Sequence determinants of polyadenylation-mediated regulation

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• The polyadenylation reaction is vital for transcription termination and mRNA processing in human cells.
• Mechanisms of polyadenylation-mediated gene expression regulation encoded in DNA remain incompletely understood.
• Polyadenylation sequences (PASs) play a critical role in these processes.

Purpose of the Study:

• To investigate the impact of diverse PAS sequences on reporter gene expression and cleavage efficiency.
• To elucidate the mechanistic relationship between cleavage efficiency and PAS mutational sensitivity.
• To develop predictive models for PAS function in gene regulation.

Main Methods:

• Massively parallel reporter assay (MPRA) with over 12,000 designed PASs.
• Scanning mutagenesis to assess cleavage efficiency and mutation effects.
• Motif discovery algorithms to identify regulatory sequence elements.
• Deep learning model training for RNA level prediction from DNA sequence.

Main Results:

• PAS sequence significantly modulates gene expression across a five-orders-of-magnitude range.
• Cleavage efficiency dictates the sensitivity and robustness of PAS to sequence mutations.
• Identification of known and novel sequence motifs governing PAS-mediated regulation.
• A deep learning model accurately predicts RNA levels from DNA sequence (R = 0.83).
• Novel methods developed for predicting cleavage sites in reporter and endogenous transcripts.

Conclusions:

• PAS sequence and its interaction with cleavage efficiency are key determinants of gene expression.
• This study provides a comprehensive resource for understanding and predicting PAS function.
• Findings advance regulatory genomics and offer tools for analyzing gene expression control.