide tails were observed at the 3'-end of all mitochondrial transcripts. A comparative genomics analysis of eight laboratory strains and 11 wild isolates of C.reinhardtii identified 2658 variants in the organellar genomes, which is 1/10th as much genetic diversity as is found in the nucleus.

NanoCAGE-XL: An Approach to High-Confidence Transcription Start Site Sequencing.

Identifying the transcription start sites (TSS) of genes is essential for characterizing promoter regions. Several protocols have been developed to capture the 5' end of transcripts via Cap-Analysis of Gene Expression (CAGE) or linker-ligation strategies such as Paired-End Analysis of Transcription Start Sites (PEAT), but often require large amounts of tissue. More recently, nanoCAGE was developed for sequencing on the Illumina GAIIx to overcome this limitation. In this chapter, we present the nanoCAGE-XL protocol, the first publicly available adaptation of nanoCAGE for sequencing on recent ultra-high-throughput platforms such as Illumina HiSeq-2000. NanoCAGE-XL provides a method for precise transcription start site identification in large eukaryotic genomes, even in cases where input total RNA quantity is very limited.

Cloning of a novel gene from Penicillium oxalicum I1 which in Escherichia coli enhances the secretion of acetic acid

Description of the subject. Organic acids play an important role in the conversion of insoluble ions into soluble ones in soil. Heterologous overexpression of a single gene in a cell is the optimal strategy for increasing the secretion of organic acids solubilizing phosphate. Objectives. In this study, we constructed a primary cDNA library of Penicillium oxalicum I1, and screened clones that can solubilize P in tricalcium phosphate (TCP) medium. We aimed to obtain the gene expressed in Escherichia coli, which can enhance organic acid secretion. Method. A primary cDNA library of Penicillium oxalicum I1 was constructed using the switching mechanism at the 5'-end of RNA transcription. The organic acid secretion ability of E. coli DH5α™ with overexpressed P. oxalicum I1gene was tested in TCP medium where glucose is the sole carbon source. Afterwards, pyruvic acid, citric acid, α-ketoglutaric acid, succinic acid, fumaric acid, and malic acid were used as sole carbon source substitutes for glucose in the TCP medium to test the organic acid secretion ability of the transformed E. coli DH5α™. Results. A total of 106 clones showed halos in TCP medium, among which clone I-2 displayed clear halo. The full-length cDNA of clone I-2 was 1,151 bp, with a complete open reading frame of 702 bp, which encoded a hypothetical protein of 233 amino acids. The cDNA sequence showed 68% identity and 73% query cover with other fungal gene sequences of which the function remains unknown. Escherichia coli containing the cloned gene secreted up to 567 mg·l-1 acetic acid within 48 h. The use of glucose, pyruvic acid, α-ketoglutaric acid, and malic acid improved the acetic acid secretion of the E. coli DH5α™ clone I-2. By contrast, the use of citric acid, succinic acid, and fumaric acid did not improve the acetic acid secretion of clone I-2 compared to a control E. coli DH5α™ strain bearing only the cloning vector without any insert. Conclusions. We obtained a novel gene from Penicillium oxalicum I1 whose overexpression in E. coli DH5α™ increased the secretion of acetic acid. This observation should help to understand what is the function of the gene isolated from P. oxalicum as well as that of its homologs found in several other species of the Penicillium genus.

Visualization of RNA 3' ends in Escherichia coli Using 3' RACE Combined with Primer Extension.

In this assay, 3' RACE (Rapid Amplification of cDNA 3' Ends) followed by PE (primer extension), abbreviated as 3' RACE-PE is used to identify the mRNA 3' ends. The following protocol describes the amplification of the mRNA 3' ends at the galactose operon in E. coli and the corresponding visualization of the PCR products through PE. In PE, the definite primer is 5' end-labeled using [γ-(32) P] ATP and T4 polynucleotide kinase, which anneals to the specific DNA molecules within the PCR product of the 3' RACE. The conventional PE can only be used to locate the 5' end of an mRNA transcript since reverse transcriptase (RTase) polymerizes only in the 5' → 3' direction. Thus, Taq polymerase is used instead of RTase, PCR is performed. Therefore, we are able to locate the 3' end of the mRNA using this assay. The relative amount of the 3' end can be directly visualized and quantified by way of separating DNA products in a denaturing 8% urea-PAGE (Polyacrylamide Gel Electrophoresis) gel. The exact position of the 3' ends can be sequenced by comparison of these final DNA products with the corresponding DNA sequencing ladder.

Characterizing Small RNAs in Filamentous Fungi Using the Rice Blast Fungus, Magnaporthe oryzae, as an Example.

The goal of this chapter is to provide a framework of sequential steps for small RNA (sRNA) analysis in filamentous fungi. Here, we present protocols for (1) comparative analysis of sRNAs in different conditions, (2) comparisons of sRNA libraries to RNAseq data and (3) identification and analysis of methylguanosine-capped and polyadenylated sRNAs (CPA-sRNAs). This species of small RNA is particularly interesting in Magnaporthe oryzae, as they map to transcription start and end sites of protein-coding genes. While we do not provide specific command lines for scripts, we provide a general framework for steps needed to carry out all three types of analyses, including relevant references, websites and free online tools. Screenshots are provided from our own customized interface using M. oryzae as an example, to assist the reader in visualizing many of the steps.

Analysis of Bacterial Transcription by "Massively Systematic Transcript End Readout," MASTER.

A systems-level view of cellular gene expression requires understanding the mechanistic principles governing each step of transcription. In this chapter, we describe a massively multiplexed method for the analysis of the relationship between nucleic acid sequence and transcription termed "MASTER," for massively systematic transcript end readout. MASTER enables parallel measurements of transcription output from at least 410 (~1,000,000) individual template sequences in vitro and in vivo. MASTER involves constructing a DNA template library of barcoded sequences, generating RNA transcripts from the library during transcription in vitro or in vivo, and analyzing the relative abundance and 5'-end sequences of the RNA transcripts by high-throughput sequencing. MASTER provides a powerful, rapid, and versatile method to identify sequence determinants of each step of transcription and to define the mechanistic basis by which these sequence determinants dictate transcription output.