To identify regulators of AU-rich element (ARE)-dependent mRNA turnover we have followed a genetic approach using a mutagenized cell line (slowC) that fails to degrade cytokine mRNA. Accordingly, a GFP reporter construct whose mRNA is under control of the ARE from interleukin-3 gives an increased fluorescence signal in slowC. Here we describe rescue of slowC by a retroviral cDNA library. Flow cytometry allowed us to isolate revertants with reconstituted rapid mRNA decay. The cDNA was identified as butyrate response factor-1 (BRF1), encoding a zinc finger protein homologous to tristetraprolin. Mutant slowC carries frame-shift mutations in both BRF1 alleles, whereas slowB with intermediate decay kinetics is heterozygous. By use of small interfering (si)RNA, independent evidence for an active role of BRF1 in mRNA degradation was obtained. In transiently transfected NIH 3T3 cells, BRF1 accelerated mRNA decay and antagonized the stabilizing effect of PI3-kinase, while mutation of the zinc fingers abolished both function and ARE-binding activity. This approach, which identified BRF1 as an essential regulator of ARE-dependent mRNA decay, should also be applicable to other cis-elements of mRNA turnover.

Butyrate response factor (BRF1) belongs to the Tis11 family of CCCH zinc-finger proteins, which bind to mRNAs containing an AU-rich element (ARE) in their 3 0 untranslated region and promote their deadenylation and rapid degradation. Independent signal transduction pathways have been reported to stabilize ARE-containing transcripts by a process thought to involve phosphorylation of ARE-binding proteins. Here we report that protein kinase B (PKB/Akt) stabilizes ARE transcripts by phosphorylating BRF1 at serine 92 (S92). Recombinant BRF1 promoted in vitro decay of ARE-containing mRNA (ARE-mRNA), yet phosphorylation by PKB impaired this activity. S92 phosphorylation of BRF1 did not impair ARE binding, but induced complex formation with the scaffold protein 14-3-3. In vivo and in vitro data support a model where PKB causes ARE-mRNA stabilization by inactivating BRF1 through binding to 14-3-3.

The underlying mechanisms are thought to be highly conserved in evolution (2, 4). RNAi in animals is initiated by doublestranded RNAs (dsRNAs) similar in sequence to the transcribed region of target genes. These dsRNAs undergo endonucleolytic cleavage to generate 21-to 23-nt-long small interfering RNAs (siRNAs), which then promote RNA degradation (5-7) Remarkably, the silent state in transgenic plants and in C. elegans can spread from cell to cell and even systemically throughout the organism, implying the existence of mobile silencing signals (2, 8). Little is known about the chemical nature of these signals, but it seems likely that the sequence-specific component is an RNA (8-11). The finding that siRNA and dsRNA accumulate in silent tissues, together with studies of informative stable transformants and PTGS induced by RNA viruses, supports the view that dsRNAs and siRNAs have key roles in plant PTGS (12-14). Nevertheless, direct evidence that these or other RNAs can induce systemic PTGS or comprise silencing signals in plants is lacking.In the present study, we used a positive marker system and real-time monitoring of green fluorescent protein (GFP) expression to show that double-stranded siRNAs, large sense, antisense, and double-stranded RNAs delivered biolistically into plant cells trigger PTGS capable of spreading locally and systemically. The introduced siRNAs trigger the production of siRNAs derived from sequences both 3Ј and 5Ј of the inducing siRNAs. Our findings support the hypothesis that siRNAs themselves or intermediates induced by siRNAs could comprise silencing signals and are generated in a self-amplifying fashion. Materials and Methods

Summary Sense and antisense tobacco chitinase (CHN) transgenes, Luciferase‐CHN transcriptional fusions, and promoterless CHN cDNAs were introduced biolistically into CHN transformants of tobacco that never exhibit spontaneous gene silencing. All of the constructs tested induced systemic silencing of the resident CHN transgene and endogenes. Nuclear run‐on transcription assays showed that local introduction of additional gene copies triggers systemic post‐transcriptional gene silencing (PTGS). Together, this provides evidence that additional transgene copies need not be either highly transcribed or produce sense transcripts to evoke production of systemic PTGS signals. CHN PTGS was transmitted by top grafting, but not by reciprocal grafting of mature stems or the exchange of tissue plugs. Thus, the commonly encountered difficulties in achieving graft‐transmission could reflect the method used. Silencing in sense but not antisense transformants was transmitted by grafting to a high‐expressing sense CHN scion suggesting that the elaboration of mobile signals may not be an essential feature of antisense‐mediated gene silencing.