Highlights d Reduced EJC abundance results in aberrant splicing using cryptic splice sites d PSAP/EJC-associated RNPS1 suppresses cryptic and reconstituted 5 0 splice sites d RNA-bound EJC core factors directly mask cryptic 3 0 splice sites d RNPS1 and the EJC protect spliced exons by preventing resplicing

β-Carotene (BC) is the most abundant carotenoid in human diet, almost solely as (all-E)-isomer. Significant amounts of (Z)-isomers of BC are present in processed food as well as in mammalian tissues. Differences are described for the activity of various BC isomers in forming retinal and protecting against cancer and cardiovascular diseases. Eccentric cleavage of BC leads to degradation products such as carotenals. A variety of negative consequences were published for the non-vitamin A active BC metabolites, such as inducing the carcinogenesis of benzo [a]pyrene, impairing mitochondrial function, or increasing CYP activity. To increase the knowledge on the antioxidant activity, a variety of BC isomers and metabolites were tested in various in vitro assays.In the present study, no ferric reducing activity (FRAP assay) was observed for the BC isomers. Between the major BC isomers (all-E, 9Z, and 13Z) no significant differences in bleaching the ABTS•+ (αTEAC assay) or in scavenging peroxyl radicals (ROO • ) generated by thermal degradation of AAPH (using a chemiluminescence assay) were detected. However, the (15Z)-isomer was less active, maybe due to its low stability. The degradation to β-apo-carotenoids increased FRAP activity and ROO • scavenging activity compared to the parent molecule. Dependence on chain length and character of the terminal function was determined in αTEAC assay with following order of increasing activity: β-apo-8'-carotenal < β-apo-8'-carotenoic acid ethyl ester < 6'-methyl-β-apo-6'-carotene-6'-one (citranaxanthin). The results indicate that BC does not lose its antioxidant activity by degradation to long chain breakdown products. OPEN ACCESSMolecules 2011, 16 1056

Eukaryotic gene expression is constantly controlled by the translation-coupled nonsense-mediated mRNA decay (NMD) pathway. Aberrant translation termination leads to NMD activation, resulting in phosphorylation of the central NMD factor UPF1 and robust clearance of NMD targets via two seemingly independent and redundant mRNA degradation branches. Here, we uncover that the loss of the first SMG5-SMG7-dependent pathway also inactivates the second SMG6-dependent branch, indicating an unexpected functional connection between the final NMD steps. Transcriptome-wide analyses of SMG5-SMG7-depleted cells confirm exhaustive NMD inhibition resulting in massive transcriptomic alterations. Intriguingly, we find that the functionally underestimated SMG5 can substitute the role of SMG7 and individually activate NMD. Furthermore, the presence of either SMG5 or SMG7 is sufficient to support SMG6-mediated endonucleolysis of NMD targets. Our data support an improved model for NMD execution that features two-factor authentication involving UPF1 phosphorylation and SMG5-SMG7 recruitment to access SMG6 activity.

The exon junction complex (EJC) is a key regulator of posttranscriptional mRNA fate and binds to mRNA during splicing. Although the composition of EJCs is well understood, the mechanism mediating splicing-dependent EJC assembly and the factor(s) recruiting the EJC remain elusive. Here, we identify CWC22 as an essential splicing factor that is required for EJC assembly. In CWC22-depleted cells, pre-mRNA splicing is impaired but is rescued by a central fragment of CWC22. We show that the MIF4G domain of CWC22 initiates EJC assembly via a direct interaction with the EJC core protein eIF4A3, and we characterize mutations in eIF4A3 that abolish binding to CWC22. These eIF4A3 mutants efficiently nucleate splicing-independent recombinant EJC core complexes, but they fail to support splicing-dependent EJC deposition. Our work establishes a direct link between the splicing machinery and the EJC, hence uncovering a molecular interaction at the center of a posttranscriptional gene regulation network.

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