In vitro cleavage requirements and specificities of mycobacterial RNase E

Regulation of RNA pools allows for adaptation to stress, which is important in pathogenic bacteria such as Mycobacterium tuberculosis (Mtb). RNA degradation is a significant contributor to RNA abundance, and Ribonuclease (RNase) E has a rate-limiting role in degradation of most mycobacterial transcripts. Many open questions remain about the RNA substrate requirements and specificities for efficient cleavage by mycobacterial RNase E. Here, we demonstrate that RNase E is only active on substrates with a minimum length of approximately 27 nt using both Mtb and Mycolicibacterium smegmatis (Msm) enzyme variants. Furthermore, we show that mycobacterial RNase E prefers substrates with 5' monophosphates to 5' triphosphates. We present that the positions of cleavage events by RNase E within substrates are dictated by both sequence and distance from the RNA ends. Finally, we show that Msm RNase E behaves similarly to Mtb RNase E, validating the use of this model organism for RNA degradation studies.