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

Boundary Formation through a Direct Threshold-Based Readout of Mobile Small RNA Gradients

Summary Small RNAs have emerged as a new class of mobile signals. Here, we investigate their mechanism of action and show that mobile small RNAs generate sharply defined domains of target gene expression through an intrinsic and direct threshold-based readout of their mobility gradients. This readout is highly sensitive to small RNA levels at the source, allowing plasticity in the positioning of a target gene expression boundary. Besides patterning their immediate targets, the readouts of opposing small RNA gradients enable specification of robust, uniformly positioned developmental boundaries. These patterning properties of small RNAs are reminiscent of those of animal morphogens. However, their mode of action and the intrinsic nature of their gradients distinguish mobile small RNAs from classical morphogens and present a unique direct mechanism through which to relay positional information. Mobile small RNAs and their targets thus emerge as highly portable, evolutionarily tractable regulatory modules through which to create pattern

The late steps of plant nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control system that identifies and degrades mRNAs containing premature termination codons (PTCs). If translation terminates at a PTC, the UPF1 NMD factor binds the terminating ribosome and recruits UPF2 and UPF3 to form a functional NMD complex, which triggers the rapid decay of the PTC-containing transcript. Although NMD deficiency is seedling lethal in plants, the mechanism of plant NMD remains poorly understood. To understand how the formation of the NMD complex leads to transcript decay we functionally mapped the UPF1 and SMG7 plant NMD factors, the putative key players of NMD target degradation. Our data indicate that the cysteine–histidine-rich (CH) and helicase domains of UPF1 are only essential for the early steps of NMD, whereas the heavily phosphorylated N- and C–terminal regions play a redundant but essential role in the target transcript degradation steps of NMD. We also show that both the N- and the C–terminal regions of SMG7 are essential for NMD. The N terminus contains a phosphoserine-binding domain that is required for the early steps of NMD, whereas the C terminus is required to trigger the degradation of NMD target transcripts. Moreover, SMG7 is a P–body component that can also remobilize UPF1 from the cytoplasm into processing bodies (P bodies). We propose that the N- and C–terminal phosphorylated regions of UPF1 recruit SMG7 to the functional NMD complex, and then SMG7 transports the PTC-containing transcripts into P bodies for degradation

Plant nonsense-mediated mRNA decay is controlled by different autoregulatory circuits and can be induced by an EJC-like complex

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control system that recognizes and degrades transcripts containing NMD cis elements in their 3'untranslated region (UTR). In yeasts, unusually long 3'UTRs act as NMD cis elements, whereas in vertebrates, NMD is induced by introns located >50 nt downstream from the stop codon. In vertebrates, splicing leads to deposition of exon junction complex (EJC) onto the mRNA, and then 3'UTR-bound EJCs trigger NMD. It is proposed that this intron-based NMD is vertebrate specific, and it evolved to eliminate the misproducts of alternative splicing. Here, we provide evidence that similar EJC-mediated intron-based NMD functions in plants, suggesting that this type of NMD is evolutionary conserved. We demonstrate that in plants, like in vertebrates, introns located >50 nt from the stop induces NMD. We show that orthologs of all core EJC components are essential for intron-based plant NMD and that plant Partner of Y14 and mago (PYM) also acts as EJC disassembly factor. Moreover, we found that complex autoregulatory circuits control the activity of plant NMD. We demonstrate that expression of suppressor with morphogenic effect on genitalia (SMG)7, which is essential for long 3'UTR- and intron-based NMD, is regulated by both types of NMD, whereas expression of Barentsz EJC component is downregulated by intron-based NMD

Inter-kingdom conservation of mechanism of nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is a quality control system that degrades mRNAs containing premature termination codons. Although NMD is well characterized in yeast and mammals, plant NMD is poorly understood. We have undertaken the functional dissection of NMD pathways in plants. Using an approach that allows rapid identification of plant NMD trans factors, we demonstrated that two plant NMD pathways coexist, one eliminates mRNAs with long 3′UTRs, whereas a distinct pathway degrades mRNAs harbouring 3′UTR-located introns. We showed that UPF1, UPF2 and SMG-7 are involved in both plant NMD pathways, whereas Mago and Y14 are required only for intron-based NMD. The molecular mechanism of long 3′UTR-based plant NMD resembled yeast NMD, whereas the intron-based NMD was similar to mammalian NMD, suggesting that both pathways are evolutionarily conserved. Interestingly, the SMG-7 NMD component is targeted by NMD, suggesting that plant NMD is autoregulated. We propose that a complex, autoregulated NMD mechanism operated in stem eukaryotes, and that despite aspect of the mechanism being simplified in different lineages, feedback regulation was retained in all kingdoms