scholarly journals Mitochondrial RNA capping: highly efficient 5’-RNA capping with NAD+ and NADH by yeast and human mitochondrial RNA polymerase

2018 ◽  
Author(s):  
Jeremy G. Bird ◽  
Urmimala Basu ◽  
David Kuster ◽  
Aparna Ramachandran ◽  
Ewa Grudzien-Nogalska ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Mitochondrial Gene ◽  
Promoter Sequence ◽  
Mitochondrial Rna ◽  
Sensors And Actuators ◽  
Mitochondrial Rna Polymerase ◽  
Mitochondrial Transcripts ◽  
Rna Capping ◽  
Reduced Forms

AbstractBacterial 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 ~10% 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 gene expression, sensing NAD+ and NADH levels and adjusting transcriptional outputs accordingly.

scholarly journals Highly efficient 5' capping of mitochondrial RNA with NAD+ and NADH by yeast and human mitochondrial RNA polymerase

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Jeremy G Bird ◽  
Urmimala Basu ◽  
David Kuster ◽  
Aparna Ramachandran ◽  
Ewa Grudzien-Nogalska ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Promoter Sequence ◽  
Mitochondrial Rna ◽  
Sensors And Actuators ◽  
Mitochondrial Rna Polymerase ◽  
Nuclear Rna ◽  
Mitochondrial Transcripts ◽  
Reduced Forms ◽  
Do So

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.

scholarly journals The N-terminal Domain of the Yeast Mitochondrial RNA Polymerase Regulates Multiple Steps of Transcription

2011 ◽  
Vol 286 (18) ◽  
pp. 16109-16120 ◽  
Author(s):  
Swaroopa Paratkar ◽  
Aishwarya P. Deshpande ◽  
Guo-Qing Tang ◽  
Smita S. Patel
Keyword(s):  
Amino Acids ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Rna Synthesis ◽  
Full Length ◽  
Mitochondrial Rna ◽  
Terminal Domain ◽  
Mitochondrial Rna Polymerase

Transcription of the yeast (Saccharomyces cerevisiae) mitochondrial (mt) genome is catalyzed by nuclear-encoded proteins that include the core RNA polymerase (RNAP) subunit Rpo41 and the transcription factor Mtf1. Rpo41 is homologous to the single-subunit bacteriophage T7/T3 RNAP. Its ∼80-kDa C-terminal domain is highly conserved among mt RNAPs, but its ∼50-kDa N-terminal domain (NTD) is less conserved and not present in T7/T3 RNAP. To understand the role of the NTD, we have biochemically characterized a series of NTD deletion mutants of Rpo41. Our studies show that NTD regulates multiple steps of transcription initiation. Interestingly, NTD functions in an autoinhibitory manner during initiation, and its partial deletion increases the efficiency of RNA synthesis. Deletion of 1–270 amino acids (DN270) reduces abortive synthesis and increases full-length to abortive RNA ratio relative to full-length (FL) Rpo41. A larger deletion of 1–380 amino acids (DN380), decreases RNA synthesis on duplex but not on premelted promoter. We show that DN380 is defective in promoter opening near the transcription start site. Most strikingly, both DN270 and DN380 catalyze highly processive RNA synthesis on the premelted promoter, and unlike the FL Rpo41, the mutants are not inhibited by Mtf1. Both mutants show weaker interactions with Mtf1, which explains many of our results, and particularly the ability of the mutants to efficiently transition from initiation to elongation. We propose that in vivo the accessory proteins that bind NTD may modulate interactions of Rpo41 with the promoter/Mtf1 to activate and allow timely release from Mtf1 for transition into elongation.

scholarly journals POLRMT regulates the switch between replication primer formation and gene expression of mammalian mtDNA

2016 ◽  
Vol 2 (8) ◽  
pp. e1600963 ◽  
Author(s):  
Inge Kühl ◽  
Maria Miranda ◽  
Viktor Posse ◽  
Dusanka Milenkovic ◽  
Arnaud Mourier ◽  
...  
Keyword(s):  
Gene Expression ◽  
Knockout Mice ◽  
Transcription Initiation ◽  
Mitochondrial Gene ◽  
Conditional Knockout ◽  
Regulatory Role ◽  
Mitochondrial Rna ◽  
Mitochondrial Transcription ◽  
Mtdna Replication

Mitochondria are vital in providing cellular energy via their oxidative phosphorylation system, which requires the coordinated expression of genes encoded by both the nuclear and mitochondrial genomes (mtDNA). Transcription of the circular mammalian mtDNA depends on a single mitochondrial RNA polymerase (POLRMT). Although the transcription initiation process is well understood, it is debated whether POLRMT also serves as the primase for the initiation of mtDNA replication. In the nucleus, the RNA polymerases needed for gene expression have no such role. Conditional knockout of Polrmt in the heart results in severe mitochondrial dysfunction causing dilated cardiomyopathy in young mice. We further studied the molecular consequences of different expression levels of POLRMT and found that POLRMT is essential for primer synthesis to initiate mtDNA replication in vivo. Furthermore, transcription initiation for primer formation has priority over gene expression. Surprisingly, mitochondrial transcription factor A (TFAM) exists in an mtDNA-free pool in the Polrmt knockout mice. TFAM levels remain unchanged despite strong mtDNA depletion, and TFAM is thus protected from degradation of the AAA+ Lon protease in the absence of POLRMT. Last, we report that mitochondrial transcription elongation factor may compensate for a partial depletion of POLRMT in heterozygous Polrmt knockout mice, indicating a direct regulatory role of this factor in transcription. In conclusion, we present in vivo evidence that POLRMT has a key regulatory role in the replication of mammalian mtDNA and is part of a transcriptional mechanism that provides a switch between primer formation for mtDNA replication and mitochondrial gene expression.

scholarly journals Cryo-EM structures reveal transcription initiation steps by yeast mitochondrial RNA polymerase

2020 ◽  
Author(s):  
Brent De Wijngaert ◽  
Shemaila Sultana ◽  
Chhaya Dharia ◽  
Hans Vanbuel ◽  
Jiayu Shen ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Large Scale ◽  
Rna Synthesis ◽  
Mitochondrial Rna ◽  
Initiation Complex ◽  
Mitochondrial Rna Polymerase ◽  
Catalytically Active ◽  
Promoter Melting ◽  
Antiviral Nucleosides

Cryo-EM structures of transcription pre-initiation complex (PIC) and initiation complex (IC) of yeast mitochondrial RNA polymerase show fully resolved transcription bubbles and explain promoter melting, template alignment, DNA scrunching, transition into elongation, and abortive synthesis. Promoter melting initiates in PIC with MTF1 trapping the −4 to −2 non-template (NT) bases in its NT-groove. Transition to IC is marked by a large-scale movement that aligns the template with RNA at the active site. RNA synthesis scrunches the NT strand into an NT-loop, which interacts with centrally positioned MTF1 C-tail. Steric clashes of the C-tail with RNA:DNA and NT-loop, and dynamic scrunching-unscrunching of DNA explain abortive synthesis and transition into elongation. Capturing the catalytically active IC-state with UTPαS poised for incorporation enables modeling toxicity of antiviral nucleosides/nucleotides.

scholarly journals Balance between Transcription and RNA Degradation Is Vital forSaccharomyces cerevisiaeMitochondria: Reduced Transcription Rescues the Phenotype of Deficient RNA Degradation

2006 ◽  
Vol 17 (3) ◽  
pp. 1184-1193 ◽  
Author(s):  
Agata T. Rogowska ◽  
Olga Puchta ◽  
Anna M. Czarnecka ◽  
Aneta Kaniak ◽  
Piotr P. Stepien ◽  
...  
Keyword(s):  
Mitochondrial Gene ◽  
Rna Degradation ◽  
Transcription Rate ◽  
Mitochondrial Rna ◽  
Rna Turnover ◽  
Loss Of Function ◽  
Mitochondrial Rna Polymerase ◽  
Genes Encoding ◽  
Mitochondrial Introns ◽  
Synthesis And Degradation

The Saccharomyces cerevisiae SUV3 gene encodes the helicase component of the mitochondrial degradosome (mtEXO), the principal 3′-to-5′ exoribonuclease of yeast mitochondria responsible for RNA turnover and surveillance. Inactivation of SUV3 (suv3Δ) causes multiple defects related to overaccumulation of aberrant transcripts and precursors, leading to a disruption of mitochondrial gene expression and loss of respiratory function. We isolated spontaneous suppressors that partially restore mitochondrial function in suv3Δ strains devoid of mitochondrial introns and found that they correspond to partial loss-of-function mutations in genes encoding the two subunits of the mitochondrial RNA polymerase (Rpo41p and Mtf1p) that severely reduce the transcription rate in mitochondria. These results show that reducing the transcription rate rescues defects in RNA turnover and demonstrates directly the vital importance of maintaining the balance between RNA synthesis and degradation.

scholarly journals Non-DNA-Templated Addition of Nucleotides to the 3′ End of RNAs by the Mitochondrial RNA Polymerase of Physarum polycephalum

2008 ◽  
Vol 28 (18) ◽  
pp. 5795-5802 ◽  
Author(s):  
Mara L. Miller ◽  
Dennis L. Miller
Keyword(s):  
Mitochondrial Dna ◽  
Rna Polymerase ◽  
Rna Editing ◽  
Physarum Polycephalum ◽  
Mitochondrial Gene ◽  
In Vitro Transcription ◽  
Open Reading Frames ◽  
Mitochondrial Rna ◽  
Mitochondrial Rna Polymerase

ABSTRACT Mitochondrial gene expression is necessary for proper mitochondrial biogenesis. Genes on the mitochondrial DNA are transcribed by a dedicated mitochondrial RNA polymerase (mtRNAP) that is encoded in the nucleus and imported into mitochondria. In the myxomycete Physarum polycephalum, nucleotides that are not specified by the mitochondrial DNA templates are inserted into some RNAs, a process called RNA editing. This is an essential step in the expression of these RNAs, as the insertion of the nontemplated nucleotides creates open reading frames for the production of proteins from mRNAs or produces required secondary structure in rRNAs and tRNAs. The nontemplated nucleotide is added to the 3′ end of the RNA as the RNA is being synthesized during mitochondrial transcription. Because RNA editing is cotranscriptional, the mtRNAP is implicated in RNA editing as well as transcription. We have cloned the cDNA for the mtRNAP of Physarum and have expressed the mtRNAP in Escherichia coli. We have used in vitro transcription assays based on the Physarum mtRNAP to identify a novel activity associated with the mtRNAP in which non-DNA-templated nucleotides are added to the 3′ end of RNAs. Any of the four ribonucleoside triphosphates (rNTPs) can act as precursors for this process, and this novel activity is observed when only one rNTP is supplied, a condition under which transcription does not occur. The implications of this activity for the mechanism of RNA editing are discussed.

scholarly journals Human Mitochondrial Transcription Factor B1 Interacts with the C-Terminal Activation Region of h-mtTFA and Stimulates Transcription Independently of Its RNA Methyltransferase Activity

2003 ◽  
Vol 23 (16) ◽  
pp. 5816-5824 ◽  
Author(s):  
Vicki McCulloch ◽  
Gerald S. Shadel
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Mitochondrial Gene ◽  
Factor H ◽  
Dual Function ◽  
Mitochondrial Rna ◽  
Methyltransferase Activity ◽  
Mitochondrial Transcription

ABSTRACT A significant advancement in understanding mitochondrial gene expression is the recent identification of two new human mitochondrial transcription factors, h-mtTFB1 and h-mtTFB2. Both proteins stimulate transcription in collaboration with the high-mobility group box transcription factor, h-mtTFA, and are homologous to rRNA methyltransferases. In fact, the dual-function nature of h-mtTFB1 was recently demonstrated by its ability to methylate a conserved rRNA substrate. Here, we demonstrate that h-mtTFB1 binds h-mtTFA both in HeLa cell mitochondrial extracts and in direct-binding assays via an interaction that requires the C-terminal tail of h-mtTFA, a region necessary for transcriptional activation. In addition, point mutations in conserved methyltransferase motifs of h-mtTFB1 revealed that it stimulates transcription in vitro independently of S-adenosylmethionine binding and rRNA methyltransferase activity. Furthermore, one mutation (G65A) eliminated the ability of h-mtTFB1 to bind DNA yet did not affect transcriptional activation. These results, coupled with the observation that h-mtTFB1 and human mitochondrial RNA (h-mtRNA) polymerase can also be coimmunoprecipitated, lead us to propose a model in which h-mtTFA demarcates mitochondrial promoter locations and where h-mtTFB proteins bridge an interaction between the C-terminal tail of h-mtTFA and mtRNA polymerase to facilitate specific initiation of transcription. Altogether, these data provide important new insight into the mechanism of transcription initiation in human mitochondria and indicate that the dual functions of h-mtTFB1 can be separated.

scholarly journals A novel intermediate in transcription initiation by human mitochondrial RNA polymerase

2014 ◽  
Vol 42 (6) ◽  
pp. 3884-3893 ◽  
Author(s):  
Yaroslav I. Morozov ◽  
Karen Agaronyan ◽  
Alan C. M. Cheung ◽  
Michael Anikin ◽  
Patrick Cramer ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Mitochondrial Rna ◽  
Mitochondrial Rna Polymerase

scholarly journals “CapZyme-Seq” comprehensively defines promoter-sequence determinants for RNA 5’ capping with NAD+

2017 ◽  
Author(s):  
Irina O. Vvedenskaya ◽  
Jeremy G. Bird ◽  
Yuanchao Zhang ◽  
Yu Zhang ◽  
Xinfu Jiao ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
High Throughput Sequencing ◽  
Promoter Sequence ◽  
Promoter Sequences ◽  
E Coli ◽  
Sequencing Method ◽  
Decapping Enzymes ◽  
General Method

SUMMARYNucleoside-containing metabolites such as NAD+ can be incorporated as “5′ caps” on RNA by serving as non-canonical initiating nucleotides (NCINs) for transcription initiation by RNA polymerase (RNAP). Here, we report “CapZyme-Seq,” a high-throughput-sequencing method that employs NCIN-decapping enzymes NudC and Rai1 to detect and quantify NCIN-capped RNA. By combining CapZyme-Seq with multiplexed transcriptomics, we determine efficiencies of NAD+ capping by Escherichia coli RNAP for ~16,000 promoter sequences. The results define preferred transcription start-site (TSS) positions for NAD+ capping and define a consensus promoter sequence for NAD+ capping: HRRASWW (TSS underlined). By applying CapZyme-Seq to E. coli total cellular RNA, we establish that sequence determinants for NCIN capping in vivo match the NAD+-capping consensus defined in vitro, and we identify and quantify NCIN-capped small RNAs. Our findings define the promoter-sequence determinants for NCIN capping with NAD+ and provide a general method for analysis of NCIN capping in vitro and in vivo.

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