NAD-RNA capping

The NAD-RNA cap stabilizes small regulatory RNAs in bacteria.

The 5'-terminal ends of cellular mRNAs contain a m7GpppN cap, in which N can be any nucleotide. In eukaryotes, the RNA helicase eIF4A, the scaffold protein translation initiation factor 4G (eIF4G) and the capping protein eIF4E are part of the complex that loads mRNAs onto the 40 S ribosomal subunit, together with eIF3. eIF4E has a crucial role in the regulation of translation. mRNA metabolism in the nucleus, such as capping, splicing, and polyadenylation, is mechanically linked to transcription. In addition, mRNA decay regulates mRNA metabolism whereas capping the 5’-end and polyadenylation of the 3’-end increases mRNAs' stability.

In the bacterium Escherichia coli, the nicotinamide adenine dinucleotide RNA (NAD-RNA) cap stabilizes small regulatory RNAs (srRNAs) in vitro against nucleotide processing by RNase E and against 5’-end modification by RNA pyrophosphohydrolase RppH. RNase E is involved in RNA decay, and RppH converts 5’-triphosphate RNA into 5’-monophosphate RNA triggering endonucleolytic processing.

As recently discovered, bacterial small RNAs, yeast and human mRNAs and non-coding RNAs contain NAD cap-like structures. Bacterial RNA polymerase (RNA Pol) can also initiate transcription in vitro by accepting nucleotide metabolites capped with flavin adenine dinucleotide (FAD), uridine diphosphate glucose (UDP-Glc), and uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Capping with NAD and UDP analogs by bacterial RNA Pol is promoter-specific and stimulates promoter escape. These recent findings suggest a role for metabolite caps in regulating gene expression. As demonstrated by Wang et al., X-ray crystallography in combination with mass spectrometry allows chracterization of cap-protein interactions.

The redox factor NAD is attached to small regulatory RNAs in bacteria as a cap.

In 2016, Hoefer et al. solved the crystal structures of the nuclear migration protein NudC from Escherichia coli in complex with the substrate NAD and the cleavage product nicotinamide mononucleotide (NMN). Crystal structures of the complexes studied revealed the catalytic residues lining the binding pocket and molecular features of substrate and product recognition.

Figure 1: NudC (NADH Pyrophosphatase) in complex with NAD (5IW4).

Höfer et al.’s study revealed that NudC is a single-strand-specific RNA decapping enzyme with a strong preference for a purine as the first nucleotide. Their biochemical experiments showed that NudC preferred NAD-RNA over NAD(H) by several orders of magnitude. Hence NAD-RNA maybe its primary biological substrate. The study results suggest that NudC can bind a diverse population of cellular RNAs in an unspecific, most likely electrostatic manner.

The E. coli Nudix hydrolase NudC hydrolyzes the pyrophosphate bond and removes the NAD cap to produce nicotinamide mononucleotide (NMN) and 5’-monophosphate RNA. NudC is also known as NAD(H) pyrophosphohydrolase. As suggested by Höfer et al. it is possible that during RNA processing, NAD capping gives the bacterium additional RNA protection by using a degradation pathway that is orthogonal to the RppH-triphosphate RNA pathway.

NudC is a critical factor in mitosis, the cell division resulting in two genetically identical daughter cells. In 2015, Chen et al, used mass spectrometry to reveal that the nuclear distribution gene C (NudC or NUDC) protein, a critical factor for the progression of mitosis, is associated with the Echinoderm microtubule-associated protein (EMAP)-like 4 (EML4). The study showed that EML4 is critical for the loading of NudC onto the mitotic spindle for mitotic progression.

As reviewed by Chen et al., in mammals, NUDCL and NUDCL2 are homologs of NudC, and both proteins are thought to have specific roles in mitosis. NUDCL is phosphorylated during mitosis, and its expression is regulated during cell cycle progression. NUDCL is localized to the centrosomes and to the midbody. The depletion of this protein induces multiple mitotic defects. During mitosis, NUDCL2 is localized to the centrosome and kinetochore. Both proteins can associate with LIS1 and the dynein/dynactin complex, however, the exact mechanisms by which NUDCL and NUDCL2 accumulate to specific sites during mitosis remain unknown. 

Figure 2: Cryo-EM structure of a single dynein tail domain bound to dynactin and BICD2N (6F3A).

In 2018, Urnavicius et al. used electron microscopy and single-molecule studies to show that adaptors can recruit a second dynein to dynactin. Dynein and its cofactor dynactin form a highly processive microtubule motor in the presence of an activating adaptor, such as BICD2. 

Dan Chen, Satoko Ito, Hong Yuan, Toshinori Hyodo, Kenji Kadomatsu, Michinari Hamaguchi & Takeshi Senga (2015) EML4 promotes the loading of NUDC to the spindle for mitotic progression, Cell Cycle, 14:10, 1529-1539. [tandfonline]

Höfer K, Li S, Abele F, Frindert J, Schlotthauer J, Grawenhoff J, Du J, Patel DJ, Jäschke A. Structure and function of the bacterial decapping enzyme NudC. Nat Chem Biol. 2016 Sep;12(9):730-4. [PMC] 

Urnavicius L, Lau CK, Elshenawy MM, Morales-Rios E, Motz C, Yildiz A, Carter AP. Cryo-EM shows how dynactin recruits two dyneins for faster movement. Nature. 2018 Feb 7;554(7691):202-206. [PMC] 

Jin Wang, Bing Liang Alvin Chew, Yong Lai, Hongping Dong, Luang Xu, Seetharamsingh Balamkundu, Weiling Maggie Cai, Liang Cui, Chuan Fa Liu, Xin-Yuan Fu, Zhenguo Lin, Pei-Yong Shi, Timothy K. Lu, Dahai Luo, Samie R. Jaffrey, Peter C. Dedon; Quantifying the RNA cap epitranscriptome reveals novel caps in cellular and viral RNA. Nucleic Acids Research, Volume 47, Issue 20, 18 November 2019, Page e130. bioRxiv 683045. [Nucleic Acids Research]

Waters LS, Storz G. Regulatory RNAs in bacteria. Cell. 2009 Feb 20;136(4):615-28. [PMC]

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