Splicing mediates the activity of four putative cellular internal ribosome entry sites

Brian T. Baranick, Nathan A. Lemp, Jill Nagashima, Kei Hiraoka, Noriyuki Kasahara, Christopher R. Logg

Research output: Contribution to journalArticle

55 Citations (Scopus)

Abstract

A growing number of cellular mRNAs are thought to possess internal ribosome entry sites (IRESs), sequences that permit translation of a transcript independent of its 5′ end and cap structure. Although dicistronic assays are the canonical method of testing sequences for IRES activity, they may produce false-positive results if unanticipated monocistronic RNAs arise from the dicistronic construct used. Using a dicistronic reporter system and a green fluorescent protein-tagged retrovirus to evaluate six previously reported cellular IRESs, we found that four contain 3′ splice sites whose activity was required for apparent IRES function and which resulted in formation of monocistronic transcripts by splicing. Bioinformatic analysis revealed that the 3′ splice sites identified in three of these putative IRESs are used in their native mRNAs and that the fourth is likely an artifactual sequence created during cDNA cloning. Our findings demonstrate a need for reexamination of other reported cellular IRESs by using careful RNA structural analysis to rule out splicing as the source of perceived IRES activity.

Original languageEnglish (US)
Pages (from-to)4733-4738
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume105
Issue number12
DOIs
StatePublished - Mar 25 2008
Externally publishedYes

Fingerprint

RNA Splice Sites
RNA
Messenger RNA
Retroviridae
Green Fluorescent Proteins
Internal Ribosome Entry Sites
Computational Biology
Organism Cloning
Complementary DNA

Keywords

  • Bicistronic
  • Dicistronic
  • RNA splicing
  • Translation

ASJC Scopus subject areas

  • General
  • Genetics

Cite this

Splicing mediates the activity of four putative cellular internal ribosome entry sites. / Baranick, Brian T.; Lemp, Nathan A.; Nagashima, Jill; Hiraoka, Kei; Kasahara, Noriyuki; Logg, Christopher R.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 105, No. 12, 25.03.2008, p. 4733-4738.

Research output: Contribution to journalArticle

Baranick, Brian T. ; Lemp, Nathan A. ; Nagashima, Jill ; Hiraoka, Kei ; Kasahara, Noriyuki ; Logg, Christopher R. / Splicing mediates the activity of four putative cellular internal ribosome entry sites. In: Proceedings of the National Academy of Sciences of the United States of America. 2008 ; Vol. 105, No. 12. pp. 4733-4738.
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