Sement, F. M. | Search bioRxiv

bioRxiv2020

Molecular connection between the TUTase URT1 and decapping activators

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.

molecular biology↗

bioRxiv2018

Transcription initiation defines kinetoplast RNA boundaries

Mitochondrial genomes are often transcribed into polycistronic primary RNAs punctuated by tRNAs whose excision defines mature RNA boundaries. Although kinetoplast DNA lacks tRNA genes, it is commonly held that monophosphorylated 5'-ends of functional molecules typify precursor partitioning by an unknown endonuclease. To the contrary, we demonstrate that in Trypanosoma brucei individual mRNAs and rRNAs are independently synthesized as 3' extended precursors. The transcription-defined 5' terminus is converted into monophosphorylated state by the 5' pyrophosphohydrolase complex, termed PPsome, which is activated by RNA editing substrate binding complex (RESC). Most guide RNAs lack PPsome recognition sites and, therefore, remain triphosphorylated. We provide evidence that both 5' pyrophosphate removal and 3' adenylation are essential for mRNA stabilization. Furthermore, we uncover a mechanism by which antisense RNA-controlled 3'-5' exonucleolytic trimming defines mRNA 3'-end. We conclude that mitochondrial mRNAs and rRNAs are transcribed and processed as insulated units irrespective of their genomic location.\n\nSignificanceIt is commonly held that in trypanosomes both mitochondrial DNA strands are transcribed into polycistronic precursors. These primary RNAs are presumably partitioned into individual pre-mRNAs by a \"cryptic\" endonuclease. We challenged the polycistronic transcription/ endonuclease model after revealing precursor processing by 3'-5' degradation. This work demonstrates individual transcription of each gene and mRNA 5'-end definition by the first incorporated nucleotide triphosphate. We have uncovered the stabilizing role of 5' triphosphate to monophosphate conversion and identified a protein complex responsible for this reaction. We have discovered antisense noncoding RNA originating near mRNA 3' end and showed that a duplex formation modulates exonuclease activity to delimit the mature 3' end. Collectively, our findings reveal mechanisms by which transcription defines both mRNA termini.

biochemistry↗