Academic journal article Genetics

A Novel Endoribonuclease, RNase LS, in Escherichia Coli

Academic journal article Genetics

A Novel Endoribonuclease, RNase LS, in Escherichia Coli

Article excerpt

ABSTRACT

The dmd gene of bacteriophage T4 is required for the stability of late-gene mRNAs. When this gene is mutated, late genes are globally silenced because of rapid degradation of their mRNAs. Our previous work suggested that a novel Escherichia coli endonuclease, RNase LS, is responsible for the rapid degradation of mRNAs. In this study, we demonstrated that rnlA (formerly yfjN) is essential for RNase LS activity both in vivo and in vitro. In addition, we investigated a role of RNase LS in the RNA metabolism of E. coli cells under vegetative growth conditions. A mutation in rnlA reduced the decay rate of many E. coli mRNAs, although there are differences in the mutational effects on the stabilization of different mRNAs. In addition, we found that a 307-nucleotide fragment with an internal sequence of 23S rRNA accumulated to a high level in rnlA mutant cells. These results strongly suggest that RNase LS plays a role in the RNA metabolism of E. coli as well as phage T4.

THE control of mRNA stability is an important aspect of gene expression. mRNA degradation has been studied extensively in both prokaryotes and eukaryotes, and such studies have revealed that cis-acting RNA elements and trans-acting proteins influence mRNA stability (SCHOENBERG and CHERNOKALSKAYA 1997; VAN HOOF and GREEN 1997; COBURN and MACKIE 1999; SCHWARTZ and PARKER 2000). The most important factor among trans-acting proteins is an RNase that plays a central role in mRNA degradation. In Escherichia coli, the degradation of mRNA is usually initiated by endonucleolytic cleavage (APIRION 1973; COBURN and MACKIE 1999; KUSHNER 2002). Under vegetative growth conditions, five endonucleases (RNases I*, III, E, G, P) are known or suggested to cleave mRNA (PORTIER et al. 1987; ARRAIANO et al 1988; BARDWELL et al 1989; CANNISTRARO and KENNELL 1991; MACKIE 1991; ALIFANO et al. 1994; UMITSUKI et al. 2001). Furthermore, two recently discovered endonucleases function under certain conditions. ReIE cleaves mRNA positioned at the ribosomal A site when bacteria are starved for amino acids (PEDERSEN et al 2003). MazF encoded by the mazEF addiction module (AIZENMAN et al. 1996) cleaves mRNAs when cells undergo programmed cell death (ZHANG et al. 2003).

We have been investigating an mRNA cleavage activity that induces late gene silencing in bacteriophage T4 (KAI et al. 1996). The T4 dmd gene is required for the regulation of mRNA stability in a stage-dependent manner during infection (UENO and YONESAKI 2001). When a T4 mutant defective in this gene infects E. coli cells at low temperatures, gene expression is globally silenced at late stages because of the rapid degradation of mRNA. The rapid degradation is caused by dmd mutant-specific cleavages of mRNA (KAI et al. 1996; KAI and YONESAKI 2002). Recently, we suggested that the host encodes an activity for performing such cleavage and we isolated E. coli std mutants that are defective in dmd mutant-specific cleavages. The loci of the std mutations as well as the effects of mutations in known RNase-encoding genes suggested that an RNA cleavage activity causing the dmd mutant-specific mRNA degradation could be attributed to a novel RNase (UTSUKA et al. 2003). Hence, we propose calling this endonuclease RNase LS (late-gene silencing in T4).

RNase LS preferentially cleaves RNA 3' to pyrimidines, but exceptions are observed. Some cleavages by RNase LS are introduced when the target RNA is translatable, while others are independent of translation (KAI et al. 1996; KAI and YONESAKI 2002). Thus, RNase LS seems to cleave RNA in a complex manner. The structural gene for RNase LS as well as its role in E. coli cells have not been described. The E. coli std-2 mutation abolishes RNase LS activity and allows T4 dmd to grow as well as wild-type phage (UTSUKA et al. 2003). As an initial step to characterizing the RNase LS, we sought to identify the gene harboring the std-2 mutation and found that yfjN is the gene. …

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