Ceg1 depletion reveals mechanisms governing degradation of non-capped RNAs in Saccharomyces cerevisiae.

Most functional eukaryotic mRNAs contain a 5' 7-methylguanosine (m7G) cap. Although capping is essential for many biological processes including mRNA processing, export and translation, the fate of uncapped transcripts has not been studied extensively. Here, we employed fast nuclear depletion of the capping enzymes in Saccharomyces cerevisiae to uncover the turnover of the transcripts that failed to be capped. We show that although the degradation of cap-deficient mRNA is dominant, the levels of hundreds of non-capped mRNAs increase upon depletion of the capping enzymes. Overall, the abundance of non-capped mRNAs is inversely correlated to the expression levels, altogether resembling the effects observed in cells lacking the cytoplasmic 5'-3' exonuclease Xrn1 and indicating differential degradation fates of non-capped mRNAs. The inactivation of the nuclear 5'-3' exonuclease Rat1 does not rescue the non-capped mRNA levels indicating that Rat1 is not involved in their degradation and consequently, the lack of the capping does not affect the distribution of RNA Polymerase II on the chromatin. Our data indicate that the cap presence is essential to initiate the Xrn1-dependent degradation of mRNAs underpinning the role of 5' cap in the Xrn1-dependent buffering of the cellular mRNA levels.

The RNA binding proteins ZFP36L1 and ZFP36L2 are dysregulated in airway epithelium in human and a murine model of asthma.

Introduction: Asthma is the most common chronic inflammatory disease of the airways. The airway epithelium is a key driver of the disease, and numerous studies have established genome-wide differences in mRNA expression between health and asthma. However, the underlying molecular mechanisms for such differences remain poorly understood. The human TTP family is comprised of ZFP36, ZFP36L1 and ZFP36L2, and has essential roles in immune regulation by determining the stability and translation of myriad mRNAs encoding for inflammatory mediators. We investigated the expression and possible role of the tristetraprolin (TTP) family of RNA binding proteins (RBPs), poorly understood in asthma. Methods: We analysed the levels of ZFP36, ZFP36L1 and ZFP36L2 mRNA in several publicly available asthma datasets, including single cell RNA-sequencing. We also interrogated the expression of known targets of these RBPs in asthma. We assessed the lung mRNA expression and cellular localization of Zfp36l1 and Zfp36l2 in precision cut lung slices in murine asthma models. Finally, we determined the expression in airway epithelium of ZFP36L1 and ZFP36L2 in human bronchial biopsies and performed rescue experiments in primary bronchial epithelium from patients with severe asthma. Results: We found ZFP36L1 and ZFP36L2 mRNA levels significantly downregulated in the airway epithelium of patients with very severe asthma in different cohorts (5 healthy vs. 8 severe asthma; 36 moderate asthma vs. 37 severe asthma on inhaled steroids vs. 26 severe asthma on oral corticoids). Integrating several datasets allowed us to infer that mRNAs potentially targeted by these RBPs are increased in severe asthma. Zfp36l1 was downregulated in the lung of a mouse model of asthma, and immunostaining of ex vivo lung slices with a dual antibody demonstrated that Zfp36l1/l2 nuclear localization was increased in the airway epithelium of an acute asthma mouse model, which was further enhanced in a chronic model. Immunostaining of human bronchial biopsies showed that airway epithelial cell staining of ZFP36L1 was decreased in severe asthma as compared with mild, while ZFP36L2 was upregulated. Restoring the levels of ZFP36L1 and ZFP36L2 in primary bronchial epithelial cells from patients with severe asthma decreased the mRNA expression of IL6, IL8 and CSF2. Discussion: We propose that the dysregulation of ZFP36L1/L2 levels as well as their subcellular mislocalization contributes to changes in mRNA expression and cytoplasmic fate in asthma.