Dynamic Membrane Localization of RNase Y in Bacillus subtilis

Abstract

Metabolic turnover of mRNA is fundamental to the control of gene expression in all organisms, notably in fast-adapting prokaryotes. In many bacteria, RNase Y initiates global mRNA decay via an endonucleolytic cleavage, as shown in the Gram-positive model organism Bacillus subtilis. This enzyme is tethered to the inner cell membrane, a pseudocompartmentalization coherent with its task of initiating mRNA cleavage/maturation of mRNAs that are translated at the cell periphery. Here, we used total internal reflection fluorescence microscopy (TIRFm) and single-particle tracking (SPT) to visualize RNase Y and analyze its distribution and dynamics in living cells. We find that RNase Y diffuses rapidly at the membrane in the form of dynamic short-lived foci. Unlike RNase E, the major decay-initiating RNase in Escherichia coli, the formation of foci is not dependent on the presence of RNA substrates. On the contrary, RNase Y foci become more abundant and increase in size following transcription arrest, suggesting that they do not constitute the most active form of the nuclease. The Y-complex of three proteins (YaaT, YlbF, and YmcA) has previously been shown to play an important role for RNase Y activity in vivo. We demonstrate that Y-complex mutations have an effect similar to but much stronger than that of depletion of RNA in increasing the number and size of RNase Y foci at the membrane. Our data suggest that the Y-complex shifts the assembly status of RNase Y toward fewer and smaller complexes, thereby increasing cleavage efficiency of complex substrates like polycistronic mRNAs. IMPORTANCE All living organisms must degrade mRNA to adapt gene expression to changing environments. In bacteria, initiation of mRNA decay generally occurs through an endonucleolytic cleavage. In the Gram-positive model organism Bacillus subtilis and probably many other bacteria, the key enzyme for this task is RNase Y, which is anchored at the inner cell membrane. While this pseudocompartmentalization appears coherent with translation occurring primarily at the cell periphery, our knowledge on the distribution and dynamics of RNase Y in living cells is very scarce. Here, we show that RNase Y moves rapidly along the membrane in the form of dynamic short-lived foci. These foci become more abundant and increase in size following transcription arrest, suggesting that they do not constitute the most active form of the nuclease. This contrasts with RNase E, the major decay-initiating RNase in E. coli, where it was shown that formation of foci is dependent on the presence of RNA substrates. We also show that a protein complex (Y-complex) known to influence the specificity of RNase Y activity in vivo is capable of shifting the assembly status of RNase Y toward fewer and smaller complexes. This highlights fundamental differences between RNase E- and RNase Y-based degradation machineries.

Phase‐separated bacterial ribonucleoprotein bodies organize mRNA decay

“…1.2 | Compositional control and evolution of BR-bodies 1.2.1 | BR-body organization stimulates mRNA decay BR-body organization helps to stimulate the rapid mRNA decay process in bacteria by organizing the RNA decay machinery together with its mRNA substrates ( Figure 6). BR-bodies dynamically assemble and disassemble during log (Hamouche et al, 2020). Red arrows mark two foci that ultimately fuse together, suggesting liquid-like properties.…”

“…Red arrows mark two foci that ultimately fuse together, suggesting liquid-like properties. Images were taken every 100 ms (Hamouche et al, 2020) growth with RNA, with mRNA decay triggering their disassembly (Al-Husini et al, 2018). RNA degradosome components are recruited into BR-bodies, requiring their specific protein-protein interaction sites for entry (Al-Husini et al, 2018).…”

The RNase J-Based RNA Degradosome Is Compartmentalized in the Gastric Pathogen Helicobacter pylori

“…In contrast, in a recent work, a fraction of RNase J2 of Streptococcus mutans was found to localize at the membrane ( 34 ). In B. subtilis , RNase Y was recently found to assemble at the membrane into dynamic short-lived foci that increased in number and size upon rifampicin treatment or deletion of the Y-complex, a protein complex that modulates RNase Y activity ( 27 ). Those data suggest that RNase Y foci represent a less active form of the enzyme in contrast to the situation of RNase E foci ( 27 ).…”

“…In S. aureus and B. subtilis , the proposed RNA degradosomes rely on the membrane-anchored RNase Y ( 24 – 26 ). In B. subtilis , an RNase Y-GFP (RNase Y-green fluorescent protein) fusion was observed to have a homogeneous membrane localization ( 25 ) and was recently found to be dynamic and to form foci similar to those of RNase E ( 27 ). Under conditions where RNase Y was less active, the foci were more abundant and showed increased size, suggesting that these structures represent a less active form of the enzyme ( 27 ).…”

“…In B. subtilis , an RNase Y-GFP (RNase Y-green fluorescent protein) fusion was observed to have a homogeneous membrane localization ( 25 ) and was recently found to be dynamic and to form foci similar to those of RNase E ( 27 ). Under conditions where RNase Y was less active, the foci were more abundant and showed increased size, suggesting that these structures represent a less active form of the enzyme ( 27 ). RNases J1 and J2 were found to localize in the cytoplasm and were excluded from the nucleoid ( 25 ).…”