Mitochondrial Transcription Elongation Research Articles

TEFM variants impair mitochondrial transcription causing childhood-onset neurological disease

Mutations in the mitochondrial or nuclear genomes are associated with a diverse group of human disorders characterized by impaired mitochondrial respiration. Within this group, an increasing number of mutations have been identified in nuclear genes involved in mitochondrial RNA biology. The TEFM gene encodes the mitochondrial transcription elongation factor responsible for enhancing the processivity of mitochondrial RNA polymerase, POLRMT. We report for the first time that TEFM variants are associated with mitochondrial respiratory chain deficiency and a wide range of clinical presentations including mitochondrial myopathy with a treatable neuromuscular transmission defect. Mechanistically, we show muscle and primary fibroblasts from the affected individuals have reduced levels of promoter distal mitochondrial RNA transcripts. Finally, tefm knockdown in zebrafish embryos resulted in neuromuscular junction abnormalities and abnormal mitochondrial function, strengthening the genotype-phenotype correlation. Our study highlights that TEFM regulates mitochondrial transcription elongation and its defect results in variable, tissue-specific neurological and neuromuscular symptoms.

I.01Congenital myasthenic syndromes

Neuromuscular junction disorders are a heterogeneous group of acquired (Myasthenia Gravis, MG) and inherited (Congenital Myasthenic Syndromes, CMS) disorders associated with distinctive clinical, electrophysiological, laboratory and ultrastructural abnormalities. The genetic defects in CMS either impair neuromuscular transmission directly or result in secondary impairments, which eventually compromise the safety margin of neuromuscular transmission. More recently, we have identified two genes (<i>DOK7, GFPT1</i>) that cause fatigable weakness of muscles in a limb-girdle distribution, but rarely affecting facial or eye muscles. Next-generation sequencing and deep phenotyping, in combination with international data sharing, reveals new genetic causes of CMS, but also unusual, overlapping clinical phenotypes, which blur the boundaries with primary myopathies and motor neuropathies. Mitochondrial dysfunction has been implicated with both myopathies and neuromuscular transmission defects. TEFM is a nuclear gene that codes for a mitochondrial transcription elongation factor – the protein responsible for prolonging activity of RNA polymerase to ensure distal transcripts of the mitochondrial genome are transcribed. We identified the first human <i>TEFM</i> variants associated with mitochondrial respiratory chain deficiency in seven patients with varying clinical presentations. While symptoms included classical features of a mitochondrial disease, some patients also presented with a treatable neuromuscular transmission defect. We will cover the significant progress made in understanding the molecular pathogenesis of CMS, which is important for both patients and clinicians in terms of reaching a definite diagnosis and selecting the most appropriate treatment.

Ribonucleotides embedded in template DNA impair mitochondrial RNA polymerase progression

Human mitochondria lack ribonucleotide excision repair pathways, causing misincorporated ribonucleotides (rNMPs) to remain embedded in the mitochondrial genome. Previous studies have demonstrated that human mitochondrial DNA polymerase γ can bypass a single rNMP, but that longer stretches of rNMPs present an obstacle to mitochondrial DNA replication. Whether embedded rNMPs also affect mitochondrial transcription has not been addressed. Here we demonstrate that mitochondrial RNA polymerase elongation activity is affected by a single, embedded rNMP in the template strand. The effect is aggravated at stretches with two or more consecutive rNMPs in a row and cannot be overcome by addition of the mitochondrial transcription elongation factor TEFM. Our findings lead us to suggest that impaired transcription may be of functional relevance in genetic disorders associated with imbalanced nucleotide pools and higher levels of embedded rNMPs.

High expression of the TEFM gene predicts poor prognosis in hepatocellular carcinoma.

Mitochondrial transcription elongation factor (TEFM) is an essential molecule that regulates the replication-transcription switch of mitochondrial DNA. TEFM modulates both transcription elongation and RNA processing in mitochondria. The purpose of the present study was to determine the association of TEFM with tumor progression and prognosis in hepatocellular carcinoma (HCC) patients. The different protein expression level of TEFM among HCC cell lines was detected by Western blotting. The gene expression profiling interactive analysis (GEPIA) was used to dynamically analyze the mRNA expression of TEFM gene in different stages of HCC. The protein and mRNA expression levels of TEFM were detected by immunohistochemistry, Western blotting and qRT-PCR. The mRNA-SeqV2 expression of TEFM and clinical information of HCC patients were downloaded from the TCGA database by using R3.6.3 software. Next, the relationships between the expression level of TEFM and clinicopathological characteristics and the prognostic value of TEFM were analyzed. A Cox regression model was used for multivariate analysis of the factors that affected the prognosis of HCC. Finally, the association between the expression levels of TEFM and other mitochondrial regulatory genes and HCC biomarker genes was analyzed by GEPIA. TEFM is upregulated in HCC cell lines compared to noncancerous liver cell line. TEFM protein and mRNA expression levels in HCC tissues were significantly upregulated compared with those in noncancerous liver tissues. In addition, the mRNA expression level of TEFM was significantly correlated with sex, serum AFP level, and vascular invasion (P<0.05). Further analysis showed that high expression level of TEFM was unfavorable in terms of the prognosis of patients with HCC. Cox multivariate regression analysis showed that patient age, vascular invasion, and TEFM expression were independent factors affecting the prognosis of HCC patients (P<0.05). The expression level of the TEFM gene was significantly positively correlated with the expression of multiple mitochondrial regulatory genes and biomarker genes of HCC (P<0.01, R>0). Our findings reveal that TEFM may play an important role in the progression of HCC. More importantly, the elevated expression of TEFM may potentially predict poor overall survival (OS) and disease-free survival (DFS) in patients with HCC.

Elevated expression of mitochondrial transcription elongation factor (TEFM) predicts poor prognosis in low grade glioma-an analysis of the Cancer Genome Atlas (TCGA) dataset.

BackgroundMitochondrial transcription elongation factor (TEFM) is a key molecule for mitochondrial DNA (mtDNA) replication-transcription switch. TEFM regulates both transcription elongation and RNA processing in mitochondria. However, the expression level and prognostic value of TEFM in low grade glioma (LGG) remain unclear. Therefore, in this study, we aimed to evaluate the clinical significance and the prognostic value of TEFM in LGG based on publicly available data.MethodsThe relative mRNA expression level of TEFM in non-tumor brain tissues and LGG tissues were retrieved from Gene Expression Profiling Interactive Analysis (GEPIA). The RNA-Seq expression of TEFM and clinical information in LGG patients were collected from the updated the Cancer Genome Atlas (TCGA) database by using R3.6.1 software. Next, the relationship between the mRNA expression of TEFM and clinicopathological characteristics were analyzed. Kaplan-Meier survival curves of overall survival (OS) and disease-free survival (DFS) were implemented for the relationship between the mRNA expression of TEFM and the prognosis of LGG patients. A Cox regression model was performed for the multivariate analysis of the factors affected the prognosis of LGG patients. GEPIA online tool was used to analyze the correlation between TEFM gene expression level and other related mitochondrial regulatory genes in LGG. Finally, The Gene Set Enrichment Analysis (GSEA) was performed to identify cell processes and molecular signaling cascades affected by TEFM.ResultsGEPIA analysis showed that the mRNA expression levels of TEFM in LGG were significantly higher than that of non-tumor tissue. Moreover, the mRNA expression of TEFM is significantly correlated with age, World Health Organization (WHO) grade, pathological types, headache history and supratentorial location (P<0.05). Kaplan-Meier analysis showed that a high expression level of TEFM mRNA indicated a poor prognosis in OS rate (log-rank, P<0.01). Multivariate Cox regression analysis showed that age, WHO grade, pathological types and supratentorial location were the independent prognostic factors of LGG patients. The mRNA expression levels of TEFM gene were positively correlated with the TFAM, TFB1M, TFB2M, MTERF1–F4 and NRF1 gene (P<0.01, R>0), but negatively correlated with the POLRMT gene (P<0.01, R=−0.18) in LGG. The GSEA revealed that genes associated with the cell cycle, RNA degradation, spliceosome, and ubiquitin mediated proteolysis signaling pathway were remarkably enriched in higher-TEFM versus lower-TEFM tumors.ConclusionsOur findings disclosed that the expression of TEFM mRNA was significantly upregulated in human LGG tissues compared to non-tumor brain tissues. More importantly, the elevated expression of TEFM mRNA may potentially predict poor OS in LGG patients.

TEFM regulates both transcription elongation and RNA processing in mitochondria.

Regulation of replication and expression of mitochondrial DNA (mtDNA) is essential for cellular energy conversion via oxidative phosphorylation. The mitochondrial transcription elongation factor (TEFM) has been proposed to regulate the switch between transcription termination for replication primer formation and processive, near genome‐length transcription for mtDNA gene expression. Here, we report that Tefm is essential for mouse embryogenesis and that levels of promoter‐distal mitochondrial transcripts are drastically reduced in conditional Tefm‐knockout hearts. In contrast, the promoter‐proximal transcripts are much increased in Tefm knockout mice, but they mostly terminate before the region where the switch from transcription to replication occurs, and consequently, de novo mtDNA replication is profoundly reduced. Unexpectedly, deep sequencing of RNA from Tefm knockouts revealed accumulation of unprocessed transcripts in addition to defective transcription elongation. Furthermore, a proximity‐labeling (BioID) assay showed that TEFM interacts with multiple RNA processing factors. Our data demonstrate that TEFM acts as a general transcription elongation factor, necessary for both gene transcription and replication primer formation, and loss of TEFM affects RNA processing in mammalian mitochondria.

TEFM Enhances Transcription Elongation by Modifying mtRNAP Pausing Dynamics

Regulation of transcription elongation is one of the key mechanisms employed to control gene expression. The single-subunit mitochondrial RNA polymerase (mtRNAP) transcribes mitochondrial genes, such as those related to ATP synthesis. We investigated how mitochondrial transcription elongation factor (TEFM) enhances mtRNAP transcription elongation using a single-molecule optical-tweezers transcription assay, which follows transcription dynamics in real time and allows the separation of pause-free elongation from transcriptional pauses. We found that TEFM enhances the stall force of mtRNAP. Although TEFM does not change the pause-free elongation rate, it enhances mtRNAP transcription elongation by reducing the frequency of long-lived pauses and shortening their durations. Furthermore, we demonstrate how mtRNAP passes through the conserved sequence block II, which is the key sequence for the switch between DNA replication and transcription in mitochondria. Our findings elucidate how both TEFM and mitochondrial genomic DNA sequences directly control the transcription elongation dynamics of mtRNAP.

The DEAD-box helicase Mss116 plays distinct roles in mitochondrial ribogenesis and mRNA-specific translation.

Members of the DEAD-box family are often multifunctional proteins involved in several RNA transactions. Among them, yeast Saccharomyces cerevisiae Mss116 participates in mitochondrial intron splicing and, under cold stress, also in mitochondrial transcription elongation. Here, we show that Mss116 interacts with the mitoribosome assembly factor Mrh4, is required for efficient mitoribosome biogenesis, and consequently, maintenance of the overall mitochondrial protein synthesis rate. Additionally, Mss116 is required for efficient COX1 mRNA translation initiation and elongation. Mss116 interacts with a COX1 mRNA-specific translational activator, the pentatricopeptide repeat protein Pet309. In the absence of Mss116, Pet309 is virtually absent, and although mitoribosome loading onto COX1 mRNA can occur, activation of COX1 mRNA translation is impaired. Mutations abolishing the helicase activity of Mss116 do not prevent the interaction of Mss116 with Pet309 but also do not allow COX1 mRNA translation. We propose that Pet309 acts as an adaptor protein for Mss116 action on the COX1 mRNA 5΄-UTR to promote efficient Cox1 synthesis. Overall, we conclude that the different functions of Mss116 in the biogenesis and functioning of the mitochondrial translation machinery depend on Mss116 interplay with its protein cofactors.

Transcription: a mitochondrial switch between transcription and replication.

A new study shows that the mitochondrial transcription elongation factor TEFM serves as a molecular switch between replication and transcription.

TEFM is a potent stimulator of mitochondrial transcription elongation in vitro.

A single-subunit RNA polymerase, POLRMT, transcribes the mitochondrial genome in human cells. Recently, a factor termed as the mitochondrial transcription elongation factor, TEFM, was shown to stimulate transcription elongation in vivo, but its effect in vitro was relatively modest. In the current work, we have isolated active TEFM in recombinant form and used a reconstituted in vitro transcription system to characterize its activities. We show that TEFM strongly promotes POLRMT processivity as it dramatically stimulates the formation of longer transcripts. TEFM also abolishes premature transcription termination at conserved sequence block II, an event that has been linked to primer formation during initiation of mtDNA synthesis. We show that POLRMT pauses at a wide range of sites in a given DNA sequence. In the absence of TEFM, this leads to termination; however, the presence of TEFM abolishes this effect and aids POLRMT in continuation of transcription. Further, we show that TEFM substantially increases the POLRMT affinity to an elongation-like DNA:RNA template. In combination with previously published in vivo observations, our data establish TEFM as an essential component of the mitochondrial transcription machinery.

Replication-transcription switch in human mitochondria

Coordinated replication and expression of the mitochondrial genome is critical for metabolically active cells during various stages of development. However, it is not known whether replication and transcription can occur simultaneously without interfering with each other and whether mitochondrial DNA copy number can be regulated by the transcription machinery. We found that interaction of human transcription elongation factor TEFM with mitochondrial RNA polymerase and nascent transcript prevents the generation of replication primers and increases transcription processivity and thereby serves as a molecular switch between replication and transcription, which appear to be mutually exclusive processes in mitochondria. TEFM may allow mitochondria to increase transcription rates and, as a consequence, respiration and adenosine triphosphate production without the need to replicate mitochondrial DNA, as has been observed during spermatogenesis and the early stages of embryogenesis.

Switching transcription and replication

Mitochondrial Biology Because mitochondrial DNA is circular, the transcription and replication machinery might be expected to collide. A single mitochondrial RNA polymerase (mtRNAP) transcribes the mitochondrial DNA and also generates primers for replication. Agaronyan et al. now show that transcription and replication are kept separate in human mitochondria, with the mitochondrial transcription elongation factor TEFM serving as a key player in the switch. In the absence of TEFM, mtRNAP terminates downstream from the promoter, forming primers to promote replication. In the presence of TEFM, the primers are not formed, and the overall processivity of mtRNAP elongation complexes is enhanced, promoting genome transcription. These mutually exclusive mechanisms allow the processes to proceed independently as needed by the cell. Science , this issue p. [548][1] [1]: /lookup/doi/10.1126/science.aaa0986

TEFM (c17orf42) is necessary for transcription of human mtDNA

Here we show that c17orf42, hereafter TEFM (transcription elongation factor of mitochondria), makes a critical contribution to mitochondrial transcription. Inactivation of TEFM in cells by RNA interference results in respiratory incompetence owing to decreased levels of H- and L-strand promoter-distal mitochondrial transcripts. Affinity purification of TEFM from human mitochondria yielded a complex comprising mitochondrial transcripts, mitochondrial RNA polymerase (POLRMT), pentatricopeptide repeat domain 3 protein (PTCD3), and a putative DEAD-box RNA helicase, DHX30. After RNase treatment only POLRMT remained associated with TEFM, and in human cultured cells TEFM formed foci coincident with newly synthesized mitochondrial RNA. Based on deletion mutants, TEFM interacts with the catalytic region of POLRMT, and in vitro TEFM enhanced POLRMT processivity on ss- and dsDNA templates. TEFM contains two HhH motifs and a Ribonuclease H fold, similar to the nuclear transcription elongation regulator Spt6. These findings lead us to propose that TEFM is a mitochondrial transcription elongation factor.

Chimeric templates and assays used to study physarum cotranscriptional insertional editing in vitro.

RNAs made in the mitochondrion of Physarum polycephalum are edited relative to their template by the precise addition of nonencoded nucleotides, while they are being synthesized. This insertional editing has been reproduced in vitro during run-on extension of RNAs initiated in vivo, within partially purified mitochondrial transcription elongation complexes (mtTECs), but it does not occur when the mitochondrial polymerase initiates transcription on exogenous cloned DNA. This chapter describes in vitro transcription systems in which mtTEC RNAs are elongated on repositioned parts of the genome or exogenous DNA, in order to investigate how the nontemplated insertions are directed. Restriction enzyme digestion and DNA ligation are used to generate the chimeric templates, and the RNA products are analyzed directly by nuclease dissection (S1 protection followed by RNase T1 digestion) or by reverse transcriptase-polymerase chain reaction (RT-PCR) followed by restriction enzyme analysis or cloning and sequencing.

Cotranscriptional editing of Physarum mitochondrial RNA requires local features of the native template.

RNAs in the mitochondrion of Physarum polycephalum are edited by the precise cotranscriptional addition of non-encoded nucleotides. Here we describe experiments to address the basis of editing specificity using a series of chimeric templates generated by either rearranging the DNA present in editing-competent mitochondrial transcription elongation complexes (mtTECs) or linking it to exogenous DNA. Notably, run-on transcripts synthesized from rearranged mtTECs are edited at the natural sites, even when different genes are ligated together, yet exogenous, deproteinized DNA does not support editing. Furthermore, the accuracy of nucleotide insertion in chimeric RNAs argues that any cis-acting determinants of cytidine insertion are limited to small regions surrounding editing sites. Taken together, these observations strongly suggest that template-associated factors affect read-out of the mitochondrial genome.