McKenney, K. M. | bioRxiv Science

bioRxiv2025

Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to RNA-binding protein-directed decay

The cytoplasmic fate of mRNAs is dictated by the balance of translation and mRNA degradation, governed in part by the 3' poly-adenosine tail and cytoplasmic poly(A)-binding proteins (PABPCs). Deadenylases remove poly(A) to initiate mRNA decay, while sequence-specific RNA-binding factors, including Pumilio proteins (PUM1 and PUM2), modulate these processes. We investigated how human PUM1&2 repress target mRNAs by accelerating their degradation. We found that the poly(A) tail plays a central role in PUM repression, dependent on the interplay of deadenylases and PABPCs. PUM-mediated repression requires the CCR4-NOT deadenylase but not the poly(A) nuclease (PAN). PUMs associate with and require PABPC1 and PABPC4 to repress. In the absence of PABPCs, both PUM targets and non-targets become unstable, bypassing PUM control. Increasing PABPC inhibits PUM activity in a concentration-dependent manner by stabilizing poly(A) mRNAs. Our results establish a Goldilocks principle wherein PABPC abundance tunes the response of mRNAs to regulatory factors through protection of poly(A) from deadenylation. Variation of PABPC levels across tissues and development suggests physiological relevance for this mechanism.

molecular biology↗

bioRxiv2023

Chemi-Northern: a versatile chemiluminescent northern blot method for analysis and quantitation of RNA molecules.

This report describes a chemiluminescence-based detection method for RNAs on northern blots, designated Chemi-Northern. This approach builds on the simplicity and versatility of northern blotting, while dispensing of the need for expensive and cumbersome radioactivity. RNAs are first separated on denaturing gel electrophoresis, transferred to a nylon membrane, and then hybridized to a biotinylated RNA or DNA antisense probe. Streptavidin conjugated with horseradish peroxidase and enhanced chemiluminescence substrate are then used to detect the probe bound to the target RNA. Our results demonstrate the versatility of this method in detecting natural and engineered RNAs expressed in cells, including messenger and noncoding RNAs. We show that Chemi-Northern detection is sensitive and fast, detecting attomole amounts of RNA in as little as 1 second, with high signal intensity and low background. The dynamic response displays excellent linearity. Using Chemi-Northern, we measure the significant, reproducible reduction of mRNA levels by human sequence-specific RNA-binding proteins, PUM1 and PUM2. Additionally, we measure the interaction of endogenous poly(A) binding protein, PABPC1, with poly-adenylated mRNA. Thus, the Chemi-Northern method provides a versatile, simple, cost-effective method to enable researchers to detect and measure changes in RNA expression, processing, binding, and decay of RNAs.

molecular biology↗