Fate of a larch unedited tRNA precursor expressed in potato mitochondria.

Abstract In higher plant mitochondria, post-transcriptional C to U conversion known as editing mostly affects mRNAs. However, three tRNAs were also shown to be edited. Among them, three editing sites were identified in larch mitochondrial tRNAHis. We have previously shown that only the edited version can undergo maturation in vitro. In this paper, we introduced via direct DNA uptake the edited or unedited version of larch mitochondrial trnH into isolated potato mitochondria and expressed them under the control of potato mitochondrial 18 S rRNA promoter. As expected, the edited form of larch mitochondrial tRNAHis precursor was processed in the isolated organelles. By contrast, no mature tRNAHis was detected when using the unedited version of trnH. However, precursor molecules could be characterized by reverse transcription-PCR. These data demonstrate that the potato mitochondrial editing machinery is not able to recognize these "foreign" editing sites and confirm that these unedited tRNA precursor molecules are not correctly processed in organello. As a consequence, the fate of these RNA precursor molecules is likely to be degradation. Indeed, we detected by PCR two 3′-end truncated precursor RNAs. Interestingly, both RNA species exhibit poly(A) tails, a hallmark of degradation in plant mitochondria. Taken together, these data suggest that, in plant mitochondria, a defective unedited RNA precursor that cannot be processed to give a mature stable tRNA, is degraded through a polyadenylation-dependent pathway

SKI2 mediates degradation of RISC 5'-cleavage fragments and prevents secondary siRNA production from miRNA targets in <i>Arabidopsis</i>

Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20–24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5′, but not 3′-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5′ to the cleavage site, but several examples of 3′-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5′-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5′-cleavage fragments

SKI2 mediates degradation of RISC 5′-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis

Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20-24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5′, but not 3′-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5′ to the cleavage site, but several examples of 3′-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5′-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5′-cleavage fragment

Respective Contributions of URT1 and HESO1 to the Uridylation of 5′ Fragments Produced From RISC-Cleaved mRNAs

In plants, post-transcriptional gene silencing (PTGS) represses gene expression by translation inhibition and cleavage of target mRNAs. The slicing activity is provided by argonaute 1 (AGO1), and the cleavage site is determined by sequence complementarity between the target mRNA and the microRNA (miRNA) or short interfering RNA (siRNA) loaded onto AGO1, to form the core of the RNA induced silencing complex (RISC). Following cleavage, the resulting 5′ fragment is modified at its 3′ end by the untemplated addition of uridines. Uridylation is proposed to facilitate RISC recycling and the degradation of the RISC 5′-cleavage fragment. Here, we detail a 3′ RACE-seq method to analyze the 3′ ends of 5′ fragments produced from RISC-cleaved transcripts. The protocol is based on the ligation of a primer at the 3′ end of RNA, followed by cDNA synthesis and the subsequent targeted amplification by PCR to generate amplicon libraries suitable for Illumina sequencing. A detailed data processing pipeline is provided to analyze nibbling and tailing at high resolution. Using this method, we compared the tailing and nibbling patterns of RISC-cleaved MYB33 and SPL13 transcripts between wild-type plants and mutant plants depleted for the terminal uridylyltransferases (TUTases) HESO1 and URT1. Our data reveal the respective contributions of HESO and URT1 in the uridylation of RISC-cleaved MYB33 and SPL13 transcripts, with HESO1 being the major TUTase involved in uridylating these fragments. Because of its depth, the 3′ RACE-seq method shows at high resolution that these RISC-generated 5′ RNA fragments are nibbled by a few nucleotides close to the cleavage site in the absence of uridylation. 3′ RACE-seq is a suitable approach for a reliable comparison of uridylation and nibbling patterns between mutants, a prerequisite to the identification of all factors involved in the clearance of RISC-generated 5′ mRNA fragments

The Elongator complex regulates hypocotyl growth in darkness and during photomorphogenesis

The Elongator complex (hereafter Elongator) promotes RNA polymerase II-mediated transcript elongation through epigenetic activities such as histone acetylation. Elongator regulates growth, development, immune response and sensitivity to drought and abscisic acid. We demonstrate that elo mutants exhibit defective hypocotyl elongation but have a normal apical hook in darkness and are hyposensitive to light during photomorphogenesis. These elo phenotypes are supported by transcriptome changes, including downregulation of circadian clock components, positive regulators of skoto-or photomorphogenesis, hormonal pathways and cell wall biogenesis-related factors. The downregulated genes LHY, HFR1 and HYH are selectively targeted by Elongator for histone H3K14 acetylation in darkness. The role of Elongator in early seedling development in darkness and light is supported by hypocotyl phenotypes of mutants defective in components of the gene network regulated by Elongator, and by double mutants between elo and mutants in light or darkness signaling components. A model is proposed in which Elongator represses the plant immune response and promotes hypocotyl elongation and photomorphogenesis via transcriptional control of positive photomorphogenesis regulators and a growth-regulatory network that converges on genes involved in cell wall biogenesis and hormone signaling. This article has an associated First Person interview with the first author of the paper

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements