What is the clinical evidence from this study?

Study design: Other. Intervention: Promoterless dicistronic vector vs. Conventional dicistronic vector. Primary outcome: Firefly to Renilla luciferase activity ratio.

What question did this study set out to answer?

To determine whether the putative internal ribosome entry site (IRES) activity in the 5'-UTR of cellular eIF4G mRNA is authentic or driven by a cryptic promoter.

October 7, 2002Molecular and Cellular BiologyOpen Access

Regulation of Gene Expression by Internal Ribosome Entry Sites or Cryptic Promoters: the eIF4G Story

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Key result

Using a novel promoterless dicistronic vector, the previously reported internal ribosome entry site (IRES) activity in the 5'-UTR of eIF4G mRNA was found to be driven by a strong cryptic promoter.

Population

HeLa, H1299, and SL2 cell lines, and rabbit reticulocyte lysate (in vitro translation system)

Comparison

Transfection with promoterless dicistronic… vs Transfection with conventional dicistronic…

Design

Preclinical

Authors

BHBaoguang HanBiogen (United States)JZJian‐Ting ZhangUniversity of Toledo

Discussion

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Implication

Apparent eIF4G IRES reflects cryptic promoter; challenges prior reports and requires promoterless vectors in cellular IRES assays.

Key Points

To determine whether the putative internal ribosome entry site (IRES) activity in the 5'-UTR of cellular eIF4G mRNA is authentic or driven by a cryptic promoter.
Engineered a novel promoterless dicistronic vector to evaluate putative cellular IRES elements without background transcription artifacts.
Assayed translation of eIF4G 5'-UTR-containing dicistronic mRNAs both in vitro and in vivo.
Evaluated transcription factor binding sites (C/EBPbeta, Sp1, Ets) and tested additional 5'-UTRs from human Sno and mouse Bad mRNAs.
Identified strong promoter activity in the eIF4G 5'-UTR driven by transcription factors C/EBPbeta, Sp1, and Ets within an essential polypyrimidine tract sequence.
Found no evidence of genuine IRES-mediated translation from the eIF4G 5'-UTR in vitro or in vivo.
Detected cryptic promoter activity in the 5'-UTRs of human Sno and mouse Bad mRNAs despite monocistronic transcripts escaping detection on Northern blots.

Structured PICO

Population

HeLa, H1299, and SL2 cell lines, and rabbit reticulocyte lysate (in vitro translation system)

Intervention

Transfection with promoterless dicistronic vectors containing the 5'-UTR of eIF4G mRNA

Comparator

Transfection with conventional dicistronic vectors or vectors containing HRV IRES

Outcome

Promoter and IRES activities measured by Renilla and firefly luciferase activities

The study demonstrates that putative IRES activity in the eIF4G mRNA is actually driven by a cryptic promoter, highlighting the need for promoterless vectors in cellular IRES research.

Limitations

Conventional Northern blot analysis may lack the sensitivity to detect low levels of monocistronic transcripts derived via cryptic promoters or splice sites.

Cite This Study

Han et al. (2002) studied this question. Promoterless dicistronic vector vs. Conventional dicistronic vector was evaluated on Firefly to Renilla luciferase activity ratio. Using a novel promoterless dicistronic vector, the previously reported internal ribosome entry site (IRES) activity in the 5'-UTR of eIF4G mRNA was found to be driven by a strong cryptic promoter.

synapsesocial.com/papers/6a94db6b2ea3449151e60f86 https://doi.org/10.1128/mcb.22.21.7372-7384.2002

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Regulation of Gene Expression by Internal Ribosome Entry Sites or Cryptic Promoters: the eIF4G Story

Discussion

Member takes

Implication

Key Points

Structured PICO

Limitations

Cite This Study

Regulation of Gene Expression by Internal Ribosome Entry Sites or Cryptic Promoters: the eIF4G Story

Discussion

Member takes

Implication

Key Points

Structured PICO

Limitations

Cite This Study

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