Abstract
Tens of thousands of chimeric RNAs have been reported. Most of them contain a short homologous sequence (SHS) at the joining site of the two partner genes but are not associated with a fusion gene. We hypothesize that many of these chimeras may be technical artifacts derived from SHS-caused mis-priming in reverse transcription (RT) or polymerase chain reactions (PCR). We cloned six chimeric complementary DNAs (cDNAs) formed by human mitochondrial (mt) 16S rRNA sequences at an SHS, which were similar to several expression sequence tags (ESTs).These chimeras, which could not be detected with cDNA protection assay, were likely formed because some regions of the 16S rRNA are reversely complementary to another region to form an SHS, which allows the downstream sequence to loop back and anneal at the SHS to prime the synthesis of its complementary strand, yielding a palindromic sequence that can form a hairpin-like structure.We identified a 16S rRNA that ended at the 4th nucleotide(nt) of the mt-tRNA-leu was dominant and thus should be the wild type. We also cloned a mouse Bcl2-Nek9 chimeric cDNA that contained a 5-nt unmatchable sequence between the two partners, contained two copies of the reverse primer in the same direction but did not contain the forward primer, making it unclear how this Bcl2-Nek9 was formed and amplified. Moreover, a cDNA was amplified because one primer has 4 nts matched to the template, suggesting that there may be many more artificial cDNAs than we have realized, because the nuclear and mt genomes have many more 4-nt than 5-nt or longer homologues. Altogether, the chimeric cDNAs we cloned are good examples suggesting that many cDNAs may be artifacts due to SHS-caused mis-priming and thus greater caution should be taken when new sequence is obtained from a technique involving DNA polymerization.
Highlights
In various DNA and RNA sequence databases, there are tens of thousands of expression sequence tags (ESTs) of human origin that are chimeric complementary DNAs, dubbed chimeras [1,2]
Cloning of chimeric complementary DNAs (cDNAs) in which two 16S rRNA sequences join at an short homologous sequence (SHS)
The anticipated DNA fragments were all abundant and visiblein agarose gel after 30 cycles of polymerase chain reactions (PCR), but we performed 35 cycles to make cloning easier.Of the bacterial colonies from each cloning procedure for each pair of primer and each cell line, 3 to 5 colonies were sequenced, which identified a total of 6 chimeric cDNAs that were formed by two regions of the 16S rRNA (Fig 2A and S1 File)
Summary
In various DNA and RNA sequence databases, there are tens of thousands of expression sequence tags (ESTs) of human origin that are chimeric complementary DNAs (cDNAs), dubbed chimeras [1,2]. Our bioinformatic analysis in 2013 identified 34770 chimeric ESTs in the National Center for Biotechnology Information (NCBI) of the United States [3], which is slightly more than the 31,005 chimeric ESTs identifed by Li et al in 2009 [4] Each of these chimeras is formed by fusion of two different genes that are coined as “two partners” of the chimera. After analyzing transcripts from about 1% of the human genome, the ENCODE project estimates that RNA transcripts from about 65% of the human genes form chimeras[5,6] These lines of information seem to imply that chimeric RNAs are omnipresent in human cells, at least in cancer cells. We wonder whether the majority of the putative chimeras documented in various databases or in the literature are technical artifacts[1,9] that may mislead the ribonomic research and lead to a great waste in time, resources and effort
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