The E. coli DEAD box ATPase CsdA, is a NAD ⁺ capped RNA binding protein

5' nicotinamide adenine dinucleotide (NAD ⁺ ) caps are one of the most common metabolites derived non-canonical caps reported on bacterial RNAs. Multiple decapping proteins are known to regulate the stability of NAD ⁺ capped transcripts. However, no other proteins have been identified that preferentially interact with these NAD ⁺ caps, and mechanistic details of the cap-dependent recognition remain poorly understood. Using an affinity capture approach, we identified multiple E. coli proteins that selectively recognize NAD ⁺ caps, including the ATP-dependent RNA helicase, CsdA. CsdA preferentially interacts directly with NAD ⁺ capped RNAs and can discriminate between 5' NAD ⁺ capped and 5' triphosphate end transcripts. Binding to NAD ⁺ capped RNA versus 5' triphosphate RNA more greatly enhances the ATPase activity of CsdA and the presence of NAD ⁺ caps on transcripts modulates the ability of CsdA to form RNA condensates. Furthermore, we find that CsdA enhances the decapping activity of the NADH hydrolase NudC, suggesting CsdA plays a role in regulating the degradation of NAD ⁺ capped transcripts. CsdA is the first identified NAD ⁺ cap reader protein and its preference for binding NAD ⁺ capped RNA provides a mechanism by which E. coli cells link RNA stability to the identity of the 5' cap.