U-rich elements drive pervasive cryptic splicing in 3' UTR massively parallel reporter assays

Area of Science:

• Molecular Biology
• Genetics
• RNA Biology

Background:

• Non-coding RNA sequences are crucial for gene expression regulation.
• Post-transcriptional regulation mechanisms, particularly those involving sequence codes, are not fully understood.
• AU-rich elements (AREs) in 3' untranslated regions (3' UTRs) have been shown to influence mRNA expression.

Purpose of the Study:

• To investigate the mechanisms behind context-dependent regulation by U-rich elements in 3' UTRs observed in previous massively parallel reporter assays (MPRAs).
• To identify the source of variable mRNA expression driven by U-rich elements.
• To understand the role of U-rich sequences in splice site selection and splicing efficiency.

Main Methods:

• Revisiting data from a prior massively parallel reporter assay (MPRA).
• Analyzing reporter gene expression under the influence of U-rich elements in 3' UTRs.
• Investigating the occurrence and impact of cryptic splicing events.
• Examining the role of U-rich sequences in regulating splice site choice.

Main Results:

• Widespread cryptic splicing was discovered, predominantly originating from an unannotated splice donor in the GFP coding sequence to various acceptor sites in 3' UTRs.
• U-rich sequences were identified as potent, position-dependent activators of cryptic splicing.
• This splicing significantly impacts reporter expression, leading to both increases and decreases through multiple mechanisms.
• Cryptic splicing affects a substantial proportion (10-50%) of measurements in other published 3' UTR MPRAs.

Conclusions:

• U-rich sequences are principal drivers of cryptic splicing.
• Cryptic splicing is a significant artifact in reporter assays, potentially confounding results from 3' UTR studies.
• Strategies to minimize cryptic splicing are essential for accurate reporter assay interpretation.
• This finding sheds light on the complex interplay between RNA sequence elements and splicing machinery in gene regulation.