Poly A- transcripts expressed in HeLa cells - PubMed (original) (raw)
Poly A- transcripts expressed in HeLa cells
Qingfa Wu et al. PLoS One. 2008.
Abstract
Background: Transcripts expressed in eukaryotes are classified as poly A+ transcripts or poly A- transcripts based on the presence or absence of the 3' poly A tail. Most transcripts identified so far are poly A+ transcripts, whereas the poly A- transcripts remain largely unknown.
Methodology/principal findings: We developed the TRD (Total RNA Detection) system for transcript identification. The system detects the transcripts through the following steps: 1) depleting the abundant ribosomal and small-size transcripts; 2) synthesizing cDNA without regard to the status of the 3' poly A tail; 3) applying the 454 sequencing technology for massive 3' EST collection from the cDNA; and 4) determining the genome origins of the detected transcripts by mapping the sequences to the human genome reference sequences. Using this system, we characterized the cytoplasmic transcripts from HeLa cells. Of the 13,467 distinct 3' ESTs analyzed, 24% are poly A-, 36% are poly A+, and 40% are bimorphic with poly A+ features but without the 3' poly A tail. Most of the poly A- 3' ESTs do not match known transcript sequences; they have a similar distribution pattern in the genome as the poly A+ and bimorphic 3' ESTs, and their mapped intergenic regions are evolutionarily conserved. Experiments confirmed the authenticity of the detected poly A- transcripts.
Conclusion/significance: Our study provides the first large-scale sequence evidence for the presence of poly A- transcripts in eukaryotes. The abundance of the poly A- transcripts highlights the need for comprehensive identification of these transcripts for decoding the transcriptome, annotating the genome and studying biological relevance of the poly A- transcripts.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. The Total RNA Detection system.
A universal RNA adaptor was firstly added to the 3′ ends of all RNA templates. The abundant 18S and 28S ribosome RNAs were then subtracted by using biotinylated ribosomal-specific probes. Small-size RNAs containing the degraded RNA intermediates were removed by size-filtration. The enriched transcripts were converted into double-strand cDNA by using the 3′ end RNA adaptor-based primer. The cDNAs were further digested by NlaIII. The 3′ cDNAs were isolated by using the streptoavidin beads. An adaptor was added to the 5′ ends of the 3′ cDNAs. The 3′ cDNAs were then amplified by PCR using the 5′ adaptor-based sense primer and the 3′ end RNA adaptor-based antisense primer. The amplified 3′ cDNAs were sequenced from the 3′ end by the 454 system. See further details in Materials and Methods.
Figure 2. Example of histone 3′ EST distribution.
Fifteen 3′ ESTs that map to the full-length histone 1H2AB cDNA sequences (NM_003513) are clustered proximal to the 3′ end of the full-length sequence. See Table 3, Table S3 and Figure S1 for the distribution of other histone 3′ ESTs.
Figure 3. Experimental verification of novel 3′ ESTs.
(A). 3′ end verification for each subtype of 3′ EST. 3′ ESTs from poly A-, poly A+, and bimorphic subtypes were selected for the confirmation. Known poly A+ transcripts were used as positive control. Random priming- generated cDNA and oligo dT-generated cDNA were used as the templates. R: cDNA generated by random priming; T: cDNA generated by oligo dT priming. See Table S7 for primer information. (B). Verification of 3′ ESTs mapped to intronic microRNA precursors. RT-PCR was used to verify the 3′ ESTs that map to intronic microRNA precursors. Amplified products were cloned and sequenced. See Table S4C for primer information. (C). northern blot verification of poly A- 3′ EST. Two poly A- 3′ ESTs were used as the probes (Table S7) and RNAs from five human cell lines were used for the detection.
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