mRNA 5' region sequence incompleteness: a potential source of systematic errors in translation initiation codon...

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Gene product amino acid sequences are typically derived from nucleotide sequences of cloned complementary DNA (cDNA).
• Standard cDNA cloning methods may struggle to capture the complete 5' region of messenger RNA (mRNA).
• This limitation can affect the accuracy of predicted protein sequences and subsequent analyses.

Purpose of the Study:

• To investigate the prevalence and impact of incomplete 5' mRNA regions on gene annotation and analysis.
• To assess the accuracy of current gene product sequence predictions based on cDNA.

Main Methods:

• Bioinformatic analysis of gene sequences from human chromosome 21 (HC 21).
• Review of cloning methods for 109 known HC 21 genes.
• Sequence analysis of 13,124 human mRNAs from the RefSeq database.

Main Results:

• 60 out of 109 HC 21 mRNAs lacked in-frame stop codons upstream of the first-AUG, indicating potential 5' end artifacts.
• Five genes on HC 21 had incompletely characterized 5' coding regions.
• Analysis of 13,124 human mRNAs revealed that 49% had upstream in-frame stop codons, while 51% might have undiscovered upstream AUGs.
• Approximately 550 human genes may be affected by this 5' end mRNA artifact.

Conclusions:

• Incomplete 5' mRNA sequencing is a significant artifact affecting gene annotation, domain identification, and functional studies.
• The findings necessitate a re-evaluation of existing gene annotations and experimental designs.
• Improved methods for 5' end mRNA capture and analysis are crucial for accurate genomic research.