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DEAD-box proteins play important roles in many RNA processes ranging from RNA synthesis to RNA decay. Furthermore, it has been reported that some bacterial DEAD-box proteins known to be components of the RNA degradosome do not cleave RNA substrates directly. However, the role of DEAD-box proteins in RNA degradation is poorly understood. The present study demonstrated that the DEAD-box protein CshA from the vancomycin-resistant Staphylococcus aureus strain Mu50 possesses RNA degradation activity, ribonuclease activity. Despite having RNA-dependent ATPase activity, CshA did not exhibit RNA helicase activity in vitro. Instead, CshA catalyzed the degradation of single-stranded RNAs of various duplex RNA substrates to form blunt-end RNA products. Thus, we suggest that the ribonuclease activity of the DEAD-box protein CshA may contribute to RNA remodeling in the bacterial RNA degradosome. To our knowledge, this study is the first to report that a DEAD-box protein from a pathogenic bacterium is implicated in multiple ATP-independent activity on RNA, such as degradation.