Növényi RNS degradációs rendszerek: a nonsense-mediated decay rendszer molekuláris biológiája = RNA degradation systems in plants: the molecular biology of nonsense-mediated decay system

A program célja a növényi Nonsense-mediated mRNA decay (NMD) rendszer molekuláris biológiájának megismerése volt. Az NMD egy ősi eukarióta minőségbiztosítási rendszer, amely felismeri és lebontja a korai stop kodonokat (PTC) tartalmazó mRNS-eket, ezáltal megelőzi a csonka, domináns-negatív mutáns fehérjék képződését. A program során kimutattuk, hogy a növényi NMD rendszer PTC-ként ismer fel minden stop kodont, amely utána 3'UTR régió szokatlanul hosszú, vagy ahol a 3'UTR-ban intron található. Azonosítottuk a növényi NMD rendszer 6 transz faktorát, és kimutattuk, hogy a kétféle NMD cisz elem felismerés csak részben átfedő génkészletet igényel. Igazoltuk, hogy a PTC tartalmú növényi transzkriptek kétféle úton bomolhatnak le, az SMG-7, illetve a UPF1 irányította útvonalon. Kimutattuk, hogy az utóbbi XRN4 5'-3' exonukleázt igényel. Munkánk során bizonyítottuk, hogy a növényi NMD autoregulált, az SMG-7 NMD faktort az NMD negatívan regulálja. Végül eredményeink alapján egy új eukarióta NMD evolúciós modellt dolgoztunk ki. | The aim of this project was to understand the molecular basis of plant Nonsense-mediated mRNA decay (NMD) system. NMD is an ancient eukaryotic quality control system that identifies and degrades mRNAs containing premature termination codons (PTC), thereby preventing the accumulation of truncated dominant-negative mutant proteins. During this project we have shown that plant NMD system identifies any stop codon as a PTC if the 3'UTR is unusually long or if the 3' UTR contains an intron. We have identified 6 NMD trans factors and shown that the two NMD cis elements identification system requires overlapping but not identical gene sets. We have demonstrated that PTC containing mRNAs can be degraded by two pathways, one is mediated by SMG-7 and another is controlled by UPF1. XRN4 exonuclease is required only for the UPF1 mediated pathway. We have shown that plant NMD is an autoregulated system as SMG-7 NMD trans factor is negatively regulated by NMD. Finally, we have elaborated a new model for the evolution of eukaryotic NMD systems

The role of RST1 and RIPR proteins in plant RNA quality control systems

To keep mRNA homeostasis, the RNA degradation, quality control and silencing systems should act in balance in plants. Degradation of normal mRNA starts with deadenylation, then deadenylated transcripts are degraded by the SKI-exosome 3 '-5 ' and/or XRN4 5 '-3 ' exonucleases. RNA quality control systems identify and decay different aberrant transcripts. RNA silencing degrades double-stranded transcripts and homologous mRNAs. It also targets aberrant and silencing prone transcripts. The SKI-exosome is essential for mRNA homeostasis, it functions in normal mRNA degradation and different RNA quality control systems, and in its absence silencing targets normal transcripts. It is highly conserved in eukaryotes, thus recent reports that the plant SKI-exosome is associated with RST1 and RIPR proteins and that, they are required for SKI-exosome-mediated decay of silencing prone transcripts were unexpected. To clarify whether RST1 and RIPR are essential for all SKI-exosome functions or only for the elimination of silencing prone transcripts, degradation of different reporter transcripts was studied in RST1 and RIPR inactivated Nicotiana benthamiana plants. As RST1 and RIPR, like the SKI-exosome, were essential for Non-stop and No-go decay quality control systems, and for RNA silencing- and minimum ORF-mediated decay, we propose that RST1 and RIPR are essential components of plant SKI-exosome supercomplex. Key message The RST1 and RIPR proteins are required for the degradation of aberrant transcripts lacking a stop codon and the 5 ' cleavage fragments of no-go decay, RNA silencing and minimum ORF

Ecotype-specific blockage of tasiARF production by two different RNA viruses in Arabidopsis

Arabidopsis thaliana is one of the most studied model organisms of plant biology with hundreds of geographical variants called ecotypes. One might expect that this enormous genetic variety could result in a differential response to pathogens. Indeed, we observed previously that the Bur ecotype develops much more severe symptoms (upward curling leaves and wavy leaf margins) upon infection with two positive strand RNA viruses of different families (turnip vein-clearing virus, TVCV, and turnip mosaic virus, TuMV). To find the genes potentially responsible for the ecotype- specific response, we performed a differential expression analysis of the mRNA and sRNA pools of TVCV and TuMV-infected Bur and Col plants along with the corresponding mock controls. We focused on the genes and sRNAs that showed an induced or reduced expression selectively in the Bur virus samples in both virus series. We found that the two ecotypes respond to the viral infection differently, yet both viruses selectively block the production of the TAS3 derived small RNA specimen called tasiARF only in the virus-infected Bur plants. The tasiARF normally forms a gradient through the adaxial and abaxial part of the leaf (being more abundant in the adaxial part) and post-transcriptionally regulates ARF4, a major leaf polarity determinant in plants. The lack of tasiARF-mediated silencing could lead to an ectopically expressed ARF4 in the adaxial part of the leaf where the misregulation of auxin-dependent signaling would result in an irregular growth of the leaf blade manifesting as upward curling leaf and wavy leaf margin. QTL mapping using Recombinant Inbred Lines (RILs) suggests that the observed symptoms are the result of a multigenic interaction that allows the symptoms to develop only in the Bur ecotype. The particular nature of genetic differences leading to the ecotype-specific symptoms remains obscure and needs further study

The nonstop decay and the RNA silencing systems operate cooperatively in plants

Translation-dependent mRNA quality control systems protect the protein homeostasis of eukaryotic cells by eliminating aberrant transcripts and stimulating the decay of their protein products. Although these systems are intensively studied in animals, little is known about the translation-dependent quality control systems in plants. Here, we characterize the mechanism of nonstop decay (NSD) system in Nicotiana benthamiana model plant. We show that plant NSD efficiently degrades nonstop mRNAs, which can be generated by premature polyadenylation, and stop codon-less transcripts, which are produced by endonucleolytic cleavage. We demonstrate that in plants, like in animals, Pelota, Hbs1 and SKI2 proteins are required for NSD, supporting that NSD is an ancient and conserved eukaryotic quality control system. Relevantly, we found that NSD and RNA silencing systems cooperate in plants. Plant silencing predominantly represses target mRNAs through endonucleolytic cleavage in the coding region. Here we show that NSD is required for the elimination of 5' cleavage product of mi- or siRNA-guided silencing complex when the cleavage occurs in the coding region. We also show that NSD and nonsense-mediated decay (NMD) quality control systems operate independently in plants

Is There a Classical Nonsense-Mediated Decay Pathway in Trypanosomes?

In many eukaryotes, messenger RNAs with premature termination codons are destroyed by a process called “nonsense-mediated decay”, which requires the RNA helicase Upf1 and also, usually, an interacting factor, Upf2. Recognition of premature termination codons may rely on their distance from either a splice site or the polyadenylation site, and long 3′-untranslated regions can trigger mRNA decay. The protist Trypanosoma brucei relies heavily on mRNA degradation to determine mRNA levels, and 3′-untranslated regions play a major role in control of mRNA decay. We show here that trypanosomes have a homologue of Upf1, TbUPF1, which interacts with TbUPF2 and (in an RNA-dependent fashion) with poly(A) binding protein 1, PABP1. Introduction of a premature termination codon in either an endogenous gene or a reporter gene decreased mRNA abundance, as expected for nonsense-mediated decay, but a dependence of this effect on TbUPF1 could not be demonstrated, and depletion of TbUPF1 by over 95% had no effect on parasite growth or the mRNA transcriptome. Further investigations of the reporter mRNA revealed that increases in open reading frame length tended to increase mRNA abundance. In contrast, inhibition of translation, either using 5′-secondary structures or by lengthening the 5′-untranslated region, usually decreased reporter mRNA abundance. Meanwhile, changing the length of the 3′-untranslated region had no consistent effect on mRNA abundance. We suggest that in trypanosomes, translation per se may inhibit mRNA decay, and interactions with multiple RNA-binding proteins preclude degradation based on 3′-untranslated region length alone

Functional and molecular characterization of the conserved Arabidopsis PUMILIO protein, APUM9

Key message Here we demonstrate that the APUM9 RNA-binding protein and its co-factors play a role in mRNA destabilization and how this activity might regulate early plant development.Abstract APUM9 is a conserved PUF RNA-binding protein (RBP) under complex transcriptional control mediated by a transposable element (TE) that restricts its expression in Arabidopsis. Currently, little is known about the functional and mechanistic details of the plant PUF regulatory system and the biological relevance of the TE-mediated repression of APUM9 in plant development and stress responses. By combining a range of transient assays, we show here, that APUM9 binding to target transcripts can trigger their rapid decay via its conserved C-terminal RNA-binding domain. APUM9 directly interacts with DCP2, the catalytic subunit of the decapping complex and DCP2 overexpression induces rapid decay of APUM9 targeted mRNAs. We show that APUM9 negatively regulates the expression of ABA signaling genes during seed imbibition, and thereby might contribute to the switch from dormant stage to seed germination. By contrast, strong TE-mediated repression of APUM9 is important for normal plant growth in the later developmental stages. Finally, APUM9 overexpression plants show slightly enhanced heat tolerance suggesting that TE-mediated control of APUM9, might have a role not only in embryonic development, but also in plant adaptation to heat stress conditions