A second look at cellular mRNA sequences said to function as internal ribosome entry sites

Area of Science:

• Molecular Biology
• Gene Expression
• RNA Biology

Background:

• Cap-dependent translation is typically reduced during mitosis and stress.
• Alternative translation initiation mechanisms, such as internal ribosome entry sequences (IRESs), have been proposed to function under these conditions.
• The evidence supporting the widespread biological relevance of IRESs is debated.

Purpose of the Study:

• To critically re-evaluate the evidence for internal ribosome entry sequences (IRESs) functioning as alternative translation initiation mechanisms.
• To identify flaws in experimental methodologies used to support the hypothesis of IRES activity.
• To assess the consistency of reported IRES efficiencies with their proposed biological roles.

Main Methods:

• Review of existing literature on IRES function.
• Critical analysis of experimental assays, including dicistronic assays and RNA transfection assays.
• Examination of common experimental vectors, such as the pRF vector, for potential artifacts.

Main Results:

• Many purported IRESs contain cryptic promoters or splice sites, invalidating their use in dicistronic assays.
• The pRF vector frequently generates spliced transcripts, confounding the interpretation of IRES activity.
• Most candidate IRESs exhibit very weak activity in RNA transfection assays.
• The low efficiency of most putative IRESs is inconsistent with their proposed biological functions.

Conclusions:

• The evidence supporting the role of IRESs in alternative translation initiation is largely based on flawed experimental data.
• Methodological artifacts, including vector-derived transcripts and cryptic promoters, significantly compromise the validity of reported IRES activities.
• The extremely low efficiency of most identified IRES candidates challenges their proposed biological significance during cellular stress or mitosis.