Regulation of ribonuclease E activity by the L4 ribosomal protein of Escherichia coli

Abstract

Whereas ribosomal proteins (r-proteins) are known primarily as components of the translational machinery, certain of these r-proteins have been found to also have extraribosomal functions. Here we report the novel ability of an r-protein, L4, to regulate RNA degradation in Escherichia coli. We show by affinity purification, immunoprecipitation analysis, and E. coli two-hybrid screening that L4 interacts with a site outside of the catalytic domain of RNase E to regulate the endoribonucleolytic functions of the enzyme, thus inhibiting RNase E-specific cleavage in vitro, stabilizing mRNAs targeted by RNase E in vivo, and controlling plasmid DNA replication by stabilizing an antisense regulatory RNA normally attacked by RNase E. Broader effects of the L4-RNase E interaction on E. coli transcripts were shown by DNA microarray analysis, which revealed changes in the abundance of 65 mRNAs encoding the stress response proteins HslO, Lon, CstA, YjiY, and YaeL, as well as proteins involved in carbohydrate and amino acid metabolism and transport, transcription/translation, and DNA/ RNA synthesis. Analysis of mRNA stability showed that the half lives of stress-responsive transcripts were increased by ectopic expression of L4, which normally increases along with other r-proteins in E. coli under stress conditions, and also by inactivation of RNase E. Our finding that L4 can inhibit RNase E-dependent decay may account at least in part for the elevated production of stress-induced proteins during bacterial adaptation to adverse environments.posttranscriptional control ͉ RNA degradation ͉ stress responses ͉ degradosome O ver the past two decades, an understanding of mRNA decay pathways in Escherichia coli has advanced significantly (for reviews, see ref. 1-3), and RNase E has emerged as a key player in mRNA turnover as well as in the processing and decay of noncoding RNAs (e.g., rRNAs [4,5], tRNAs [6,7], M1 RNA [8], and 6S RNA [9]). RNase E is a multifunctional endoribonuclease (10) known to preferentially cleave RNA within AU-rich singlestranded regions (11, 12) enriched in specific sequence determinants (13). The level of this enzyme in vivo is controlled via autoregulation of its own synthesis (14-16).In addition to its N-terminal catalytic domain (N-RNase E), RNase E contains a C-terminal region (C-RNase E) that serves as a scaffold (17, 18) for association with polynucleotide phosphorylase (PNPase), RhlB RNA helicase, and the glycolytic enzyme enolase to form the RNA-degrading complex known as the ''degradosome' ' (19, 20). C-terminal truncation of RNase E, which prevents degradosome assembly, leads to accumulation of RNase E-targeted mRNAs (21, 22), suggesting that degradosome assembly and functional interactions of degradosome components are necessary for normal mRNA turnover in E. coli.Although ribosomal proteins (r-proteins) function primarily as components of the translation machinery, some prokaryotic and eukaryotic r-proteins also have extraribosomal functions (23). For example, L4, an essential r-protein encoded by...

The regulatory protein RraA modulates RNA-binding and helicase activities of the E. coli RNA degradosome

“…The association of RraA with the RNA-binding site in the CTD of RNase E interferes with its interactions with tRNA or a 27-mer single-stranded RNA. A similar masking effect has been reported for the L4 ribosomal protein on RNase E, which may occlude the RNA-binding site (Singh et al 2009). …”

Rapid Degradation of Host mRNAs by Stimulation of RNase E Activity by Srd of Bacteriophage T4

“…Furthermore, it would be also valuable to measure the kinetics of RNA cleavage by RNase E with or without Srd in vitro. To date, three E. coli factors to regulate RNase E activity, RraA, RraB, and ribosomal protein L4, have been identified, but all of them regulate RNase E activity negatively (Lee et al 2003;Gao et al 2006;Yeom et al 2008a,b;Singh et al 2009). Apart from these factors in E. coli, only one viral factor has been reported to regulate the degradation of mRNAs by RNase E. A protein kinase of bacteriophage T7 phosphorylates RNase E at its CTH and stabilizes a subset of RNase E substrates (Marchand et al 2001).…”

Unraveling new roles for minor components of the E. coli RNA degradosome

“…However, different stress conditions (e.g., amino acid or carbon starvation) can induce ribosome disassembly and the accumulation of significant amounts of free ribosomal proteins. Our recent finding 24 that RNase E activity towards a selected group of stress-responsive transcripts is inhibited by ribosome-free L4 may account at least in part for increased levels of stress-induced proteins (right) important for bacterial resistance to several environmental stresses.…”

“…24 As the release of free L4 along with other ribosomal proteins is known to occur during ribosome disassembly under starvation for amino acids (stringent response) or carbon sources, 25 the L4-mediated inhibition of RNase E activity and resulting accumulation of stress-related proteins seem to be critical for bacterial adaptation to different stresses. The putative model depicting this regulatory mechanism is presented in Figure 2.…”

“…2). 24 Unlike the mode of L4 action, two recently discovered global regulators, RraA and RraB (middle) can bind to the C-terminal domain and exert their inhibitory functions via remodeling the structure of the degradosome complex, thereby affecting a larger group of E. coli transcripts. 17,18 In contrast to L4, RraA and RraB, which have an inhibitory effect on RNase E, the RNA chaperone Hfq can enhance the efficiency of RNase E cleavage through interaction with the C-terminal domain of this endoribonuclease.…”