Nonsense-mediated mRNA decay immunity can help identify human polycistronic transcripts

Area of Science:

• Molecular Biology
• Genomics
• Transcriptomics

Background:

• Eukaryotic polycistronic transcription units are exceptionally rare, with most examples discovered serendipitously.
• Nonsense-mediated mRNA decay (NMD) typically degrades aberrant transcripts, posing a challenge for identifying functional polycistronic mRNAs.

Purpose of the Study:

• To computationally screen the human transcriptome for novel polycistronic transcripts.
• To investigate the role of NMD insensitivity as a characteristic feature of polycistronic transcripts.
• To experimentally validate the existence and functionality of predicted polycistronic transcripts.

Main Methods:

• Computational screening of the human RefSeq transcriptome using NMD elicitation and ORF rescue criteria.
• Domain-based analysis to assess the protein-coding potential of candidate ORFs.
• Experimental validation using NMD-inhibition studies (GEO datasets) and global translation initiation sequencing data.

Main Results:

• Prediction of forty-nine novel polycistronic transcripts based on computational analysis.
• Experimental verification confirmed NMD insensitivity in known and eleven of twelve predicted polycistronic transcripts.
• Validation of translation initiation sites for rescuing upstream ORFs (uORFs) in several predicted polycistronic genes.

Conclusions:

• NMD-immune architecture is a key parameter for identifying eukaryotic polycistronic genes.
• The study provides evidence for NMD-mediated regulation in the production of proteins from polycistronic transcripts.
• This work expands the known landscape of eukaryotic gene expression and regulation.