The human transcriptome is enriched for miRNA-binding sites located in cooperativity-permitting distance.

MiRNAs are short, non-coding RNAs that regulate gene expression post-transcriptionally through specific binding to mRNA. Deregulation of miRNAs is associated with various diseases and interference with miRNA function has proven therapeutic potential. Most mRNAs are thought to be regulated by multiple miRNAs and there is some evidence that such joint activity is enhanced if a short distance between sites allows for cooperative binding. Until now, however, the concept of cooperativity among miRNAs has not been addressed in a transcriptome-wide approach. Here, we computationally screened human mRNAs for distances between miRNA binding sites that are expected to promote cooperativity. We find that sites with a maximal spacing of 26 nucleotides are enriched for naturally occurring miRNAs compared with control sequences. Furthermore, miRNAs with similar characteristics as indicated by either co-expression within a specific tissue or co-regulation in a disease context are predicted to target a higher number of mRNAs cooperatively than unrelated miRNAs. These bioinformatic data were compared with genome-wide sets of biochemically validated miRNA targets derived by Argonaute crosslinking and immunoprecipitation (HITS-CLIP and PAR-CLIP). To ease further research into combined and cooperative miRNA function, we developed miRco, a database connecting miRNAs and respective targets involved in distance-defined cooperative regulation (mips.helmholtz-muenchen.de/mirco). In conclusion, our findings suggest that cooperativity of miRNA-target interaction is a widespread phenomenon that may play an important role in miRNA-mediated gene regulation.

Nitrosative stress treatment of E. coli targets distinct set of thiol-containing proteins.

Reactive nitrogen species (RNS) function as powerful antimicrobials in host defence, but so far little is known about their bacterial targets. In this study, we set out to identify Escherichia coli proteins with RNS-sensitive cysteines. We found that only a very select set of proteins contain cysteines that undergo reversible thiol modifications upon nitric oxide (NO) treatment in vivo. Of the 10 proteins that we identified, six (AtpA, AceF, FabB, GapA, IlvC, TufA) have been shown to harbour functionally important thiol groups and are encoded by genes that are considered essential under our growth conditions. Media supplementation studies suggested that inactivation of AceF and IlvC is, in part, responsible for the observed NO-induced growth inhibition, indicating that RNS-mediated modifications play important physiological roles. Interestingly, the majority of RNS-sensitive E. coli proteins differ from E. coli proteins that harbour H2O2-sensitive thiol groups, implying that reactive oxygen and nitrogen species affect distinct physiological processes in bacteria. We confirmed this specificity by analysing the activity of one of our target proteins, the small subunit of glutamate synthase. In vivo and in vitro activity studies confirmed that glutamate synthase rapidly inactivates upon NO treatment but is resistant towards other oxidative stressors.