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SAGE2Splice: Unmapped SAGE Tags Reveal Novel Splice Junctions  [PDF]
Byron Yu-Lin Kuo,Ying Chen,Slavita Bohacec,?jvind Johansson,Wyeth W Wasserman,Elizabeth M Simpson
PLOS Computational Biology , 2006, DOI: 10.1371/journal.pcbi.0020034
Abstract: Serial analysis of gene expression (SAGE) not only is a method for profiling the global expression of genes, but also offers the opportunity for the discovery of novel transcripts. SAGE tags are mapped to known transcripts to determine the gene of origin. Tags that map neither to a known transcript nor to the genome were hypothesized to span a splice junction, for which the exon combination or exon(s) are unknown. To test this hypothesis, we have developed an algorithm, SAGE2Splice, to efficiently map SAGE tags to potential splice junctions in a genome. The algorithm consists of three search levels. A scoring scheme was designed based on position weight matrices to assess the quality of candidates. Using optimized parameters for SAGE2Splice analysis and two sets of SAGE data, candidate junctions were discovered for 5%–6% of unmapped tags. Candidates were classified into three categories, reflecting the previous annotations of the putative splice junctions. Analysis of predicted tags extracted from EST sequences demonstrated that candidate junctions having the splice junction located closer to the center of the tags are more reliable. Nine of these 12 candidates were validated by RT-PCR and sequencing, and among these, four revealed previously uncharacterized exons. Thus, SAGE2Splice provides a new functionality for the identification of novel transcripts and exons. SAGE2Splice is available online at http://www.cisreg.ca.
Identifying novel genes in C. elegans using SAGE tags
Matthew J Nesbitt, Donald G Moerman, Nansheng Chen
BMC Molecular Biology , 2010, DOI: 10.1186/1471-2199-11-96
Abstract: In this project, we have developed a method of reconstructing full-length cDNA sequences based on short expressed sequence tags which is called sequence tag-based amplification of cDNA ends (STACE). Expressed tags are used as anchors for retrieving full-length transcripts in two rounds of PCR amplification. We have demonstrated the application of STACE in reconstructing full-length cDNA sequences using expressed tags mined in an array of serial analysis of gene expression (SAGE) of C. elegans cDNA libraries. We have successfully applied STACE to recover sequence information for 12 genes, for two of which we found isoforms. STACE was used to successfully recover full-length cDNA sequences for seven of these genes.The STACE method can be used to effectively reconstruct full-length cDNA sequences of genes that are under-represented in cDNA sequencing projects and have been missed by existing gene prediction methods, but their existence has been suggested by short sequence tags such as SAGE tags.The nematode Caenorhabditis elegans, which is a well-established model organism for biomedical research [1], is the first metazoan whose genome was subject to whole-genome sequencing [2]. Its gene models were first predicted using the gene prediction program Genefinder (P. Green, unpublished). Over the dozen years since the completion of the C. elegans genome sequencing project [2], the C. elegans gene set has been curated by the C. elegans research community and by WormBase curators [1,3-5]. However, the C. elegans gene set is still far from complete for the following reasons: First, because Genefinder, like other gene prediction programs, was developed based on an incomplete understanding of gene structures, it suffers from both false positive and false negative predictions; second, many bona fide genes, especially those of unknown character, have been missed. In WormBase http://www.wormbase.org webcite, the official database for the biology and genomics of C. elegans, less th
Overlapping Antisense Transcription in the Human Genome  [PDF]
M. E. Fahey,T. F. Moore,D. G. Higgins
Comparative and Functional Genomics , 2002, DOI: 10.1002/cfg.173
Abstract: Accumulating evidence indicates an important role for non-coding RNA molecules in eukaryotic cell regulation. A small number of coding and non-coding overlapping antisense transcripts (OATs) in eukaryotes have been reported, some of which regulate expression of the corresponding sense transcript. The prevalence of this phenomenon is unknown, but there may be an enrichment of such transcripts at imprinted gene loci. Taking a bioinformatics approach, we systematically searched a human mRNA database (RefSeq) for complementary regions that might facilitate pairing with other transcripts. We report 56 pairs of overlapping transcripts, in which each member of the pair is transcribed from the same locus. This allows us to make an estimate of 1000 for the minimum number of such transcript pairs in the entire human genome. This is a surprisingly large number of overlapping gene pairs and, clearly, some of the overlaps may not be functionally significant. Nonetheless, this may indicate an important general role for overlapping antisense control in gene regulation. EST databases were also investigated in order to address the prevalence of cases of imprinted genes with associated non-coding overlapping, antisense transcripts. However, EST databases were found to be completely inappropriate for this purpose.
RNAi Screen Indicates Widespread Biological Function for Human Natural Antisense Transcripts  [PDF]
Mohammad Ali Faghihi,Jannet Kocerha,Farzaneh Modarresi,P?r G. Engstr?m,Alistair M. Chalk,Shaun P. Brothers,Eric Koesema,Georges St. Laurent III,Claes Wahlestedt
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0013177
Abstract: Natural antisense transcripts represent a class of regulatory RNA molecules, which are characterized by their complementary sequence to another RNA transcript. Extensive sequencing efforts suggest that natural antisense transcripts are prevalent throughout the mammalian genome; however, their biological significance has not been well defined. We performed a loss-of-function RNA interference (RNAi) screen, which targeted 797 evolutionary conserved antisense transcripts, and found evidence for a regulatory role for a number of natural antisense transcripts. Specifically, we found that natural antisense transcripts for CCPG1 and RAPGEF3 may functionally disrupt signaling pathways and corresponding biological phenotypes, such as cell viability, either independently or in parallel with the corresponding sense transcript. Our results show that the large-scale siRNA screen can be applied to evaluate natural antisense transcript modulation of fundamental cellular events.
Unexpected observations after mapping LongSAGE tags to the human genome
Céline Keime, Marie Sémon, Dominique Mouchiroud, Laurent Duret, Olivier Gandrillon
BMC Bioinformatics , 2007, DOI: 10.1186/1471-2105-8-154
Abstract: Using a published error rate in SAGE libraries, we first removed the tags likely to result from sequencing errors. We then observed that an unexpectedly large number of the remaining tags still did not match the genome sequence. Some of these correspond to parts of human mRNAs, such as polyA tails, junctions between two exons and polymorphic regions of transcripts. Another non-negligible proportion can be attributed to contamination by murine transcripts and to residual sequencing errors. After filtering out our data with these screens to ensure that our dataset is highly reliable, we studied the tags that map once to the genome. 31% of these tags correspond to unannotated transcripts. The others map to known transcribed regions, but many of them (nearly half) are located either in antisense or in new variants of these known transcripts.We performed a comprehensive study of all publicly available human LongSAGE tags, and carefully verified the reliability of these data. We found the potential origin of many tags that did not match the human genome sequence. The properties of the remaining tags imply that the level of sequencing error may have been under-estimated. The frequency of tags matching once the genome sequence but not in an annotated exon suggests that the human transcriptome is much more complex than shown by the current human genome annotations, with many new splicing variants and antisense transcripts. SAGE data is appropriate to map new transcripts to the genome, as demonstrated by the high rate of cross-validation of the corresponding tags using other methods.Serial Analysis of Gene Expression (SAGE) [1] is a widely used method for transcriptome analysis. This technique has been successfully used for the analysis of a variety of biological phenomena, by investigating the expression level of previously characterized mRNAs [2]. It has also permitted the study of important structural characteristics of the human genome such as co-expressed gene clusters [
Genome-wide analysis of antisense transcription with Affymetrix exon array
Xijin Ge, Wendy S Rubinstein, Yong-chul Jung, Qingfa Wu
BMC Genomics , 2008, DOI: 10.1186/1471-2164-9-27
Abstract: Here we show that Affymetrix Exon arrays, which were designed to detect conventional transcripts in the sense orientation, can be used to monitor antisense expression across all exonic loci in mammalian genomes. Through modification of the cDNA synthesis protocol, we labeled single-strand cDNA in the reverse orientation as in the standard protocol, thus enabling the detection of antisense transcripts using the same array. Applying this technique to human Jurkat cells, we identified antisense transcription at 2,088 exonic loci of 1,516 UniGene clusters. Many of these antisense transcripts were not observed previously and some were validated by orientation-specific RT-PCR.Our results suggest that with a modified protocol Affymetrix human, mouse and rat Exon arrays can be used as a routine method for genome-wide analysis of antisense transcription in these genomes.Genome-wide analyses of mRNA and cDNA sequences have revealed large-scale antisense transcription in various animal and plant species. Based on analysis of mouse full length cDNA generated by FANTOM2 consortium, Okazaki et al. identified 2,481 pairs of overlapping sense/antisense transcripts [1]. An updated analysis using FANTOM3 cDNAs and mouse cDNA at GenBank showed that there are 4,520 transcription units forming sense and antisense pairs on exons [2]. For human, Yelin et al. [3] and Chen et al. [4] reported 2,667 and 2,940 pairs, respectively. The prevalence of natural antisense transcripts (NATs) is also supported by data derived by sequence tag-based technologies such as serial analysis of gene expression (SAGE) [5-7] and massively parallel signature sequencing (MPSS) [8].Microarray-based studies also confirmed the prevalence of antisense transcripts. Using strand-specific oligonucleotide probes Yelin et al. studied the expression of both strands of 264 genomic loci in 19 human cell lines and detected antisense transcription in 112 (42.5%) of these loci [3]. Kiyosawa et al. studied the expression of 1,9
Deep and comparative analysis of the mycelium and appressorium transcriptomes of Magnaporthe grisea using MPSS, RL-SAGE, and oligoarray methods
Malali Gowda, RC Venu, Mohan B Raghupathy, Kan Nobuta, Huameng Li, Rod Wing, Eric Stahlberg, Sean Couglan, Christian D Haudenschild, Ralph Dean, Baek-Hie Nahm, Blake C Meyers, Guo-Liang Wang
BMC Genomics , 2006, DOI: 10.1186/1471-2164-7-310
Abstract: The MPSS analyses identified 12,531 and 12,927 distinct significant tags from mycelia and appressoria, respectively, while the RL-SAGE analysis identified 16,580 distinct significant tags from the mycelial library. When matching these 12,531 mycelial and 12,927 appressorial significant tags to the annotated CDS, 500 bp upstream and 500 bp downstream of CDS, 6,735 unique genes in mycelia and 7,686 unique genes in appressoria were identified. A total of 7,135 mycelium-specific and 7,531 appressorium-specific significant MPSS tags were identified, which correspond to 2,088 and 1,784 annotated genes, respectively, when matching to the same set of reference sequences. Nearly 85% of the significant MPSS tags from mycelia and appressoria and 65% of the significant tags from the RL-SAGE mycelium library matched to the M. grisea genome. MPSS and RL-SAGE methods supported the expression of more than 9,000 genes, representing over 80% of the predicted genes in M. grisea. About 40% of the MPSS tags and 55% of the RL-SAGE tags represent novel transcripts since they had no matches in the existing M. grisea EST collections. Over 19% of the annotated genes were found to produce both sense and antisense tags in the protein-coding region. The oligoarray analysis identified the expression of 3,793 mycelium-specific and 4,652 appressorium-specific genes. A total of 2,430 mycelial genes and 1,886 appressorial genes were identified by both MPSS and oligoarray.The comprehensive and deep transcriptome analysis by MPSS and RL-SAGE methods identified many novel sense and antisense transcripts in the M. grisea genome at two important growth stages. The differentially expressed transcripts that were identified, especially those specifically expressed in appressoria, represent a genomic resource useful for gaining a better understanding of the molecular basis of M. grisea pathogenicity. Further analysis of the novel antisense transcripts will provide new insights into the regulation and functio
Gene Isoform Specificity through Enhancer-Associated Antisense Transcription  [PDF]
Courtney S. Onodera, Jason G. Underwood, Sol Katzman, Frank Jacobs, David Greenberg, Sofie R. Salama, David Haussler
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0043511
Abstract: Enhancers and antisense RNAs play key roles in transcriptional regulation through differing mechanisms. Recent studies have demonstrated that enhancers are often associated with non-coding RNAs (ncRNAs), yet the functional role of these enhancer:ncRNA associations is unclear. Using RNA-Sequencing to interrogate the transcriptomes of undifferentiated mouse embryonic stem cells (mESCs) and their derived neural precursor cells (NPs), we identified two novel enhancer-associated antisense transcripts that appear to control isoform-specific expression of their overlapping protein-coding genes. In each case, an enhancer internal to a protein-coding gene drives an antisense RNA in mESCs but not in NPs. Expression of the antisense RNA is correlated with expression of a shorter isoform of the associated sense gene that is not present when the antisense RNA is not expressed. We demonstrate that expression of the antisense transcripts as well as expression of the short sense isoforms correlates with enhancer activity at these two loci. Further, overexpression and knockdown experiments suggest the antisense transcripts regulate expression of their associated sense genes via cis-acting mechanisms. Interestingly, the protein-coding genes involved in these two examples, Zmynd8 and Brd1, share many functional domains, yet their antisense ncRNAs show no homology to each other and are not present in non-murine mammalian lineages, such as the primate lineage. The lack of homology in the antisense ncRNAs indicates they have evolved independently of each other and suggests that this mode of lineage-specific transcriptional regulation may be more widespread in other cell types and organisms. Our findings present a new view of enhancer action wherein enhancers may direct isoform-specific expression of genes through ncRNA intermediates.
Evidence for Antisense Transcription Associated with MicroRNA Target mRNAs in Arabidopsis  [PDF]
Qing-Jun Luo,Manoj P. Samanta,Fatih K?ksal,Jaroslav Janda,David W. Galbraith,Casey R. Richardson,Fangqian Ou-Yang,Christopher D. Rock
PLOS Genetics , 2009, DOI: 10.1371/journal.pgen.1000457
Abstract: Antisense transcription is a pervasive phenomenon, but its source and functional significance is largely unknown. We took an expression-based approach to explore microRNA (miRNA)-related antisense transcription by computational analyses of published whole-genome tiling microarray transcriptome and deep sequencing small RNA (smRNA) data. Statistical support for greater abundance of antisense transcription signatures and smRNAs was observed for miRNA targets than for paralogous genes with no miRNA cleavage site. Antisense smRNAs were also found associated with MIRNA genes. This suggests that miRNA-associated “transitivity” (production of small interfering RNAs through antisense transcription) is more common than previously reported. High-resolution (3 nt) custom tiling microarray transcriptome analysis was performed with probes 400 bp 5′ upstream and 3′ downstream of the miRNA cleavage sites (direction relative to the mRNA) for 22 select miRNA target genes. We hybridized RNAs labeled from the smRNA pathway mutants, including hen1-1, dcl1-7, hyl1-2, rdr6-15, and sgs3-14. Results showed that antisense transcripts associated with miRNA targets were mainly elevated in hen1-1 and sgs3-14 to a lesser extent, and somewhat reduced in dcl11-7, hyl11-2, or rdr6-15 mutants. This was corroborated by semi-quantitative reverse transcription PCR; however, a direct correlation of antisense transcript abundance in MIR164 gene knockouts was not observed. Our overall analysis reveals a more widespread role for miRNA-associated transitivity with implications for functions of antisense transcription in gene regulation. HEN1 and SGS3 may be links for miRNA target entry into different RNA processing pathways.
Extensive Polycistronism and Antisense Transcription in the Mammalian Hox Clusters  [PDF]
Ga?ll Mainguy, Jan Koster, Joost Woltering, Hans Jansen, Antony Durston
PLOS ONE , 2007, DOI: 10.1371/journal.pone.0000356
Abstract: The Hox clusters play a crucial role in body patterning during animal development. They encode both Hox transcription factor and micro-RNA genes that are activated in a precise temporal and spatial sequence that follows their chromosomal order. These remarkable collinear properties confer functional unit status for Hox clusters. We developed the TranscriptView platform to establish high resolution transcriptional profiling and report here that transcription in the Hox clusters is far more complex than previously described in both human and mouse. Unannotated transcripts can represent up to 60% of the total transcriptional output of a cluster. In particular, we identified 14 non-coding Transcriptional Units antisense to Hox genes, 10 of which (70%) have a detectable mouse homolog. Most of these Transcriptional Units in both human and mouse present conserved sizeable sequences (>40 bp) overlapping Hox transcripts, suggesting that these Hox antisense transcripts are functional. Hox clusters also display at least seven polycistronic clusters, i.e., different genes being co-transcribed on long isoforms (up to 30 kb). This work provides a reevaluated framework for understanding Hox gene function and dys-function. Such extensive transcriptions may provide a structural explanation for Hox clustering.
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