Biology subjects

Stowell, J. A.

Publications and source records attributed to Stowell, J. A..

3 recordsLinked to original sources

bioRxiv2025

RNA-binding proteins provide specificity to the PAN2-PAN3 mRNA deadenylation complex

Cytoplasmic shortening of mRNA poly(A) tails represses eukaryotic gene expression by inhibiting efficient translation and committing an mRNA to decay. The CCR4-NOT deadenylase machinery interacts with sequence-specific RNA-binding proteins (RBPs), termed RNA adaptors, to target specific transcripts for deadenylation. In contrast, the PAN2-PAN3 deadenylation complex is thought to be predominantly recruited to mRNAs via interaction with the poly(A) binding protein, raising the question of whether it acts in a transcript-specific manner. Here, using biochemical reconstitution, we show that PAN2-PAN3 can also be recruited to specific RNAs via RNA adaptors, including MEX3, YTHDF and ZFP36 proteins. In cells, we find that a diverse range of RNA adaptors interact with both major deadenylation complexes. Thus, our data suggest that, in addition to CCR4-NOT, PAN2-PAN3 also contributes to the specificity of mRNA degradation and the robustness of post-transcriptional regulation of gene expression.

molecular biology↗

bioRxiv2024

Phosphorylation-dependent tuning of mRNA deadenylation rates

mRNA decay is a major determinant of gene regulation that is controlled through shortening of mRNA poly(A) tails by the Ccr4-Not complex. The specificity of deadenylation can be mediated through RNA adaptors - RNA-binding proteins that tether substrate mRNAs to Ccr4-Not in a regulated and context-specific manner. Interaction with Ccr4-Not is mediated by intrinsically disordered regions (IDRs) within the RNA adaptors. Due to the difficulty in studying large IDR-containing complexes, the determinants of specificity and their regulation remain unclear. Here we use structural biology and biochemical reconstitution to show that dispersed segments within IDRs of RNA adaptors bind to several distinct binding sites on Ccr4-Not through multivalent interactions. We further demonstrate that binding can be modulated by phosphorylation, altering the consequent deadenylation rate in a continuously tunable manner. This mechanism is broadly applicable in evolutionarily divergent IDRs from multiple RNA adaptors including fission yeast Puf3, and human Pumilio/PUM1 and Tristetraprolin/TTP. Together, our work suggests that multivalent interactions and phosphorylation represent conserved strategies for regulating gene expression. Thus, in response to cellular cues, mRNA decay can be regulated by a graded mechanism, rather than a bistable on/off switch, rationalizing how post-transcriptional gene expression is fine-tuned.

molecular biology↗

bioRxiv2022

Specific recognition and ubiquitination of slow-moving ribosomes by human CCR4-NOT

Eukaryotic messenger RNA (mRNA) decay is generally initiated by removal of the 3 polyadenosine (poly(A)) tail by the CCR4-NOT complex. Yeast Ccr4-Not binds and ubiquitinates ribosomes stalled on mRNAs with sub-optimal codons to trigger deadenylation and decay of the associated transcript. However, the mammalian ortholog of the E3 ubiquitin ligase subunit, CNOT4, is not a constitutive component of human CCR4-NOT. It therefore remains unclear how the mammalian deadenylation machinery targets stalled ribosomes. Here, we reconstitute translational stalling on non-optimal codons. We find that human CCR4-NOT recognizes translating mammalian ribosomes and is required for stable CNOT4 association. Our cryoEM structure reveals that the CNOT3 subunit detects slow translation and locks the L1 stalk of the ribosome in an open conformation to impede further elongation. Using crosslinking mass spectrometry, we show that CNOT4 and CNOT11 also bind in the vicinity of the E site. Overall, our work defines how CCR4-NOT enforces ribosomal stalling in response to low codon optimality.

molecular biology↗