The anticancer compound JTE-607 reveals hidden sequence specificity of the mRNA 3' processing machinery.

JTE-607 is an anticancer and anti-inflammatory compound and its active form, compound 2, directly binds to and inhibits CPSF73, the endonuclease for the cleavage step in pre-messenger RNA (pre-mRNA) 3' processing. Surprisingly, compound 2-mediated inhibition of pre-mRNA cleavage is sequence specific and the drug sensitivity is predominantly determined by sequences flanking the cleavage site (CS). Using massively parallel in vitro assays, we identified key sequence features that determine drug sensitivity. We trained a machine learning model that can predict poly(A) site (PAS) relative sensitivity to compound 2 and provide the molecular basis for understanding the impact of JTE-607 on PAS selection and transcription termination genome wide. We propose that CPSF73 and associated factors bind to the CS region in a sequence-dependent manner and the interaction affinity determines compound 2 sensitivity. These results have not only elucidated the mechanism of action of JTE-607, but also unveiled an evolutionarily conserved sequence specificity of the mRNA 3' processing machinery.

The anti-cancer compound JTE-607 reveals hidden sequence specificity of the mRNA 3' processing machinery.

JTE-607 is a small molecule compound with anti-inflammation and anti-cancer activities. Upon entering the cell, it is hydrolyzed to Compound 2, which directly binds to and inhibits CPSF73, the endonuclease for the cleavage step in pre-mRNA 3' processing. Although CPSF73 is universally required for mRNA 3' end formation, we have unexpectedly found that Compound 2- mediated inhibition of pre-mRNA 3' processing is sequence-specific and that the sequences flanking the cleavage site (CS) are a major determinant for drug sensitivity. By using massively parallel in vitro assays, we have measured the Compound 2 sensitivities of over 260,000 sequence variants and identified key sequence features that determine drug sensitivity. A machine learning model trained on these data can predict the impact of JTE-607 on poly(A) site (PAS) selection and transcription termination genome-wide. We propose a biochemical model in which CPSF73 and other mRNA 3' processing factors bind to RNA of the CS region in a sequence-specific manner and the affinity of such interaction determines the Compound 2 sensitivity of a PAS. As the Compound 2-resistant CS sequences, characterized by U/A-rich motifs, are prevalent in PASs from yeast to human, the CS region sequence may have more fundamental functions beyond determining drug resistance. Together, our study not only characterized the mechanism of action of a compound with clinical implications, but also revealed a previously unknown and evolutionarily conserved sequence-specificity of the mRNA 3' processing machinery.

Genetic removal of synaptic Zn2+ impairs cognition, alters neurotrophic signaling and induces neuronal hyperactivity.

Vesicular Zn2+ (zinc) is released at synapses and has been demonstrated to modulate neuronal responses. However, mechanisms through which dysregulation of zinc homeostasis may potentiate neuronal dysfunction and neurodegeneration are not well-understood. We previously reported that accumulation of soluble amyloid beta oligomers (AßO) at synapses correlates with synaptic loss and that AßO localization at synapses is regulated by synaptic activity and enhanced by the release of vesicular Zn2+ in the hippocampus, a brain region that deteriorates early in Alzheimer's disease (AD). Significantly, drugs regulating zinc homeostasis inhibit AßO accumulation and improve cognition in mouse models of AD. We used both sexes of a transgenic mouse model lacking synaptic Zn2+ (ZnT3KO) that develops AD-like cognitive impairment and neurodegeneration to study the effects of disruption of Zn2+ modulation of neurotransmission in cognition, protein expression and activation, and neuronal excitability. Here we report that the genetic removal of synaptic Zn2+ results in progressive impairment of hippocampal-dependent memory, reduces activity-dependent increase in Erk phosphorylation and BDNF mRNA, alters regulation of Erk activation by NMDAR subunits, increases neuronal spiking, and induces biochemical and morphological alterations consistent with increasing epileptiform activity and neurodegeneration as ZnT3KO mice age. Our study shows that disruption of synaptic Zn2+ triggers neurodegenerative processes and is a potential pathway through which AßO trigger altered expression of neurotrophic proteins, along with reduced hippocampal synaptic density and degenerating neurons, neuronal spiking activity, and cognitive impairment and supports efforts to develop therapeutics to preserve synaptic zinc homeostasis in the brain as potential treatments for AD.