Cytoplasmic mRNA degradation controls gene expression to help eliminate pathogens during infection. However, it has remained unclear whether such regulation also extends to nuclear RNA decay. Here, we show that 145 unstable nuclear RNAs, including enhancer RNAs (eRNAs) and long noncoding RNAs (lncRNAs) such as NEAT1v2, are stabilized upon infection in HeLa cells. In uninfected cells, the RNA exosome, aided by the Nuclear EXosome Targeting (NEXT) complex, degrades these labile transcripts. Upon infection, the levels of the exosome/NEXT components, RRP6 and MTR4, dramatically decrease, resulting in transcript stabilization. Depletion of lncRNAs, NEAT1v2, or eRNA07573 in HeLa cells triggers increased susceptibility to infection concomitant with the deregulated expression of a distinct class of immunity-related genes, indicating that the accumulation of unstable nuclear RNAs contributes to antibacterial defense. Our results highlight a fundamental role for regulated degradation of nuclear RNA in the response to pathogenic infection.

The role of the alpha subunit of Escherichia coli RNA polymerase in transcription activation by the OxyR protein was investigated using in-vitro-reconstituted RNA polymerase containing alpha subunits carrying C-terminal truncations or an amino acid substitution. Mutant RNA polymerases failed to respond to transcription activation of the E. coli OxyR-dependent promoters. DNase I footprinting analysis indicates that the OxyR protein exerts a co-operative effect on the binding of wild-type RNA polymerase, but not the mutant RNA polymerases, to the katG promoter. Together, these results suggest that direct protein-protein contact between the OxyR protein and the C-terminal contact site I region of the RNA polymerase alpha subunit plays an essential role in transcription activation at the OxyR-dependent promoters.

The Escherichia coli OxyR protein requires the C-terminal contact site I region of the RNA polymerase ␣ subunit for cooperative interaction with and transcription activation at OxyR-dependent promoters, suggesting direct protein-protein contact between OxyR and the C-terminal region of the ␣ subunit. To determine the precise location of the OxyR protein contact site(s) in this region, we carried out mutational analysis of the 3 half of E. coli rpoA, the gene encoding the ␣ subunit of RNA polymerase. We isolated a number of rpoA mutants defective in oxyR-dependent transcription activation at the E. coli katG promoter. Nucleotide sequence analysis of the rpoA gene from these mutants revealed that the mutations showing clear phenotypes are all clustered at two narrow regions (amino acid residues 265 to 269 and 293 to 300) within the C terminus of the ␣ subunit. Reconstituted RNA polymerases containing the mutant ␣ subunits were unable to respond to transcription activation in vitro at the katG, ahpC, and oxyX promoters by OxyR. These results suggest that these two regions comprise the contact surfaces on the ␣ subunit for OxyR.Several lines of evidence indicate that transcription activation by DNA-binding transcription factors involves a direct protein-protein contact between those factors and RNA polymerase (1, 13). With RNA polymerases containing mutant ␣ subunits with C-terminal truncation, we have demonstrated that the C-terminal region of ␣ is involved in direct molecular communication with a group of transcription activator proteins (reviewed in reference 6). Together with other genetic, immunochemical, and biochemical evidence (15), it is proposed that the C-terminal one-third of the RNA polymerase ␣ subunit (contact site I) makes direct contact with a group of transcription activator proteins (class I activator proteins), most of which bind upstream of basic promoter elements (5, 6). Besides the role in class I activator protein-dependent transcription, recent studies indicate that the C-terminal region of the ␣ subunit is also involved in protein activator-independent transcription stimulation at several strong promoters, each containing an AT-rich sequence upstream of the Ϫ35 sequence (UP element) (14).Recently, we showed that OxyR protein, an activator protein for hydrogen peroxide-inducible genes, activates transcription initiation in vitro from the katG, ahpC, and oxyX promoters by wild-type RNA polymerase but not by mutant RNA polymerases containing a C-terminal-truncated ␣ subunit (19). Furthermore, we demonstrated that OxyR exerts cooperative binding to promoter DNA with wild-type RNA polymerase but not with the mutant RNA polymerases. These observations suggest that direct protein-protein contact between the OxyR protein and the C-terminal region of the ␣ subunit plays an essential role in transcription activation at OxyR-dependent promoters.To carry out detailed mapping of the contact site with the OxyR protein in the C-terminal contact site I region of the ␣ subunit, we isolated a number of rpoA ...

Treatment of Escherichia coli and Salmonella typhimurium cells with a low dose of hydrogen peroxide induces expression of a large number of genes, and confers resistance to oxidative stresses. The oxyR gene encodes a positive regulatory protein for a subset of these genes involved in the defense against oxidative damage. We cloned a DNA fragment that contains the E. coli oxyR region on a plasmid vector, and analyzed the nucleotide sequence of the gene. The amino acid sequence of OxyR protein, deduced from the nucleotide sequence, shows a high degree of homology to the sequences of a number of bacterial activator proteins including LysR, CysB, IlvY, MetR and NodD. The product of the oxyR gene identified by the maxicell procedure was a 34 kDa protein, which agrees with the size predicted from the nucleotide sequence of the gene.