mRNA 3' end modifications by nucleotide addition: impact on translation and mRNA decay in Arabidopsis thaliana – 3'modRN

The main technical originality of the 3'modRN project is the use of novel transcriptome-wide methods to determine the modifications by nucleotide additions at the 3' extremities of mRNAs. We develop protocols for the construction of libraries suitable for Illumina sequencing and their analysis in various genetic backgrounds of Arabidopsis plants grown under control conditions or confronted to a heat stress. We compare the global dynamics of mRNA end modifications both for the total population of mRNAs and for those engaged in translation.

The main results of the on-going 3'modRN project are the identification of terminal nucleotidylransferases (TNTases) that modify mRNA 3' ends in Arabidopsis. These TNTases include terminal uridylyltransferases that uridylate mRNAs. Interestingly, distinct TUTases target different sub-populations of mRNAs and uridylation of these different sub-populations has a different impact on mRNA’s fate. The differential interaction network of these TUTases is being characterized to identify new factors involved in the metabolism of uridylated mRNAs. Other key components of the mRNA uridylation pathway are being identified through a forward genetic strategy. The molecular and phenotypic characterization of the corresponding mutants under control growth conditions and in response to heat stress aims at identifying new components of the stress response linked to RNA uridylation.

The identification of new components implicated in the heat stress response and of new regulatory roles linked to mRNA uridylation constitute the main prospects of this on-going project.

Scheer H, Zuber H, De Almeida C and Gagliardi D (2016) Uridylation earmarks mRNAs for degradation… and more
Trends in Genetics 32:607-619. doi: 10.1016/j.tig.2016.08.003

mRNA stability and translation are key interrelated checkpoints of gene expression that need to be massively reprogrammed across developmental transitions or during an acclimation response. A multitude of RNA binding proteins and miRNAs are key effectors controlling mRNAs' fate but recently, mRNA modifications and their modulation also emerged as new potent regulatory processes. mRNA modifications include base modifications (e.g. adenosine methylation) and the addition of nucleotides (e.g. uridylation) 3’ to the poly(A) tails. The recent development of innovative high-throughput technologies has revealed the prevalence of mRNA modifications in eukaryotes but their dynamics, as well as mechanistic insights into their roles, remain mostly to be discovered.
The overall objective of the 3'modRN project is to foster knowledge on how mRNA 3' end modifications by nucleotide addition participate in the control and reprogramming of gene expression in response to stress, using the model organism Arabidopsis thaliana. We recently identified two types of mRNA uridylation with distinct roles in Arabidopsis, a unique case so far in eukaryotes. Only URT1-mediated uridylation protects oligoadenylated mRNA 3' ends from excessive deadenylation and prevents the formation of 3' truncated transcripts, while the second type of uridylation likely plays an antagonistic role by triggering mRNA decay. We also recently showed that heat stress, which blocks translation initiation, also triggers ribosome pausing, provoking the degradation of 25% of Arabidopsis mRNAs either in the cytosol or in polysomes. Co-translational decay involves the 5'-3' exoribonuclease XRN4. Interestingly, URT1-mediated uridylation protects mRNA 3' ends engaged in polysomes, indicating that URT1 could favour the 5’-3’ polarity of degradation for polysomal mRNAs. In addition, Arabidopsis mutants impaired in mRNA uridylation lose their ability to overcome a moderate heat stress, display a premature senescence phenotype and have severely reduced seed sets. Taken together, these observations point to an important role of mRNA uridylation in regulating gene expression during development or in response to adverse conditions. However, our current knowledge on how distinct types of uridylation and other 3’ modifications affect mRNA stability, translation and co-translational decay remains fragmentary in multicellular organisms.
In the course of the 3’modRN project, we will establish transcriptome-wide maps of both URT1-mediated and URT1-independent uridylation, as well as other nucleotide addition to mRNA 3’ ends. We will use a novel high-throughput sequencing method, called TAIL-seq. TAIL-seq was recently developed by Narry Kim’s team, which will provide support for data analysis. Importantly, the landscape and dynamics of mRNA 3' end modification will be established by analysing both total and polysomal RNA in control conditions or in response to heat stress (WP1). We will also identify new factors involved in mRNA 3' end modifications by both reverse and forward genetic strategies (WP2). We will determine the impact of mRNA 3' end modifications on mRNA decay and translational activity in response to heat stress (WP3) and finally define their role during co-translational decay (WP4).
Both partners of the consortium made recent and seminal contributions to the field of mRNA uridylation and heat-induced cytosolic and co-translational mRNA decay. Their complementary expertise and common interest in the field of RNA degradation will contribute to the success of this project. In conclusion, the 3'modRN project will provide novel and crucial insights on mRNA 3' end modifications by identifying new actors of these regulatory pathways and by addressing their functional importance on translation and mRNA decay control in the context of an abiotic stress.