Discovery of ANTAR-RNAs and their Mechanism of Action in Mycobacteria
Authors: Arati Ramesh
Anjali Koottathazhath
Dolly Mehta
Journal: Journal of Molecular Biology (2020)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: ANTAR regulation; RNA:protein complex; regulatory RNA; structured RNA.
Affiliations: National Centre for Biological Sciences, GKVK Post, Bellary Road, Bangalore 560065, India; SASTRA University, Tirumalaisamudram, Thanjavur 613401, India., National Centre for Biological Sciences, GKVK Post, Bellary Road, Bangalore 560065, India. Electronic address: .

Abstract:  Non-coding RNAs play pivotal roles in bacterial signaling. However, RNAs from certain phyla (specially high-GC actinobacteria) still remain elusive. Here, by re-engineering the existing genome-wide search approach, we discover a family of structurally conserved RNAs that are present ubiquitously across actinobacteria, including mycobacteria. In vitro analysis shows that RNAs belonging to this family bind response-regulator proteins that contain the widely prevalent ANTAR domain. The Mycobacterium tuberculosis ANTAR protein gets phosphorylated by a histidine kinase and interacts with RNA only in its phosphorylated state. These newly identified RNAs reside only in certain transcripts and typically overlap with the ribosome-binding site, regulating translation of these transcripts. In this way, the RNAs directly link signaling pathways to translational control, thus expanding the mechanistic tool kit available for ANTAR-based control of gene expression. In mycobacteria, we find that RNAs targeted by ANTAR proteins majorly encode enzymes of lipid metabolism and associated redox pathways. This now allows us to identify the key genes that mediate ANTAR-dependent control of lipid metabolism. Our study establishes the identity and wide prevalence of ANTAR-target RNAs in mycobacteria, bringing RNA-mediated regulation in these bacteria to the center stage. 

Retracted: No

PubPeer comments:
 Figure 6C and Supplemental Figure S7B. * These two panels, albeit representing different lacZ reporter assays, appear to look remarkably similar if one of them is rotated differently. ![file] , 
 #1 Indeed, that is curious. It is also worth examining the handwritten labeling (in blue) on the three sectors of each plate. For one sector, the labeling appears identical between the two plates (and says something like  pssA008 ), as do the blue lines dividing the three sectors. For the other two sectors, the handwritten labeling is clearly different on the two plates. It is difficult to imagine how this could happen, assuming the labeling is on the bottom of the plates (not the lids). , 
 #2  It is difficult to imagine how this could happen, assuming the labeling is on the bottom of the plates (not the lids). The plates are always kept inverted that is the lid of the plate is facing the bottom while the bottom part of the plate (which contains the agar) is facing upwards. Markings on the plate are done with a marker pen on the bottom side of the plate which upon keeping inverted comes on the top. These marking are generally done with a marker. The markings can thus be removed or re-written by wiping them off with the help of 70% ethanol. The image posted by Dr. Bik in #1 shows a difference in light reflection patterns of both the plates which further suggests that they are two different photographs of the same plate taken by slightly rotating the plate (Fig. S7B). I can imagine what could have happened in this case. After drawing the 3 quadrants on the plate and labelling them according to Fig 6C, the authors might have mislabeled the same plate due to ignorance or other reasons such as the plate getting mixed up with other plates resulting in this apparent reuse. I really don t think that the labeling or plate itself are tempered digitally by using photoshop or any similar software. , 
 As someone whou2019s handled a fair share of agar plates during cloning, we rarely ever label plates AFTER culturing. The plates are generally labeled prior and imaged afterwards. Even if the labels were not digitally altered, itu2019s confusing why the authors felt the need to erase and re-label, especially when the plate is exactly the same. Mix-up can happen when you image the same plate twice instead of imaging two different plates. In this case the labels would be the same. Itu2019s hard to argue for a simple mix-up here when the plates are similar but labels are different. , 
 #3 : I should have been more explicit. What I meant was that it is difficult to imagine how this could happen ***under normal circumstances***. Erasing and relabeling would be an example of a ***not*** normal circumstance (although it is perhaps the most likely explanation here). #4: Exactly. It is one thing to realize a mistake and relabel. It is quite another thing to take a photo first, then relabel afterward, then take a second photo, and then use **BOTH** photos in the paper and portray them as different experiments. Clearly this situtation calls for an explanation from the authors. , 
 Correction, November 2021, found here: https: www.sciencedirect.com science article pii S0022283621005441  The authors would like to make 3 corrections and a clarification. 1. We would like to correct the labels in Fig. 6C to that shown below. The labels for the two blue strains (u201cno protein + wt RNAu201d and u201cRv1626D65E + mut RNAu201d) were inadvertently swapped for each other. This labeling error is corrected here. 2. The authors would also like to make a correction in Fig. S7B. In the paper we inadvertently added another image of a plate actually belonging to Fig. 6C. We apologize for this error. We would like to correct the image for Fig. S7B to that shown below (experiment by Anirudh KN and Akshay Das Adhikari), Here the u201cno protein + mut RNAu201d control experiment is plated alongside the u201cno protein + wtRNAu201d for comparison (both blue colonies). The results and the underlying conclusions made with regard to the translational control of Rv1936 by the ANTAR protein remains unchanged. The legend for Fig. S7B should now read: u201cM. smegmatis strains containing lacZ reporter fusions to the Mtb Rv1936 5u2019UTR RNA are visualized on X-gal containing plates. Strains and RNA details are as shown in Fig. 6C. Both u201cno protein + mut RNA (mutations made in the RNA to abolish ANTAR binding) and u201cno protein + wt RNAu201d controls show high lacZ expression (blue colonies).u201d 3. We correct a typographical error in the Fig. 4C sub-panel label for Mtb EspE 5u2032UTR and Mtb Rv1936alt 5u2032UTR. The label is corrected to u201cRv1626D65E + and ++u201d instead of u201cu2212 and +u201d. 4. We would like to clarify that the image for Fig. 4C sub-panel Mtb Rv1936 5u2032UTR in the paper appears to be of poor quality, hence we provide the full gel below. Lanes 1,2,3 (red box) of the below gel are shown in the paper for Mtb Rv1936 5u2032UTR. The authors would like to apologise for any inconvenience caused. ,