Alternative cleavage and polyadenylation generates downstream uncapped RNA isoforms with translation potential

Area of Science:

• Molecular Biology
• Transcriptomics
• RNA Biology

Background:

• Alternative promoter usage, splicing, and cleavage and polyadenylation (APA) contribute to mRNA isoform diversity.
• The full extent of transcriptome complexity arising from these mechanisms remains incompletely understood.

Purpose of the Study:

• To identify and characterize novel RNA species generated through alternative APA.
• To investigate the stability, translational potential, and biological functions of these newly discovered transcripts.

Main Methods:

• Bioinformatic analysis of transcriptomic data to identify 5' uncapped and polyadenylated transcripts (5' UPTs).
• RNA structure probing and epitranscriptomic analysis (N6-methyladenosine mapping).
• Polysome profiling and functional assays to assess translational activity and biological impact.

Main Results:

• Thousands of previously undescribed 5' UPTs were identified, arising downstream of APA sites.
• These 5' UPTs exhibit resistance to exonucleases, attributed to RNA structure and N6-methyladenosine modification.
• APA activation induces 5' UPTs, which are enriched in polysomes, indicating translational potential.
• 5' UPTs facilitate downstream translation initiation, non-canonical protein production, and altered cell surface peptide presentation.
• The anti-apoptotic gene Bcl2 serves as an example where its coding sequence functions as a 5' UPT.

Conclusions:

• Alternative cleavage and polyadenylation (APA) is a significant source of novel, uncapped, polyadenylated transcripts (5' UPTs).
• These 5' UPTs are translationally active and possess biological relevance, influencing protein output and cell surface characteristics.
• APA contributes not only to transcript termination but also to the generation of functional RNA molecules, thereby increasing transcriptome complexity and biological impact.