MRNA deadenylation modeling at permissive and stress conditions reveals complex relations to decay
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ABSTRACT: The prevailing model postulates that complete cytoplasmic polyadenosine tail (pA-tail) deadenylation is essential for initiating mRNA decapping and subsequent degradation. To investigate this, we conducted direct RNA sequencing of yeast mRNAs derived from steady-state and stress condition chase experiments. Subsequently, we developed a numerical model based on a modified gamma distribution function, which estimated the transcriptomic deadenylation rate at 10 A/min. A simplified independent method, based on the delineation of quantile pA-tail values, showed a correlation between the decay and deadenylation rates of individual mRNA, which appeared consistent within functional transcript groups and associated with to codon optimality. Notably, these rates varied during the stress response. Detailed analysis of ribosomal protein-coding mRNAs (RPG mRNAs), constituting 40 % of the transcriptome, singled out this transcript group. While RPG mRNA deadenylation and decay accelerated under heat stress, their degradation could proceed even when deadenylation was blocked, depending entirely on ongoing nuclear export. Our findings support the general primary function of deadenylation in dictating decapping onset, while also demonstrating complex relations between these processes.
Project description:For all but a few mRNAs, the dynamics of metabolism are unknown. Here, we developed an experimental and analytical framework for examining these dynamics for mRNAs from thousands of genes. mRNAs of mouse fibroblasts exit the nucleus with diverse intragenic and intergenic poly(A)-tail lengths. Once in the cytoplasm, they have a broad (1000-fold) range of deadenylation rates, which correspond to cytoplasmic lifetimes. Indeed, degradation appears to occur primarily through deadenylation-linked mechanisms, with little contribution from endonucleolytic cleavage or deadenylation-independent decapping. Most mRNA molecules degrade only after their tail lengths fall below 25 nt. Decay rates of short-tailed mRNAs vary broadly (1000-fold) and are more rapid for short-tailed mRNAs that had previously undergone more rapid deadenylation. This coupling helps clear rapidly deadenylated mRNAs, enabling the large range in deadenylation rates to impart a similarly large range in stabilities.
Project description:Uridylation is a widespread modification destabilizing eukaryotic mRNAs. Yet, molecular mechanisms underlying TUTase-mediated mRNA degradation remain mostly unresolved. Here, we report that the Arabidopsis TUTase URT1 participates in a molecular network connecting several translational repressors/decapping activators. URT1 directly interacts with DECAPPING 5 (DCP5), the Arabidopsis ortholog of human LSM14 and yeast Scd6, and this interaction connects URT1 to additional decay factors like DDX6/Dhh1-like RNA helicases. Nanopore direct RNA sequencing reveals a global role of URT1 in shaping poly(A) tail length, notably by preventing the accumulation of excessively deadenylated mRNAs. Based on in vitro and in planta data, we propose a model that explains how URT1 could reduce the accumulation of oligo(A)-tailed mRNAs both by favoring their degradation and because 3’ terminal uridines intrinsically hinder deadenylation. Importantly, preventing the accumulation of excessively deadenylated mRNAs avoids the biogenesis of illegitimate siRNAs that silence endogenous mRNAs and perturb Arabidopsis growth and development.
Project description:The general pathways of eukaryotic mRNA decay occur via deadenylation followed by 3’ to 5’ degradation or decapping, although some endonuclease sites have been identified in metazoan mRNAs. To determine the role of endonucleases in mRNA degradation in Saccharomyces cerevisiae, we mapped 5’ monophosphate ends on mRNAs in wild-type and dcp2∆ xrn1∆ yeast cells, wherein mRNA endonuclease cleavage products are stabilized. This led to three important observations. First, only few mRNAs that undergo low level endonucleotyic cleavage were observed suggesting that endonucleases are not a major contributor to yeast mRNA decay. Second, independent of known decapping enzymes, we observed low levels of 5’ monophosphates on some mRNAs suggesting that an unknown mechanism can generate 5' exposed ends, although for all substrates tested Dcp2 was the primary decapping enzyme. Finally, we identified debranched lariat intermediates from intron-containing genes, demonstrating a significant discard pathway for mRNAs during the second step of pre-mRNA splicing, which is a potential new step to regulate gene expression. 5' monophosphorylated ends of poly(A) RNA from wild-type and dcp2D xrn1D strains were identified in duplicates and triplicates, respectively.
Project description:MicroRNAs regulate gene expression through deadenylation, repression and mRNA decay. However, the contribution of each mechanism in non-steady-state situations remains unclear. We monitored the impact of miR-430 on ribosome occupancy of endogenous mRNAs in wild type and dicer mutants lacking mature miR-430. Our results indicate that miR-430 reduces the number of ribosomes on target mRNAs before causing mRNA decay. Translational repression occurs before complete deadenylation, and disrupting deadenylation using an internal poly(A) tail did not block target repression. Finally, we observe that ribosome density along the length of the target mRNA remains constant, suggesting that translational repression occurs by reducing the initiation rate rather than reducing elongation or causing ribosomal drop-off. In summary, our results show that miR-430 regulates translation initiation before inducing mRNA decay. Time course parallel ribosome profiling and input mRNA quantification in wildtype and MZdicer mutant embryos
Project description:Uridylation is a widespread modification destabilizing eukaryotic mRNAs. Yet, molecular mechanisms underlying TUTase-mediated mRNA degradation remain mostly unresolved. Here, we report that the Arabidopsis TUTase URT1 participates in a molecular network connecting several translational repressors/decapping activators including DECAPPING 5 (DCP5), the Arabidopsis ortholog of human LSM14 and yeast Scd6. A conserved Helical Leucine-rich Motif (HLM) within an intrinsically disordered region of URT1 binds to the LSm domain of DCP5. This interaction connects URT1 to additional decay factors like DDX6/Dhh1-like RNA helicases. The combination of in planta and in vitro analyses supports a model that explains how URT1 reduces the accumulation of oligo(A)-tailed mRNAs: first, by connecting decapping factors and second, because 3’ terminal uridines can intrinsically hinder deadenylation. Importantly, preventing the accumulation of excessively deadenylated mRNAs in Arabidopsis avoids the biogenesis of illegitimate siRNAs that silence endogenous mRNAs and perturb plant growth and development.
Project description:The general pathways of eukaryotic mRNA decay occur via deadenylation followed by 3’ to 5’ degradation or decapping, although some endonuclease sites have been identified in metazoan mRNAs. To determine the role of endonucleases in mRNA degradation in Saccharomyces cerevisiae, we mapped 5’ monophosphate ends on mRNAs in wild-type and dcp2∆ xrn1∆ yeast cells, wherein mRNA endonuclease cleavage products are stabilized. This led to three important observations. First, only few mRNAs that undergo low level endonucleotyic cleavage were observed suggesting that endonucleases are not a major contributor to yeast mRNA decay. Second, independent of known decapping enzymes, we observed low levels of 5’ monophosphates on some mRNAs suggesting that an unknown mechanism can generate 5' exposed ends, although for all substrates tested Dcp2 was the primary decapping enzyme. Finally, we identified debranched lariat intermediates from intron-containing genes, demonstrating a significant discard pathway for mRNAs during the second step of pre-mRNA splicing, which is a potential new step to regulate gene expression.
Project description:In eukaryotes, mRNA decay is thought to occur in an ordered manner, with an initial deadenylation step that triggers decapping and RNA degradation. We performed SLAM-seq experiments in yeast strains depleted either for the decapping or for the major deadenylase enzymes to evaluate changes in the half-life of mRNA. Our results suggest that deadenylation and decapping of mRNA are not necessarily linked for mRNA degradation, since the effects of inhibiting the enzymes on RNA stability were not correlated.
Project description:MicroRNAs regulate gene expression through deadenylation, repression and mRNA decay. However, the contribution of each mechanism in non-steady-state situations remains unclear. We monitored the impact of miR-430 on ribosome occupancy of endogenous mRNAs in wild type and dicer mutants lacking mature miR-430. Our results indicate that miR-430 reduces the number of ribosomes on target mRNAs before causing mRNA decay. Translational repression occurs before complete deadenylation, and disrupting deadenylation using an internal poly(A) tail did not block target repression. Finally, we observe that ribosome density along the length of the target mRNA remains constant, suggesting that translational repression occurs by reducing the initiation rate rather than reducing elongation or causing ribosomal drop-off. In summary, our results show that miR-430 regulates translation initiation before inducing mRNA decay.
Project description:The oocyte-to-embryo transition (OET) occurs in the absence of new transcription and relies on post-transcriptional gene regulation, including translational control by mRNA poly(A) tail regulation, where cytoplasmic polyadenylation activates translation and deadenylation leads to translational repression and decay. However, how the transcriptome-wide landscape of mRNA poly(A) tails shapes translation across the OET in mammals remains unknown. Here, we performed long-read RNA sequencing to uncover poly(A) tail lengths and mRNA abundance transcriptome-wide in mice across five stages of development from oocyte to embryo. Integrating these data with recently published ribosome profiling data, we demonstrate that poly(A) tail length is coupled to translational efficiency across the entire OET. We uncover an extended wave of global deadenylation during fertilization, which sets up a switch in translation control between the oocyte and embryo. In the oocyte, short-tailed maternal mRNAs that resist deadenylation in the oocyte are translationally activated, whereas large groups of mRNAs deadenylated without decay in the oocyte are later readenylated to drive translation activation in the early embryo. Our findings provide an important resource and insight into the mechanisms by which cytoplasmic polyadenylation and deadenylation dynamically shape poly(A) tail length in a stage-specific manner to orchestrate development from oocyte to embryo in mammals.
Project description:RNA turnover in eukaryotes is triggered by decapping, believed to be activated mostly after deadenylation. To investigate simultaneous changes in RNA levels and poly(A) tail size we performed Nanopore RNA sequencing on total RNA extracted from S. cerevisiae strains in which the decapping enzyme Dcp2, or the deadenylation factors Ccr4 and Pop2 were depleted through an inducible degron system. Two fully independent replicate experiments were performed for wild type and the two mutant conditions.