Highly efficient 5\u27 capping of mitochondrial RNA with NAD+ and NADH by yeast and human mitochondrial RNA polymerase

Bacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD+ and NADH, using NAD+ and NADH as non-canonical initiating nucleotides for transcription initiation. Here, we show that mitochondrial RNAPs (mtRNAPs) cap RNA with NAD+ and NADH, and do so more efficiently than nuclear RNAPs. Direct quantitation of NAD+- and NADH-capped RNA demonstrates remarkably high levels of capping in vivo: up to ~60% NAD+ and NADH capping of yeast mitochondrial transcripts, and up to ~15% NAD+ capping of human mitochondrial transcripts. The capping efficiency is determined by promoter sequence at, and upstream of, the transcription start site and, in yeast and human cells, by intracellular NAD+ and NADH levels. Our findings indicate mtRNAPs serve as both sensors and actuators in coupling cellular metabolism to mitochondrial transcriptional outputs, sensing NAD+ and NADH levels and adjusting transcriptional outputs accordingly. © 2018, Bird et al

Regulatory components that determine the cell type specificity of the immunoglobulin heavy chain enhancer and the human liver/bone/kidney alkaline phosphatase gene

Cell type specific expression of both the murine immunoglobulin heavy chain enhancer and human liver/bone/kidney alkaline phosphatase gene were analyzed. The enhancer was dissected into two distinct functional domains, each retaining the cell type preference of the entire enhancer. The functional significance of the $\mu$E1, $\mu$E2, $\mu$E3, $\mu$E4 and the octanucleotide sites were determined. An additional element located between the $\mu$E1 and $\mu$E2 sites was identified and the transcription factor that binds to this region was partially characterized. The interaction of this trans factor with the protein Id (inhibitor of DNA-binding) was characterized. The level and mechanism of regulation of the human liver/bone/kidney alkaline phosphatase gene were delineated in osteoblast (high expressing) and non-osteoblast (low expressing) cell lines. This gene is regulated post-transcriptionally since the same promoter sequences are utilized to initiate transcription and the activity of this promoter is the same in both osteoblast and non-osteoblast cells. The level of steady state of this gene is much higher in osteoblast cells even though the cytosolic stability is the same. Furthermore, splicing and nucleocytoplasmic transport does not appear to be the determinant of cell type specific regulation. The results indicate that the levels of liver/bone/kidney alkaline phosphatase mRNA in different cell types are established by differential nuclear stability of nascent RNA

Poly(A)-binding-protein-mediated regulation of hDcp2 decapping in vitro

Regulation of mRNA decapping is a critical determinant for gene expression. We demonstrate that the poly(A) tail-mediated regulation of mRNA decapping observed in humans can be recapitulated in vitro by the cytoplasmic poly(A)-binding protein PABP through a direct and specific binding to the 5′ end of capped mRNA. The specific association of PABP with the cap occurred only within the context of the RNA whereby a cap attached to an RNA moiety served as the high-affinity substrate but not the cap structure or RNA alone. Binding of PABP to the RNA 5′ end required the presence of the cap and was accentuated by the N7 methyl moiety of the cap. Interestingly, conditions that enhanced hDcp2 decapping activity reduced the affinity of PABP for cap association and consequently its ability to inhibit decapping, suggestive of a regulated association of PABP with the cap. These observations reveal a novel direct involvement of human PABP in the stabilization of mRNA by protecting the 5′ end from decapping

A View to a Kill: Structure of the RNA Exosome

The exosome is a 3′ to 5′ exoribonuclease central to many cellular processes, including mRNA decay. Liu et al. (2006) now present the biochemical reconstitution and crystal structure of the eukaryotic exosome. This remarkable achievement provides key insights into the composition and assembly of the human and yeast exosomes, revealing functions of individual subunits

Scavenger Decapping Activity Facilitates 5′ to 3′ mRNA Decay

mRNA degradation occurs through distinct pathways, one primarily from the 5′ end of the mRNA and the second from the 3′ end. Decay from the 3′ end generates the m(7)GpppN cap dinucleotide, which is subsequently hydrolyzed to m(7)Gp and ppN in Saccharomyces cerevisiae by a scavenger decapping activity termed Dcs1p. Although Dcs1p functions in the last step of mRNA turnover, we demonstrate that its activity modulates earlier steps of mRNA decay. Disruption of the DCS1 gene manifests a threefold increase of the TIF51A mRNA half-life. Interestingly, the hydrolytic activity of Dcs1p was essential for the altered mRNA turnover, as Dcs1p, but not a catalytically inactive Dcs1p mutant, complemented the increased mRNA stability. Mechanistic analysis revealed that 5′ to 3′ exoribonucleolytic activity was impeded in the dcs1Δ strain, resulting in the accumulation of uncapped mRNA. These data define a new role for the Dcs1p scavenger decapping enzyme and demonstrate a novel mechanism whereby the final step in the 3′ mRNA decay pathway can influence 5′ to 3′ exoribonucleolytic activity

Functional characterization of the mammalian mRNA decapping enzyme hDcp2

Regulation of decapping is a critical determinant of mRNA stability. We recently identified hDcp2 as a human decapping enzyme with intrinsic decapping activity. This activity is specific to N(7)-methylated guanosine containing RNA. The hDcp2 enzyme does not function on the cap structure alone and is not sensitive to competition by cap analog, suggesting that hDcp2 requires the RNA for cap recognition. We now demonstrate that hDcp2 is an RNA-binding protein and its recognition and hydrolysis of the cap substrate is dependent on an initial interaction with the RNA moiety. A biochemical characterization of hDcp2 revealed that a 163 amino acid region containing two evolutionarily conserved regions, the Nudix fold hydrolase domain and the adjacent Box B region contained methyl-cap-specific hydrolysis activity. Maximum decapping activity for wild-type as well as truncation mutants of hDcp2 required Mn(2+) as a divalent cation. The demonstration that hDcp2 is an RNA-binding protein with an RNA-dependent decapping activity will now provide new approaches to identify specific mRNAs that are regulated by this decapping enzyme as well as provide novel avenues to control mRNA decapping and turnover by influencing the RNA-binding property of hDcp2