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.

BRF1 posttranscriptionally regulates mRNA levels by targeting ARE-bearing transcripts to the decay machinery. We previously showed that protein kinase B (PKB) phosphorylates BRF1 at Ser92, resulting in binding to 14-3-3 and impairment of mRNA decay activity. Here we identify an additional regulatory site at Ser203 that cooperates in vivo with Ser92. In vitro kinase labeling and wortmannin sensitivity indicate that Ser203 phosphorylation is also performed by PKB. Mutation of both serines to alanine uncouples BRF1 from PKB regulation, leading to constitutive mRNA decay even in the presence of stabilizing signals. BRF1 protein is labile because of proteasomal degradation (half-life, <3 h) but becomes stabilized upon phosphorylation and is less stable in PKB␣ ؊/؊ cells. Surprisingly, phosphorylation-dependent protein stability is also regulated by Ser92 and Ser203, with parallel phosphorylation required at these sites. Phosphorylation-dependent binding to 14-3-3 is abolished only when both sites are mutated. Cell compartment fractionation experiments support a model in which binding to 14-3-3 sequesters BRF1 through relocalization and prevents it from executing its mRNA decay activity, as well as from proteasomal degradation, thereby maintaining high BRF1 protein levels that are required to reinstate decay upon dissipation of the stabilizing signal.

Aim: As a biosafety laboratory, we take samples from surfaces in microbiological laboratories to survey the handling of micro-organisms. Whereas contaminations with other micro-organisms were rare, Staphylococcus aureus was found in the working environment of many laboratories. As 20-60% of the healthy population are carriers of S. aureus we wanted to asses the effect of carriers on our sampling results. Methods and Results: Nasal swabs of staff members in nonmicrobiological laboratories and offices as well as surface samples from their personal work environment were taken and analysed for S. aureus DNA. In addition S. aureus strains were isolated using S. aureus-specific agar plates and analysed by randomly amplified polymorphic DNA (RAPD)-PCR and multilocus sequence typing (MLST). Our data show that contaminations with S. aureus in nonmicrobiological environments are common with 29% of the surface samples containing S. aureus DNA. In the working environment of carriers, the number of contaminations was significantly increased compared to the environment of noncarriers. Conclusion: The carrier status of staff members significantly affects the number of contaminations on laboratory surfaces. Therefore, even in the absence of intentional handling of S. aureus, contaminations can be detected on a substantial amount of surfaces. Significance and Impact of the Study: Sampling procedures need to be adapted based on these results with respect to the locations where samples are taken and the threshold for significant contaminations. Because of its wide distribution, S. aureus can serve as a marker for hygienic standards in laboratories.

Aims:  As a biosafety laboratory, we survey the handling of adenovirus type 5 (Ad5) and HIV1‐derived lentivirus in contained‐use facilities in Switzerland to identify insufficiencies of the safety precautions taken by the laboratories. Methods and Results:  In the past 9 years, we took 430 swab samples from various types of surfaces in research laboratories. Samples were examined for Ad5 contaminations by real‐time PCR and infectivity assay or for the presence of lentivirus (HIV1) nucleic acids by real‐time (RT) PCR. Samples collected from centrifuges did not only contain Ad5 DNA more frequently but also exhibited higher numbers of Ad5 and lentiviral (HIV1) DNA copies than swabs from any other area of sampling. Five of ten samples containing infectious Ad5 particles or lentivirus (HIV1) RNA were found in samples taken from centrifuges. Ad5 contamination rates were higher in the tube holder and lower on the inner wall of the rotor chamber in centrifuges that were fitted with aerosol tight covers compared to centrifuges without covers. Conclusions:  Our results allowed the comparison of hygiene standards of different laboratories and lead to the identification of centrifuges as hotspots for contaminations. Significance and Impact of the Study:  Based on our results, we propose to use the collected data as a tool for rating future swab results. Furthermore, the amount of Ad5 and HIV1‐derived lentivirus DNA could serve as an indicator of the level of good laboratory practice in contained‐use laboratories handling these viral vectors.