Dataset Information

Mapping the 5' and 3' ends of Tetrahymena thermophila mRNAs using RNA ligase mediated amplification of cDNA ends (RLM-RACE).

ABSTRACT: A procedure is described for mapping the ends of RNAs. Using T4 RNA ligase, a DNA (3' end) or RNA (5' end) oligonucleotide is ligated to RNA ends followed by cDNA synthesis, PCR amplification, cloning and sequencing. This method determines 5' ends, 3' polyadenylation sites and the size of poly(A) tails, and should be applicable to non-polyadenylated mRNAs and to non-message RNAs. Analysis of four Tetrahymena thermophila histone mRNAs revealed multiple, closely spaced 5' ends consistent with those determined by other methods. Except for a 'CCAAT' box in either orientation 100-200 nucleotides upstream of the transcription start site, no conserved sequence elements were observed in the untranslated 5' region or in sequences immediately flanking the transcription start site. Analysis of the 3' ends of mRNAs encoding four histones, two tubulins and the Tetrahymena TATA binding protein confirmed the observations that Tetrahymena histone messages are polyadenylated and that poly(A) tails in this organism are short (approximately 50 nt). No canonical poly(A) addition signal was identified. The four histone messages analyzed have contained three sequence elements, TGTGT-TAA-AAGTATT, not found in non-histone messages. Two non-histone messages contained GCATT(N)15ATACC near the poly(A) addition site.

SUBMITTER: Liu X

PROVIDER: S-EPMC311412 | biostudies-other | 1993 Oct

REPOSITORIES: biostudies-other

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Mapping the 5' and 3' ends of Tetrahymena thermophila mRNAs using RNA ligase mediated amplification of cDNA ends (RLM-RACE).

Liu X X Gorovsky M A MA

Nucleic acids research 19931001 21

A procedure is described for mapping the ends of RNAs. Using T4 RNA ligase, a DNA (3' end) or RNA (5' end) oligonucleotide is ligated to RNA ends followed by cDNA synthesis, PCR amplification, cloning and sequencing. This method determines 5' ends, 3' polyadenylation sites and the size of poly(A) tails, and should be applicable to non-polyadenylated mRNAs and to non-message RNAs. Analysis of four Tetrahymena thermophila histone mRNAs revealed multiple, closely spaced 5' ends consistent with thos ...[more]

PMID: 8177745

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Project description:BACKGROUND: The ciliated protozoan Tetrahymena thermophila is a well-studied single-celled eukaryote model organism for cellular and molecular biology. However, the lack of extensive T. thermophila cDNA libraries or a large expressed sequence tag (EST) database limited the quality of the original genome annotation. METHODOLOGY/PRINCIPAL FINDINGS: This RNA-seq study describes the first deep sequencing analysis of the T. thermophila transcriptome during the three major stages of the life cycle: growth, starvation and conjugation. Uniquely mapped reads covered more than 96% of the 24,725 predicted gene models in the somatic genome. More than 1,000 new transcribed regions were identified. The great dynamic range of RNA-seq allowed detection of a nearly six order-of-magnitude range of measurable gene expression orchestrated by this cell. RNA-seq also allowed the first prediction of transcript untranslated regions (UTRs) and an updated (larger) size estimate of the T. thermophila transcriptome: 57 Mb, or about 55% of the somatic genome. Our study identified nearly 1,500 alternative splicing (AS) events distributed over 5.2% of T. thermophila genes. This percentage represents a two order-of-magnitude increase over previous EST-based estimates in Tetrahymena. Evidence of stage-specific regulation of alternative splicing was also obtained. Finally, our study allowed us to completely confirm about 26.8% of the genes originally predicted by the gene finder, to correct coding sequence boundaries and intron-exon junctions for about a third, and to reassign microarray probes and correct earlier microarray data. CONCLUSIONS/SIGNIFICANCE: RNA-seq data significantly improve the genome annotation and provide a fully comprehensive view of the global transcriptome of T. thermophila. To our knowledge, 5.2% of T. thermophila genes with AS is the highest percentage of genes showing AS reported in a unicellular eukaryote. Tetrahymena thus becomes an excellent unicellular model eukaryote in which to investigate mechanisms of alternative splicing.

Dataset Information

Mapping the 5' and 3' ends of Tetrahymena thermophila mRNAs using RNA ligase mediated amplification of cDNA ends (RLM-RACE).

Publications

Mapping the 5' and 3' ends of Tetrahymena thermophila mRNAs using RNA ligase mediated amplification of cDNA ends (RLM-RACE).

Similar Datasets

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