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Cryptic promoter activity in the DNA sequence corresponding to the pim-1 5'-UTR

Zeping Wang1, Matt Weaver, Nancy S Magnuson

  • 1School of Molecular Biosciences, Washington State University Pullman, WA 99164-4234, USA.

Nucleic Acids Research
|April 22, 2005
PubMed

Insights

The pim-1 5'-untranslated region (UTR) was re-examined for internal ribosomal entry site (IRES) activity. Researchers found strong cryptic promoter activity instead of IRES activity, suggesting previous findings were due to promoter function.

Area of Science:

  • Molecular Biology
  • Gene Regulation
  • RNA Biology

Background:

  • The serine/threonine kinase pim-1 mRNA features a G/C-rich 5 acronym{'-untranslated region (UTR).
  • Previous studies proposed an internal ribosomal entry site (IRES) within the pim-1 5 acronym{'-UTR for cap-independent translation initiation.
  • Concerns exist that some reported eukaryotic IRES elements may possess cryptic promoter activity.

Purpose of the Study:

  • To rigorously investigate the presence of IRES activity or cryptic promoter activity in the pim-1 5 acronym{'-UTR.
  • To resolve conflicting reports regarding the translational control mechanism of pim-1 mRNA.

Main Methods:

  • Utilized stringent test procedures, including promoterless dicistronic assays and Northern blot analysis.
  • Performed in vitro and in vivo translation assays with dicistronic mRNAs containing the pim-1 5 acronym{'-UTR.
  • Employed RNase protection assays and 5 acronym{'-RACE (Rapid Amplification of cDNA Ends) to analyze endogenous pim-1 transcripts.

Main Results:

  • Demonstrated strong cryptic promoter activity within the DNA sequence of the pim-1 5 acronym{'-UTR.
  • Transcripts were detected originating from this cryptic promoter across multiple cell types (Cos-7, NIH3T3, HEK293, Jurkat, K562).
  • No IRES activity was detected for the pim-1 5 acronym{'-UTR in translation assays, while control IRES elements showed robust activity.

Conclusions:

  • The previously reported IRES activity in the pim-1 5 acronym{'-UTR is attributed to cryptic promoter activity.
  • Endogenous pim-1 transcripts possess shorter 5 acronym{'-UTRs than previously assumed.
  • The findings necessitate a re-evaluation of translational regulation mechanisms for pim-1 kinase.

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