In plants, RNA silencing (RNA interference) is an efficient antiviral system, and therefore successful virus infection requires suppression of silencing. Although many viral silencing suppressors have been identified, the molecular basis of silencing suppression is poorly understood. It is proposed that various suppressors inhibit RNA silencing by targeting different steps. However, as double-stranded RNAs (dsRNAs) play key roles in silencing, it was speculated that dsRNA binding might be a general silencing suppression strategy. Indeed, it was shown that the related aureusvirus P14 and tombusvirus P19 suppressors are dsRNA-binding proteins. Interestingly, P14 is a size-independent dsRNA-binding protein, while P19 binds only 21-nucleotide ds-sRNAs (small dsRNAs having 2-nucleotide 3 overhangs), the specificity determinant of the silencing system. Much evidence supports the idea that P19 inhibits silencing by sequestering silencing-generated viral ds-sRNAs. In this study we wanted to test the hypothesis that dsRNA binding is a general silencing suppression strategy. Here we show that many plant viral silencing suppressors bind dsRNAs. Beet yellows virus Peanut P21, clump virus P15, Barley stripe mosaic virus ␥B, and Tobacco etch virus HC-Pro, like P19, bind ds-sRNAs size-selectively, while Turnip crinkle virus CP is a size-independent dsRNA-binding protein, which binds long dsRNAs as well as ds-sRNAs. We propose that size-selective ds-sRNA-binding suppressors inhibit silencing by sequestering viral ds-sRNAs, whereas size-independent dsRNA-binding suppressors inactivate silencing by sequestering long dsRNA precursors of viral sRNAs and/or by binding ds-sRNAs. The findings that many unrelated silencing suppressors bind dsRNA suggest that dsRNA binding is a general silencing suppression strategy which has evolved independently many times.RNA silencing (termed RNA interference [RNAi] in animals) is an RNA-based eukaryotic gene regulatory system that plays essential roles in many biological processes. RNA silencing is induced by accumulation of double-stranded RNAs (dsRNAs). dsRNAs are first processed by an RNase III-like nuclease called DICER (in plants it is termed DICER-LIKE [DCL]) into (21-to 25-nucleotide [nt]) small dsRNAs (ds-sRNAs) having 2-nt 3Ј overhangs, and then these sRNAs incorporate into different silencing effector complexes. In the active effector complexes sRNAs are present as single-stranded molecules, which guide these complexes to the complementary nucleic acids for suppression (2, 3, 22, 61).In plants, different dsRNA precursors are processed by distinct DCLs into functionally different short (21-to 22-nt) and long (23-to 25-nt) sRNAs (24, 25, 67). Short sRNAs guide a multicomponent nuclease (RNA-induced silencing complex [RISC]) to homologous mRNAs for suppression. RISC cleaves targeted mRNA in the case of (near) perfect base pairing between mRNA and guide RNA. When the guide RNA is only partially complementary to the mRNA, RISC mediates translational repression. Short sRNAs could also provide ...

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.

Cytochrome c heme lyases encoded by the Sinorhizobium meliloti cycHJKL operon are responsible for generating the covalent bond between the heme prosthetic group and apocytochromes c. The CycH protein with its presumably membrane-associated N-terminal and periplasmic C-terminal parts is thought to be responsible for binding apocytochrome and presenting it to the heme ligation machinery. We propose that these two modules of CycH play roles in different functions of the protein. The N-terminal 96 amino acids represent an active subdomain of the protein, which is able to complement the protoporphyrin IX (PPIX) accumulation phenotype of the cycH mutant strain AT342, suggesting that it is involved in the final steps of heme C biosynthesis. Furthermore, three tetratricopeptide (TPR) domains have been identified in the C-terminal periplasmic region of the CycH protein. TPR domains are known to mediate protein-protein interactions. Each of these CycH domains is absolutely required for protein function, since plasmid constructs carrying cycH genes with in-frame TPR deletions were not able to complement cycH mutants for their nitrate reductase (Rnr-) and nitrogen-fixing (Fix-) phenotypes. We also found that the 309-amino acid N-terminal portion of the CycH, which includes all the TPR domains, is able to mediate the assembly of the c-type cytochromes required for the Rnr+ phenotype. In contrast, only the full-length protein confers the ability to fix nitrogen.