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

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 3' 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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Lemp NA,Hiraoka K,Kasahara N,Logg CR

doi

10.1093/nar/gks451

subject

Has Abstract

pub_date

2012-08-01 00:00:00

pages

7280-90

issue

15

eissn

0305-1048

issn

1362-4962

pii

gks451

journal_volume

40

pub_type

杂志文章

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