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Abstract
Bacteria are dependent on rapid alterations in gene expression. A prerequisite for rapid adaptations is efficient RNA turnover, with endonuclease RNase Y playing a crucial role in mRNA stability as well as in maturation. In Bacillus subtilis, RNase Y in turn interacts with the so-called “Y-complex” consisting of three proteins, which play important functions in sporulation, natural transformation and biofilm formation. It is thought that the Y-complex acts as an accessory factor in RNase Y regulation but might also have independent functions. Using single-molecule tracking, we show that all three Y-complex proteins exhibit three distinct mobilities, including movement through the cytosol and confined motion, predominantly at membrane-proximal sites but also within the cell center. A transcriptional arrest leads to a strong change in localization and dynamics of YmcA, YlbF and YaaT, supporting their involvement in global RNA degradation. However, Y-complex proteins show distinguishable protein dynamics, and the deletion of yaaT or ylbF shows a minor effect on the dynamics of YmcA. Cell fractionation reveals that YaaT displays a mixture of membrane association and presence in the cytosol, while YlbF and YmcA do not show direct membrane attachment. Taken together, our experiments reveal membrane-associated and membrane-independent activities of Y-complex proteins and a dynamic interplay between them with indirect membrane association of YmcA and YlbF via YaaT.
Highlights
Control of messenger RNA stability is a central part of gene regulation in all cells
We addressed the question of how Y-complex proteins can affect processes such as messenger RNA (mRNA) maturation, which we would expect to take place on the nucleoids, where mRNA is synthesized [21], unless non-matured mRNAs move from the nucleoids into the membrane-attached RNase Y foci
We assayed for the processing of the cggR-gapA mRNA, which depends on the activity of RNase Y as well as that of the Y-complex proteins [17]
Summary
Control of messenger RNA (mRNA) stability is a central part of gene regulation in all cells. For bacteria, the regulation of mRNA turnover allows cells to control gene expression at a post-transcriptional level and react quickly to changing growth conditions [4]. In addition to biofilm formation, competence and sporulation (here, the complex acts on the phosphorelay, this is currently under debate), it was shown that the Ycomplex plays a role global in mRNA stability and is necessary for the processing of the cggR-gapA transcript in B. subtilis [17]. Because of its crucial role in mRNA cleavage in vivo, Y-complex proteins have been proposed to act as accessory factors that regulate RNase Y activity [17]. A functional RNase Y-GFP fusion forms distinct foci at the cell membrane [19], and the dynamics of these foci have recently been shown to be influenced by. Our data considerably revise and refine our understanding of Y-complex dynamics in cells and reveal distinct biophysical properties of the three proteins
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