Intragenic suppressors of temperature-sensitive rne mutations lead to the dissociation of RNase E activity on mRNA and tRNA substrates in Escherichia coli

RNase E of Escherichia coli is an essential endoribonuclease that is involved in many aspects of RNA metabolism. Point mutations in the S1 RNA-binding domain of RNase E (rne-1 and rne-3071) lead to temperature-sensitive growth along with defects in 5S rRNA processing, mRNA decay and tRNA maturation. However, it is not clear whether RNase E acts similarly on all kinds of RNA substrates. Here we report the isolation and characterization of three independent intragenic second-site suppressors of the rne-1 and rne-3071 alleles that demonstrate for the first time the dissociation of the in vivo activity of RNase E on mRNA versus tRNA and rRNA substrates. Specifically, tRNA maturation and 9S rRNA processing were restored to wild-type levels in each of the three suppressor mutants (rne-1/172, rne-1/186 and rne-1/187), while mRNA decay and autoregulation of RNase E protein levels remained as defective as in the rne-1 single mutant. Each single amino acid substitution (Gly→Ala at amino acid 172; Phe → Cys at amino acid 186 and Arg → Leu at amino acid 187) mapped within the 5′ sensor region of the RNase E protein. Molecular models of RNase E suggest how suppression may occur

The role of nuclear factor-κB essential modulator (NEMO) in B cell development and survival

The transcription factor nuclear factor-κB (NF-κB) is essential for immune and inflammatory responses. NF-κB essential modulator (NEMO) is a scaffolding component of the IκB kinase complex required for NF-κB activation in vitro. Because NF-κB activation is involved in B cell development and function, we set out to determine whether NEMO is required for these processes. NEMO(−/−) mice die very early during embryogenesis, and fetal livers from NEMO(−/−) embryos can not reconstitute either B or T lymphopoiesis in irradiated host mice. We therefore used NEMO(−/−) embryonic stem cells and the OP9 in vitro differentiation system to demonstrate that NEMO is not required for B cell development but plays an important role in B cell survival