Cryptic transcripts from a ubiquitous plasmid origin of replication confound tests for cis-regulatory function

Nathan A. Lemp, Kei Hiraoka, Noriyuki Kasahara, Christopher R. Logg

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

A vast amount of research on the regulation of gene expression has relied on plasmid reporter assays. In this study, we show that plasmids widely used for this purpose constitutively produce substantial amounts of RNA from a TATA-containing cryptic promoter within the origin of replication. Readthrough of these RNAs into the intended transcriptional unit potently stimulated reporter activity when the inserted test sequence contained a 3' splice site (ss). We show that two human sequences, originally reported to be internal ribosome entry sites and later to instead be promoters, mimic both types of element in dicistronic reporter assays by causing these cryptic readthrough transcripts to splice in patterns that allow efficient translation of the downstream cistron. Introduction of test sequences containing 30 ss into monocistronic luciferase reporter vectors widely used in the study of transcriptional regulation also created the false appearance of promoter function via the same mechanism. Across a large number of variants of these plasmids, we found a very highly significant correlation between reporter activity and levels of such spliced readthrough transcripts. Computational estimation of the frequency of cryptic 3' ss in genomic sequences suggests that misattribution of cis-regulatory function may be a common occurrence.

Original languageEnglish (US)
Pages (from-to)7280-7290
Number of pages11
JournalNucleic Acids Research
Volume40
Issue number15
DOIs
StatePublished - Aug 2012
Externally publishedYes

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Genetic Complementation Test
RNA Splice Sites
Replication Origin
Plasmids
RNA
Gene Expression Regulation
Luciferases
Research
Genes

ASJC Scopus subject areas

  • Genetics

Cite this

Cryptic transcripts from a ubiquitous plasmid origin of replication confound tests for cis-regulatory function. / Lemp, Nathan A.; Hiraoka, Kei; Kasahara, Noriyuki; Logg, Christopher R.

In: Nucleic Acids Research, Vol. 40, No. 15, 08.2012, p. 7280-7290.

Research output: Contribution to journalArticle

Lemp, Nathan A. ; Hiraoka, Kei ; Kasahara, Noriyuki ; Logg, Christopher R. / Cryptic transcripts from a ubiquitous plasmid origin of replication confound tests for cis-regulatory function. In: Nucleic Acids Research. 2012 ; Vol. 40, No. 15. pp. 7280-7290.
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