A hőmérséklet hatása a növényi RNS interferencia hatékonyságára = The effect of temperature on RNA interference in higher plants

A hőmérséklet hatása a növényi RNS interferencia hatékonyságára Programunk célja a növényi RNS interferencia (RNAi) rendszer hőmérsékletfüggésének vizsgálata volt. Igazoltuk, hogy a növényi RNAi rendszer egyes útvonalai erősen hőmérsékletfüggőek, így pl. a transzgén-, illetve vírus-indukálta RNAi válaszok alacsony hőmérsékleten alig működnek, míg normál hőmérsékleten ezek a rendszerek igen aktívak. Kimutattuk, hogy a transzgénekről, illetve vírusokról származó, az RNAi rendszer által generált rövid RNS-ek (siRNS-ek) szintje a hőmérséklet emelésével gyorsan nő. Ismert, hogy a növény-vírus kapcsolatok jelentős részében a tünetek alacsony hőmérsékleten erősek, míg magas hőmérsékleten gyengék. Igazoltuk, hogy ennek oka az, hogy a vírus-indukálta RNAi rendszer -a növények leghatékonyabb antivirális rendszere- hidegben alig működik, így a tünetek felerősödnek, a víruskártétel jelentős. Ugyanakkor melegben a hatékony RNAi rendszer tünetcsökkenéshez, vírusellenállósághoz vezet. A transzgénikus növények jelentős részénél a transzgénikus fenotípus a transzgén-indukálta RNAi rendszeren alapszik. Bizonyítottuk, hogy alacsony hőmérsékleten a transzgén-indukálta RNAi rendszeren alapuló transzgénikus fenotípusok, pl antiszensz gátlás, vírusellenállóság, elveszhetnek. Kimutattuk azt is, hogy megfelelő transzgén konstrukciók (fordított ismétlődést tartalmazó konstrukciók) használatával ez a veszély csökkenthető, azaz a transzgénikus fenotípusok alacsony hőmérsékleten is stabilak maradnak. | The effect of temperature on RNA interference in higher plants The aim of our project was to study the effect of temperature on efficiency of RNA interference (RNAi) system of higher plants. We have shown that certain plant RNAi pathways, as transgene- or virus- induced RNAi pathways are inhibited at low temperature, while these patways work efficiently at normal temperature. Indeed, the levels of RNAi generated transgene or virus derived short RNAs (siRNAs) are dramatically reduced at low temperature. Previously, it has been reported that in cold, viral infections of plants lead to strong symptoms and that outbreaks of viral diseases are frequent. By contrast, at high temperature the symptoms are masked, plants recover quickly. We show that viral symptoms are strong in cold because virus induced plant RNAi (the most important antiviral sytem of plants) is inefficient, whereas at high temperature the efficient RNAi can protect plants. Transgenic phenotypes of many transgenic plants are depend on transgene induced RNAi. We have demonstrated that in cold, transgenic phenotypes -as antisense inactivation or virus resistance- that depend on trangene induced RNAi are dramatically weakened. Importantly, if inverted repeat containing constructs are used, trangenic phenotypes are stable even at low temperature

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

Bulbous perennials precisely detect the length of winter and adjust flowering dates

In order to identify the most relevant environmental parameters that regulate flowering time of bulbous perennials, first flowering dates of 329 taxa over 33 yr are correlated with monthly and daily mean values of 16 environmental parameters (such as insolation, precipitation, temperature, soil water content, etc.) spanning at least 1 yr back from flowering. A machine learning algorithm is deployed to identify the best explanatory parameters because the problem is strongly prone to overfitting for traditional methods: if the number of parameters is the same or greater than the number of observations, then a linear model can perfectly fit the dependent variable (observations). Surprisingly, the best proxy of flowering date fluctuations is the daily snow depth anomaly, which cannot be a signal itself, however it should be related to some integrated temperature signal. Moreover, daily snow depth anomaly as proxy performs much better than mean soil temperature preceding the flowering, the best monthly explanatory parameter. Our findings support the existence of complicated temperature sensing mechanisms operating on different timescales, which is a prerequisite to precisely observe the length and severity of the winter season and translate for example, 'lack of snow' information to meaningful internal signals related to phenophases

Expression of the translation termination factor eRF1 is autoregulated by translational readthrough and 3'UTR intron-mediated NMD in Neurospora crassa

Eukaryotic release factor 1 (eRF1) is a translation termination factor that binds to the ribosome at stop codons. The expression of eRF1 is strictly controlled, since its concentration defines termination efficiency and frequency of translational readthrough. Here, we show that eRF1 expression in Neurospora crassa is controlled by an autoregulatory circuit that depends on the specific 3'UTR structure of erf1 mRNA. The stop codon context of erf1 promotes readthrough that protects the mRNA from its 3'UTR-induced nonsense-mediated mRNA decay (NMD). High eRF1 concentration leads to inefficient readthrough, thereby allowing NMD-mediated erf1 degradation. We propose that eRF1 expression is controlled by similar autoregulatory circuits in many fungi and seed plants and discuss the evolution of autoregulatory systems of different translation termination factors

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

Both introns and long 3′-UTRs operate as cis-acting elements to trigger nonsense-mediated decay in plants

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control mechanism that identifies and eliminates aberrant mRNAs containing a premature termination codon (PTC). Although, key trans-acting NMD factors, UPF1, UPF2 and UPF3 are conserved in yeast and mammals, the cis-acting NMD elements are different. In yeast, short specific sequences or long 3′-untranslated regions (3′-UTRs) render an mRNA subject to NMD, while in mammals' 3′-UTR located introns trigger NMD. Plants also possess an NMD system, although little is known about how it functions. We have elaborated an agroinfiltration-based transient NMD assay system and defined the cis-acting elements that mediate plant NMD. We show that unusually long 3′-UTRs or the presence of introns in the 3′-UTR can subject mRNAs to NMD. These data suggest that both long 3′-UTR-based and intron-based PTC definition operated in the common ancestors of extant eukaryotes (stem eukaryotes) and support the theory that intron-based NMD facilitated the spreading of introns in stem eukaryotes. We have also identified plant UPF1 and showed that tethering of UPF1 to either the 5′- or 3′-UTR of an mRNA results in reduced transcript accumulation. Thus, plant UPF1 might bind to mRNA in a late, irreversible phase of NMD