The analysis of rpsO
mRNA half-life in 3′ exonuclease mutants (Table and Fig. ) indicated that 3′-to-5′ exoribonucleases have a minor role in determining this mRNA half-life. We observed slight but not statistically significant increases in the rpsO
mRNA half-life in strains containing only PNPase or only RNase PH and <2-fold increases in the rpsO
mRNA half-life in strains containing only YhaM, only RNase R, or none of the known 3′ exonucleases. These results suggested that 3′ exonucleases have some effect on the half-life of full-length rpsO
mRNA, but endonuclease cleavage was likely more important for initiation of decay. We have no good explanation at present for the greater increase in the rpsO
mRNA half-life in the strains containing only YhaM or RNase R, especially since the increase was greater than that in the strain containing none of the four known exoribonucleases (Table ). Perhaps particular perturbations in the exoribonuclease complement of the cell indirectly affect endoribonuclease activity; recent evidence for a putative B. subtilis
degradosome complex that includes PNPase, RNase J1, and RNase Y (7
) may be indicative of other interactions between RNases.
Endonuclease cleavage in the rpsO
mRNA decay pathway was also inferred from the detection of multiple RNA fragments that were different sizes but all contained the 3′ end (Fig. ). We found that the patterns were similar for the wild-type and exoribonuclease mutant strains examined (Fig. ), despite the enormous differences between these strains when the patterns detected with the 5′-proximal probe were examined (Fig. ). The contrast between the complete lack of 5′-proximal decay intermediates in the strain containing only PNPase (Fig. ) and the presence of 3′-proximal decay intermediates in the same strain (Fig. , lane A) is particularly striking and is consistent with initiation of decay by endonuclease cleavage. The 3′-terminal fragments may be direct products of endonuclease cleavage, or they may be secondary products of RNase J1 5′ exonuclease activity that proceeds from the 5′ end(s) generated by endonuclease cleavage. The low level of 3′-end-containing fragments in the wild-type strain likely occurs because RNase J1 is capable of degrading through the secondary structure in the 5′-to-3′ direction (14
). Indeed, depleting the cell severely (without IPTG) or moderately (with IPTG) of RNase J1 resulted in increased intensity of the 3′-end-containing decay intermediates (Fig. ).
We found in assays of mRNA half-lives and of accumulation of full-length RNA and decay intermediates that RNase J1 was not responsible for determining the stability of rpsO
mRNA. Although RNase Y, the product of the ymdA
gene, was suspected long ago of being an RNase (1
), only in the last year have data on the role of RNase Y in RNA processing been described. Meinken and colleagues obtained evidence that cleavage of gapA
operon mRNA at a particular site (30
) was due to RNase Y (7
). Shahbabian and colleagues demonstrated that cleavage of the yitJ
riboswitch RNA, as well as other S
-adenosylmethionine-dependent riboswitches, could be attributed to RNase Y (38
). They also found that depletion of RNase Y resulted in an increase in the half-life of bulk mRNA. Thus, we turned our attention to RNase Y and showed, for the first time, that RNase Y has an effect on decay of a specific B. subtilis
mRNA. We observed a >2-fold increase in the rpsO
mRNA half-life in the RNase Y conditional mutant (Fig. ). This suggests that there is a strong dependence on RNase Y for initiation of decay, since we assumed that the RNase Y conditional mutant grown with 1 mM IPTG contains a significant level of the enzyme. We have not proven that RNase Y acts directly on rpsO
mRNA, which would require in vitro
tests, and it is possible that a deficiency in RNase Y has indirect effects on initiation of mRNA decay. Nevertheless, the simplest interpretation of our results is that RNase Y cleaves rpsO
mRNA, and for the discussion below we assume that this is the case.
The pbac system was useful for demonstrating faster accumulation of full-length rpsO mRNA in the RNase Y mutant strain (Fig. , zero-time lane), presumably because initiation of decay was slower due to the lower level of RNase Y. We hypothesize that the 180-nt RNA results from endonuclease cleavage followed by 3′ exonuclease activity up to the strong stem-loop structure that ends at nt 172. Thus, if RNase Y cleavage is required to generate this RNA, accumulation of the RNA should be slower in the RNase Y mutant, and this was indeed the case (Fig. and ). Other bands, in addition to the full-length and 180-nt bands, were detected (Fig. ). Some of these bands were faint and were present throughout the time course, and these bands likely represent nonspecific hybridization. We speculate that the band at around 270 nt may be a prematurely terminated transcription product or a processing product of a different RNase acting on full-length RNA.
From the current analysis of rpsO mRNA decay intermediates, it was not clear if RNase Y cleaves once or several times in the body of the message. Even a single endonuclease cleavage could give rise to numerous decay intermediates due to subsequent 3′-to-5′ exonuclease and 5′-to-3′ exonuclease activities and hindrance of these activities by RNA structure. In any event, the results obtained to date allow construction of a preliminary model for the complete turnover of rpsO mRNA, which begins with endonuclease cleavage by RNase Y and is completed by the 3′ exonuclease activity of PNPase on upstream products and the 5′ exonuclease activity of RNase J1 on downstream products (Fig. ). As RNase Y is essential, we expect that RNase Y catalyzes decay-initiating cleavage of many B. subtilis mRNAs.
Model for the pathway of rpsO mRNA decay. Only a single RNase Y endonuclease cleavage site is shown, but there may be additional cleavage sites.
Unlike RNase J1, which has robust endonuclease activity with 5′ triphosphorylated substrates (14
), RNase Y endonuclease activity is sensitive to the nature of the 5′ end, and the in vitro
activity on RNA with a 5′ monophosphorylated end is significantly higher (38
). If this is also true of RNase Y activity in vivo
, it has major consequences for models of RNase Y-dependent initiation of decay. Figure shows that decay intermediates detected in the PNPase-deficient strains were extremely stable, and their intensities did not decrease throughout the experiment. This suggests that degradation by RNase J1 5′ exonucleolytic activity from the native 5′ end did not occur, even though the data in Fig. indicate that the same 5′ exonuclease activity degraded the 3′-end-containing fragments. This suggests that the 5′ triphosphate group of the initial rpsO
transcription product is not removed, making 5′-end-containing decay intermediates resistant to RNase J1 5′ exonucleolytic decay. However, if this is the case and if RNase Y is sensitive to the 5′ triphosphate end, then it is hard to understand how internal cleavage by RNase Y resulting in a relatively short half-life (3.2 min) occurs. Much work is needed to understand the basis of endonucleolytic cleavage by RNase J1 and RNase Y and to determine why particular messages are subject to one of the activities or perhaps both activities.