Escherichia coli endoribonuclease E has a major influence on gene expression. It is essential for the maturation of ribosomal and transfer RNA as well as the rapid degradation of messenger RNA. The latter ensures that translation closely follows programming at the level of transcription. Recently, one of the hallmarks of RNase E, i.e. its ability to bind via a 5′-monophosphorylated end, was shown to be unnecessary for the initial cleavage of some polycistronic tRNA precursors. Here we show using RNA-seq analyses of ribonuclease-deficient strains in vivo and a 5′-sensor mutant of RNase E in vitro that, contrary to current models, 5′-monophosphate-independent, ‘direct entry’ cleavage is a major pathway for degrading and processing RNA. Moreover, we present further evidence that direct entry is facilitated by RNase E binding simultaneously to multiple unpaired regions. These simple requirements may maximize the rate of degradation and processing by permitting multiple sites to be surveyed directly without being constrained by 5′-end tethering. Cleavage was detected at a multitude of sites previously undescribed for RNase E, including ones that regulate the activity and specificity of ribosomes. A potentially broad role for RNase G, an RNase E paralogue, in the trimming of 5′-monophosphorylated ends was also revealed.

The best characterized pathway for the initiation of mRNA degradation in Escherichia coli involves the removal of the 5′-terminal pyrophosphate to generate a monophosphate group that stimulates endonucleolytic cleavage by RNase E. We show here however, using well-characterized oligonucleotide substrates and mRNA transcripts, that RNase E can cleave certain RNAs rapidly without requiring a 5′-monophosphorylated end. Moreover, the minimum substrate requirement for this mode of cleavage, which can be categorized as ‘direct’ or ‘internal’ entry, appears to be multiple single-stranded segments in a conformational context that allows their simultaneous interaction with RNase E. While previous work has alluded to the existence of a 5′ end-independent mechanism of mRNA degradation, the relative simplicity of the requirements identified here for direct entry suggests that it could represent a major means by which mRNA degradation is initiated in E. coli and other organisms that contain homologues of RNase E. Our results have implications for the interplay of translation and mRNA degradation and models of gene regulation by small non-coding RNAs.

The RNase E family is renowned for being central to the processing and decay of all types of RNA in many species of bacteria, as well as providing the first examples of endonucleases that can recognize 5′-monophosphorylated ends thereby increasing the efficiency of cleavage. However, there is increasing evidence that some transcripts can be cleaved efficiently by Escherichia coli RNase E via direct entry, i.e. in the absence of the recognition of a 5′-monophosphorylated end. Here, we provide biochemical evidence that direct entry is central to the processing of transfer RNA (tRNA) in E. coli, one of the core functions of RNase E, and show that it is mediated by specific unpaired regions that are adjacent, but not contiguous to segments cleaved by RNase E. In addition, we find that direct entry at a site on the 5′ side of a tRNA precursor triggers a series of 5′-monophosphate-dependent cleavages. Consistent with a major role for direct entry in tRNA processing, we provide additional evidence that a 5′-monophosphate is not required to activate the catalysis step in cleavage. Other examples of tRNA precursors processed via direct entry are also provided. Thus, it appears increasingly that direct entry by RNase E has a major role in bacterial RNA metabolism.