Decay of the IS 10 antisense RNA by 3′ exoribonucleases: evidence that RNase II stabilizes RNA‐OUT against PNPase attack

Abstract

RNA-OUT, the 69-nucleotide antisense RNA that regulates Tn10/IS10 transposition folds into a simple stem-loop structure. The unusually high metabolic stability of RNA-OUT is dependent, in part, on the integrity of its stem-domain: mutations that disrupt stem-domain structure (Class II mutations) render RNA-OUT unstable, and restoration of structure restores stability. Indeed, there is a strong correlation between the thermodynamic and metabolic stabilities of RNA-OUT. We show here that stem-domain integrity determines RNA-OUT's resistance to 3' exoribonucleolytic attack: Class II mutations are almost completely suppressed in Escherichia coli cells lacking its principal 3' exoribonucleases, ribonuclease II (RNase II) and polynucleotide phosphorylase (PNPase). RNase II and PNPase are individually able to degrade various RNA-OUT species, albeit with different efficiencies: RNA-OUT secondary structure provides greater resistance to RNase II than to PNPase. Surprisingly, RNA-OUT is threefold more stable in wild-type cells than in cells deficient for RNase II activity, suggesting that RNase II somehow lessens PNPase attack on RNA-OUT. We discuss how this might occur. We also show that wild-type RNA-OUT stability changes only two-fold across the normal range of physiological growth temperatures (30-44 degrees C) in wild-type cells, which has important implications for IS10 biology.

Action of RNase II and Polynucleotide Phosphorylase against RNAs Containing Stem-Loops of Defined Structure

“…To a large extent, the determinants of these enzymes' abilities to attack different substrates are quite similar. Neither enzyme is able to attack RNA stem-loops containing a 3Ј-terminal extension of six residues, in agreement with previous data in vitro (6) and in vivo (3,28). However, comparably stable RNAs in the SLxA or SLxR class are readily shortened by both enzymes.…”

“…This finding has been interpreted to indicate that these exonucleases are functionally redundant but collectively essential. Other data, however, suggest that RNase II and PNPase are not functionally equivalent but are differentially sensitive to RNA secondary structure (3,7,8,16,19,24,28). In such cases, PNPase is required for the degradation of highly structured RNAs and RNase II cannot substitute (7,8,19).…”

“…In such cases, PNPase is required for the degradation of highly structured RNAs and RNase II cannot substitute (7,8,19). Moreover, RNase II, but not PNPase, may actually stabilize some RNAs (3,28). In addition, while RNase II behaves as a soluble monomeric enzyme (31), PNPase can be assembled into a multienzyme complex, the degradosome (5, 27, 29).…”

ISAfe1, an ISL3 Family Insertion Sequence fromAcidithiobacillus ferrooxidansATCC 19859

“…The function of ORF 2 remains unknown (26). Several other insertion sequences, such as IS30 (2), IS10 (8,9,33), and pot2 (25), have been shown to encode antisense RNA on the opposite strand to the TPase. The antisense RNA of IS30 contains an ORF which has been shown not to be translated at detectable levels (2).…”

Polyadenylation of mRNA in bacteria

“…It must be noted that the effects of pcnB deletions described in the preceding paragraph were observed only in r e mutants and not in a wild-type background and that mRNA destabilization by polyadenylation was much less pronounced (O'Hara e t al., 1995) or absent (Hajnsdorf e t al., 1995) in a wild-type background. Moreover, the observation that the absence of RNase I1 can stabilize transcripts (Hajnsdorf e t al., 1994;Pepe et al, 1994;Py e t al., 1996), the discovery of specific complexes involving RNase E and PNPase (Carpousis et al, 1994;Py et al, 1994), as well as the fact that mRNA degradation occurs efficiently even in the absence of RNase E, PNPase and RNase I1 (Hajnsdorf et al, 1995;O'Hara et al, 1995) suggests that additional factors must be taken into consideration before the roles of PAPs and ribonucleases in mRNA degradation can be fully understood.…”