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.

中文翻译：

---

枯草芽孢杆菌中RNase Y的动态膜定位。

mRNA的代谢转换对于控制所有生物中的基因表达至关重要，特别是在快速适应的原核生物中。如革兰氏阳性模型生物枯草芽孢杆菌所示，在许多细菌中，RNase Y通过内切核酸酶裂解来启动整体mRNA衰变。这种酶被束缚在细胞内膜上，是一种伪小隔室化，与其启动mRNA的裂解/成熟有关。在细胞外围翻译的。在这里，我们使用全内反射荧光显微镜（TIRFm）和单粒子跟踪（SPT）可视化RNase Y，并分析其在活细胞中的分布和动力学。我们发现，RNase Y以动态短寿命病灶的形式在膜上迅速扩散。与RNase E不同，大肠杆菌中主要的引发衰变的RNase 病灶的形成不取决于RNA底物的存在。相反，RNase Y焦点在转录停止后变得更加丰富，并且大小增加，这表明它们并不构成核酸酶的最活跃形式。先前已显示三种蛋白质（YaaT，YlbF和YmcA）的Y复合物在体内对RNase Y的活性起着重要作用。我们证明Y复合物突变的作用类似于但强于消耗RNA增加了膜上RNase Y焦点的数量和大小。我们的数据表明，Y-复合物使RNase Y的装配状态向越来越少的复合物转移，从而提高了复杂底物（如多顺反子mRNA）的切割效率。重要信息所有生物都必须降解mRNA，以使基因表达适应不断变化的环境。在细菌中，mRNA衰减的启动通常通过内切核酸酶裂解而发生。在革兰氏阳性模型生物枯草芽孢杆菌和可能的许多其他细菌中，用于此任务的关键酶是RNase Y，其锚定在细胞内膜上。虽然这种伪分隔区的出现与主要发生在细胞边缘的翻译是连贯的，但是我们对活细胞中RNase Y的分布和动力学的知识非常匮乏。在这里，我们显示了RNase Y以动态短寿命病灶的形式沿着膜快速移动。这些病灶变得更丰富，并且在转录停滞后大小增加，表明它们不构成核酸酶的最活跃形式。这与RNase E（大肠杆菌中主要的引起衰变的RNase）相反，后者显示病灶的形成取决于RNA底物的存在。我们还表明，已知在体内影响RNase Y活性特异性的蛋白质复合物（Y-复合物）能够将RNase Y的组装状态向越来越少的复合物转移。这突显了基于RNase E和RNase Y的降解机制之间的根本差异。