Post-transcriptional Processes Research Articles

Biomolecular condensates in immune cell fate.

Fate decisions during immune cell development require temporally precise changes in gene expression. Evidence suggests that the dynamic modulation of these changes is associated with the formation of diverse, membrane-less nucleoprotein assemblies that are termed biomolecular condensates. These condensates are thought to orchestrate fate-determining transcriptional and post-transcriptional processes by locally and transiently concentrating DNA or RNA molecules alongside their regulatory proteins. Findings have established a link between condensate formation and the gene regulatory networks that ensure the proper development of immune cells. Conversely, condensate dysregulation has been linked to impaired immune cell fates, including ageing and malignant transformation. This Review explores the putative mechanistic links between condensate assembly and the gene regulatory frameworks that govern normal and pathological development in the immune system.

A mitochondria-to-nucleus regulation mediated by the nuclear-translocated mitochondrial lncRNAs.

A bidirectional nucleus-mitochondria communication is essential for homeostasis and stress. By acting as critical molecules, the nuclear-encoded lncRNAs (nulncRNAs) have been implicated in the nucleus-to-mitochondria anterograde regulation. However, role of mitochondrial-derived lncRNAs (mtlncRNAs) in the mitochondria-to-nucleus retrograde regulation remains elusive. Here, we identify functional implication of the mtlncRNAs MDL1AS, lncND5 and lncCyt b in retrograde regulation. Mediated by HuR and PNPT1 proteins, the mtlncRNAs undergo a mitochondria-to-nucleus traveling and then regulate a network of nuclear genes. Moreover, as an example of the functional consequence, we showed that the nuclear-translocated lncCyt b cooperates with the splicing factor hnRNPA2B1 to influence several aspects of cell metabolism including glycolysis, possibly through their regulatory effect on the post-transcriptional processing of related nuclear genes. This study advances our knowledge in mitochondrial biology and provides new insights into the role of mtlncRNAs in mitochondria-nucleus communications.

MRNA Transcript Variants Expressed in Mammalian Cells

Gene expression or gene regulation studies often assume one gene expresses one mRNA. However, contrary to the conventional idea, a single gene in mammalian cells can express multiple transcript variants translated into several different proteins. The transcript variants are generated through transcription from alternative start sites and alternative post-transcriptional processing of the precursor mRNA (pre-mRNA). In addition, gene mutations and RNA editing further enhance the diversity of the transcript variants. The transcript variants can encode proteins with various domains, expanding the functional repertoire of a single gene. Some transcript variants may not encode proteins but function as non-coding RNAs and regulate gene expression. The expression level of the transcript variants may vary between cell types or within the same cells under different biological conditions. Transcript variants are characteristic of cell differentiation in a particular tissue, and the variants may play a key role in normal development and aging. Studies also reported that some transcript variants may have roles in disease pathogenesis. The biological significances urge studying the complexity of gene expression at the transcript level. This article updates the molecular basis of transcript variants in mammalian cells, including the formation mechanisms and potential roles in host biology. Gaining insight into the transcript variants will not only identify novel mechanisms of gene regulation but also unravel the role of the variants in health and disease.

Identification of robust RT-qPCR reference genes for studying changes in gene expression in response to hypoxia in breast cancer cell lines

Hypoxia is common in breast tumours and is linked to therapy resistance and advanced disease. To understand hypoxia-driven breast cancer progression, RT-qPCR is a widely used technique to quantify transcriptional changes that occur during malignant transformation. Reference genes (RGs) are endogenous RT-qPCR controls used to normalise mRNA levels, allowing accurate assessment of transcriptional changes. However, hypoxia reprograms transcription and post-transcriptional processing of RNA such that favoured RGs including GAPDH or PGK1 are unsuitable for this purpose. To address the need for robust RGs to study hypoxic breast cancer cell lines, we identified 10 RG candidates by analysing public RNA-seq data of MCF-7 and T-47D (Luminal A), and, MDA-MB-231 and MDA-MB-468 (triple negative breast cancer (TNBC)) cells cultured in normoxia or hypoxia. We used RT-qPCR to determine RG candidate levels in normoxic breast cancer cells, removing TBP and EPAS1 from downstream analysis due to insufficient transcript abundance. Assessing primer efficiency further removed ACTB, CCSER2 and GUSB from consideration. Following culture in normoxia, acute, or chronic hypoxia, we ascertained robust non-variable RGs using RefFinder. Here we present RPLP1 and RPL27 as optimal RGs for our panel of two Luminal A and two TNBC cell lines cultured in normoxia or hypoxia. Our result enables accurate evaluation of gene expression in selected hypoxic breast cancer cell lines and provides an essential resource for assessing the impact of hypoxia on breast cancer progression.

New Genetic Variations in RNA-binding Protein Gene and Breast Cancer Risk: A Case-Control Study.

LIN28, a highly conserved RNA-binding protein, regulate a wide variety of post-transcriptional cellular processes. The current study aimed to identify genetic variants of five single nucleotide polymorphisms (SNPs) in the LIN28B gene (rs221634, rs22163, rs314276, rs9404590, and rs12194974) and their association with Breast cancer. 220 patients and 230 controls were genotyped by the RFLP assay for Lin28B gene variants. Odds ratio analysis was used to determine the association between Lin28B variants and breast cancer. Haplotype analysis was performed to determine the combined impact of the investigated variants on BC. Novel in-silico analysis were performed to predict the potential functions of these polymorphisms, as well. Patients carrying all variant genotypes for lin28B rs221634 (codominant, dominant, recessive, and allelic inheritance models), rs221635 (codominant and dominant genotypes), and rs9404590 (codominant, dominant, and inheritance model). Significant associations between reduced cancer risk and rs12194974 and rs314276 were found in codominant, dominant, recessive, and allele inheritance models. According to haplotype analysis of rs9404590, rs12194974, rs314276, rs221634, and rs221635 SNPs ,the GGCTT, GGCAT, TGCAC, TGCTC, GGCAC, GGCTC, and GGAAC haplotypes are associated with an increased risk of BC, whereas the TACAT and TAAAT haplotypes were associated with a decreased risk of BC. The splicing enhancers (ESE) binding site was found to be altered by the SNPs rs9404590, rs12194974, and rs314276, according to in-silico analysis. Breast cancer susceptibility appears to be linked to genetic variations in the Lin28B gene, and haplotypes in this region have been linked to increased risk.

Post-transcriptional regulation of behavior plasticity in social insects.

Social insects often show remarkable behavioral plasticity, which is closely associated with their respective castes. The underpinnings of this plasticity are complex, involving genetic differences among individuals within a colony and regulation of gene expression at multiple levels. Post-transcriptional regulation, which increases the complexity of the transcriptome, plays a crucial role in the multilayer regulatory network that influences social insect behavior. We provide an overview of the impact of three post-transcriptional regulatory processes on the reproductive division of labor and worker division of labor in social insects: alternative splicing, RNA modifications and non-coding RNAs. We also discuss the relationship between post-transcriptional regulation and chromatin modification.

UBL5 and Its Role in Viral Infections.

Unlike other ubiquitin-like family members, UBL5 is structurally and functionally atypical, and a novel role in various biological processes and diseases has been discovered. UBL5 can stabilize the structure of the spliceosome, can promote post-transcriptional processing, and has been implicated in both DNA damage repair and protein unfolding reactions, as well as cellular mechanisms that are frequently exploited by viruses for their own proliferation during viral infections. In addition, UBL5 can inhibit viral infection by binding to the non-structural protein 3 of rice stripe virus and mediating its degradation. Therefore, UBL5 is an important link between viral infections and immunity, and its study will be beneficial for the prevention and treatment of viral infections in the future. However, a review of the current findings on the role of UBL5 in viral infection has not been undertaken. Therefore, in this review, we summarize the recent progress in understanding the functions of UBL5 and discuss its putative role in viral infections.

SLAM-seq reveals independent contributions of RNA processing and stability to gene expression in African trypanosomes.

Gene expression is a multi-step process that converts DNA-encoded information into proteins, involving RNA transcription, maturation, degradation, and translation. While transcriptional control is a major regulator of protein levels, the role of post-transcriptional processes such as RNA processing and degradation is less well understood due to the challenge of measuring their contributions individually. To address this challenge, we investigated the control of gene expression in Trypanosoma brucei, a unicellular parasite assumed to lack transcriptional control. Instead, mRNA levels in T. brucei are controlled by post-transcriptional processes, which enabled us to disentangle the contribution of both processes to total mRNA levels. In this study, we developed an efficient metabolic RNA labeling approach and combined ultra-short metabolic labeling with transient transcriptome sequencing (TT-seq) to confirm the long-standing assumption that RNA polymerase II transcription is unregulated in T. brucei. In addition, we established thiol (SH)-linked alkylation for metabolic sequencing of RNA (SLAM-seq) to globally quantify RNA processing rates and half-lives. Our data, combined with scRNA-seq data, indicate that RNA processing and stability independently affect total mRNA levels and contribute to the variability seen between individual cells in African trypanosomes.

Intron RPS25Ai, a Novel DNA Element, Has Global Effects on Synthetic Pathway Engineering by Empowering Protein Synthesis.

Classical genetic components in synthetic biology encompass essential elements of promoters, transcription factors, protein-coding genes, and terminators while both academic and industrial needs require novel engineering tools. Our study explores the potential of introns as versatile, novel biological DNA elements. Using intron RPS25Ai from Saccharomyces cerevisiae, the expression of mCherry was enhanced by 18.4-fold, demonstrating spatiotemporal regulatory patterns at both transcriptional and translational levels. A molecular mechanism study shows that this distinctive fine-tuning control relies on correct splicing events and extends to post-transcriptional processes. Intron RPS25Ai was applied to a heterologous metabolic pathway in engineered yeast, increasing β-carotene production by 4.29-fold. RPS25Ai functioned as a multilevel regulatory genetic element, enabling the increase in the expression of crtYB both at the pre-mRNA (99%) and mature RNA level (64%), with a splicing efficiency of 82%. Furthermore, the intron-engineered strain achieved a genome-scale regulation, upregulating 67% of "intron-containing" genes, with an average expression increase of 27%, compared with the upregulation of only 37% of "no-intron" genes. In addition, RPS25Ai induced a comprehensive rearrangement of ribosomal components, with the expression of 89% of ribosomal genes being upregulated, further empowering protein synthesis in the β-carotene-producing yeast cell factory.

Pentatricopeptide repeat proteins in plants: Cellular functions, action mechanisms and potential applications.

Pentatricopeptide repeat (PPR) proteins are involved in nearly all aspects of post-transcriptional processing in plant mitochondria and plastids, where they play a vital role in plant growth, development, cytoplasmic male sterility (CMS) restoration, and response to biotic and abiotic stresses. Through research in the last three decades, PPR functions and the primary mechanisms by which PPR proteins mediate post-transcriptional processing have been uncovered. Here, we aim to summarize the advances in PPR research with highlighting on the mechanisms of how PPR proteins mediate RNA editing, intron splicing, and RNA maturation in the context of their role in organellar gene expression. We also present the latest progress toward PPR engineering and an outlook for their potential applications as biotechnological tools. Additionally, we discuss further questions that need to be answered.

Alternative splicing regulation by tumor suppressing subtransferable candidate 4: a pathway to tumor suppression.

RNA splicing is a crucial posttranscriptional process that governs gene expression, and defects in alternative splicing contribute to various diseases, including cancer. Tumor suppressing subtransferable candidate 4 (TSSC4) is a known tumor suppressor and has been identified as part of the U5 small nuclear ribonucleoprotein (snRNP), which is involved in tri-snRNP biogenesis. However, the precise role of TSSC4 in regulating alternative splicing and its impact on tumor growth remain unclear. To explore the link between splicing modulation and tumor suppression driven by TSSC4, we conducted transcriptome sequencing (RNA-seq) on TSSC4-knockout and wild-type HeLa cells. Additionally, we analyzed alternative splicing and gene expression in various cancer cell lines, including TSSC4-knockout A549 cells and TSSC4-knockdown PANC-1, MDA-MB-231, and MCF-7 cells. Splicing patterns and gene expression profiles were compared between TSSC4-deficient and control cells. Our RNA-seq analysis revealed that TSSC4 deficiency in HeLa cells results in widespread alterations in splicing patterns and gene expression. Specifically, the loss of TSSC4 led to abnormal alternative splicing events and dysregulation of tumor-associated genes, including several oncogenes. This effect was confirmed across multiple cancer cell lines, highlighting a consistent role of TSSC4 in splicing regulation. These findings demonstrate that TSSC4 plays a crucial role in regulating RNA splicing, particularly in controlling the splicing of many oncogenes. Our results reveal a novel mechanism by which TSSC4 mediates tumor suppression through the modulation of alternative splicing, which could provide implications for understanding TSSC4's role in cancer biology.

Advances and Prospects of Plant Mitochondrial Pentatricopeptide Repeat Proteins in Post-transcriptional Processing

Advances and Prospects of Plant Mitochondrial Pentatricopeptide Repeat Proteins in Post-transcriptional Processing

Differences in Splicing Factors may predict Type 2 Diabetes Remission in the CORDIOPREV study

Alternative splicing is a post-transcriptional process resulting in multiple protein isoforms from a single gene. Abnormal splicing may lead to metabolic diseases, including type 2 diabetes mellitus (T2DM). To identify splicing factor expression predicts T2DM remission in coronary heart disease (CHD) patients, we identified newly diagnosed T2DM at baseline (n=190) from the CORDIOPREV study. Patients were classified as Responders (T2DM remission during 5 years without antidiabetic drugs) or non-Responders. Baseline dysregulation in 5 splicing factors (MBNL1, RBM5, hnRNP G/RBMX, CD44, NT5E) distinguished Responders from non-Responders. Adding these factors to clinical variables [AUC=0.67], insulin resistance and beta-cell indexes [AUC=0.76], improved T2DM remission prediction [AUC=0.80]. Cox regression analysis showed those with higher remission scores had a 2.63-fold increased remission probability. To conclude, a set of splicing factors contribute to predicting T2DM remission in patients with CHD has been identified. Further research is needed to elucidate these findings’ clinical relevance.

RNA stability is regulated by both RNA polyadenylation and ATP levels, linking RNA and energy metabolisms in Escherichia coli.

The post-transcriptional process of RNA polyadenylation sits at the crossroads of energy metabolism and RNA metabolism. RNA polyadenylation is catalyzed by poly(A) polymerases, which use ATP as a substrate to add adenine to the 3' end of RNAs, which can alter their stability. In Escherichia coli, RNA polyadenylation mediated by the major poly(A) polymerase was previously shown to facilitate degradation of individual RNAs. In this study, we performed the first genome-wide study of RNA stability in the absence of PAP I. Inactivation of the pcnB gene coding for PAP I led to the stabilization of more than a thousand of E. coli RNAs in the form of full-length functional molecules or non-functional fragments. The absence of PAP I altered the energy metabolism, with an almost 20% reduction in ATP levels. To better understand how RNA and energy metabolisms are interconnected, we investigated the role of ATP levels in regulating RNA stability. When we lowered intracellular ATP levels below 0.5 mM, many RNAs were stabilized, demonstrating the causal link between ATP levels and RNA stability for the first time in E. coli. Above this concentration, changes in ATP levels had no impact on RNA stability. We also demonstrated that some RNAs were stabilized when PAP I was inactivated by low ATP availability. These results clearly demonstrate that PAP I mediates an energy-dependent RNA stabilization, which may contribute to cell energy homeostasis under energy-limited conditions.IMPORTANCEPoly(A) polymerases are prime targets for understanding the interactions between RNA polyadenylation, RNA stability, and cellular energy. These enzymes catalyze the process of RNA polyadenylation, which involves ATP hydrolysis and addition of poly(A) tails to the 3' end of RNAs. 3' end poly(A) extensions potentially facilitate RNA degradation in bacteria. In this study, we inactivated the pcnB gene encoding PAP I, the major poly(A) polymerase in E. coli, and investigated the effects on RNA stability and energy levels. Our results show for the first time in E. coli a genome-wide RNA stabilization in the absence of PAP I associated with a decrease in ATP levels. We provide the first evidence in E. coli of a link between ATP levels and RNA stabilization and demonstrate that this is mediated in some cases by PAP I. PAP I-mediated RNA stabilization at low ATP levels could be a means of energy conservation under energy-limited conditions.

Nanopore direct RNA sequencing reveals N6-methyladenosine and polyadenylation landscapes on long non-coding RNAs in Arabidopsis thaliana

BackgroundLong non-coding RNAs (lncRNAs) play important roles in various biological processes, including stage development in plants. N6-methyladenosine (m6A) modification and polyadenylation are noteworthy regulatory processes that impact transcript functions by modulating their abundance. However, the specific landscapes of m6A modification and polyadenylation on lncRNAs remain largely unexplored. The advent of nanopore direct RNA sequencing (DRS) provides unprecedented opportunities for directly detecting m6A modifications and estimating polyadenine (poly[A]) tail lengths on individual RNA molecules.ResultsHere we utilized nanopore DRS to identify lncRNAs and map the transcriptome-wide m6A modification and polyadenylation landscapes in the model plant Arabidopsis thaliana. Leveraging the Low-abundance Aware Full-length Isoform clusTEr (LAFITE) assembly pipeline, we identified 1149 novel lncRNAs in seventeen nanopore DRS datasets from the wild-type Columbia-0. Through the precise detection of 2381 m6A modification sites on lncRNAs at single-base resolution, we observed that lncRNAs exhibited lower methylation levels compared to protein-coding RNAs, and m6A modification facilitated lncRNA abundance. Additionally, we estimated the poly(A) tail lengths of individual lncRNAs and found that poly(A) tails contributed to lncRNA stability, while their effect was not length-dependent. Furthermore, by comparing lncRNA abundance between 2-week seedlings and 5-week floral buds, we revealed the dynamic expression patterns of lncRNAs during the transition from the vegetative stage to the reproductive stage. These observations provided insights into their potential roles in specific tissues or stages in Arabidopsis, including regulating stage development. Moreover, by integrating information on m6A modification, we unveiled a positive correlation between methylation variances and differential expressions of lncRNAs during stage development.ConclusionsThese findings highlighted the significance of epigenetic modification and post-transcriptional processing in shaping lncRNA expression and their functions during Arabidopsis stage development, contributing to the growing field of lncRNA research in plants.Clinical trial numberNot applicable.

RNA Modifications in Pathogenic Viruses: Existence, Mechanism, and Impacts.

RNA modification is a key posttranscriptional process playing various biological roles, and one which has been reported to exist extensively in cellular RNAs. Interestingly, recent studies have shown that viral RNAs also contain a variety of RNA modifications, which are regulated dynamically by host modification machinery and play critical roles in different stages of the viral life cycle. In this review, we summarize the reports of four typical modifications reported on viral RNAs, including N6-methyladenosine (m6A), 5-methylcytosine (m5C), N4-acetylcytosine (ac4C), and N1-methyladenosine (m1A), describe the molecular mechanisms of these modification processes, and illustrate their impacts on viral replication, pathogenicity, and innate immune responses. Notably, we find that RNA modifications in different viruses share some common features and mechanisms in their generation, regulation, and function, highlighting the potential for viral RNA modifications and the related host machinery to serve as the targets or bases for the development of antiviral therapeutics and vaccines.

A Systematic Identification of RNA-Binding Proteins (RBPs) Driving Aberrant Splicing in Cancer.

Alternative Splicing (AS) is a post-transcriptional process that allows a single RNA to produce different mRNA variants and, in some cases, multiple proteins. Various processes, many yet to be discovered, regulate AS. This study focuses on regulation by RNA-binding proteins (RBPs), which are not only crucial for splicing regulation but also linked to cancer prognosis and are emerging as therapeutic targets for cancer treatment. CLIP-seq experiments help identify where RBPs bind on nascent transcripts, potentially revealing changes in splicing status that suggest causal relationships. Selecting specific RBPs for CLIP-seq experiments is often driven by a priori hypotheses. We developed an algorithm to detect RBPs likely related to splicing changes between conditions by integrating several CLIP-seq databases and a differential splicing detection algorithm. This work refines a previous study by improving splicing event prediction, testing different enrichment statistics, and performing additional validation experiments. The new method provides more accurate predictions and is included in the Bioconductor package EventPointer 3.14. We tested the algorithm in four experiments involving knockdowns of seven different RBPs. The algorithm accurately assessed the statistical significance of these RBPs using only splicing alterations. Additionally, we applied the algorithm to study sixteen cancer types from The Cancer Genome Atlas (TCGA) and three from TARGET. We identified relationships between RBPs and various cancer types, including alterations in CREBBP and MBNL2 in adenocarcinomas of the lung, liver, prostate, rectum, stomach, and colon. Some of these findings are validated in the literature, while others are novel. The developed algorithm enhances the ability to predict and understand RBP-related splicing changes, offering more accurate predictions and novel insights into cancer-related splicing alterations. This work highlights the potential of RBPs as therapeutic targets and contributes to the broader understanding of their roles in cancer biology.

Current insights on m6A RNA modification in acute leukemia: therapeutic targets and future prospects.

RNA modification is the critical mechanism for regulating post-transcriptional processes. There are more than 150 RNA modifications reported so far, among which N6-Methyladenosine is the most prevalent one. M6A RNA modification complex consists of 'writers', 'readers' and 'erasers' which together in a group catalyze, recognize and regulate the methylation process of RNA and thereby regulate the stability and translation of mRNA. The discovery of erasers also known as demethylases, revolutionized the research on RNA modifications as it revealed that this modification is reversible. Since then, various studies have focused on discovering the role of m6A modification in various diseases especially cancers. Aberrant expression of these 'readers', 'writers', and 'erasers' is found to be altered in various cancers resulting in disturbance of cellular homeostasis. Acute leukemias are the most common cancer found in pediatric patients and account for 20% of adult cases. Dysregulation of the RNA modifying complex have been reported in development and progression of hematopoietic malignancies. Further, targeting m6A modification is the new approach for cancer immunotherapy and is being explored extensively. This review provides detailed information about current information on the role of m6A RNA modification in acute leukemia and their therapeutic potential.

The Roles of RNA Modifications in Regulating Chloroplast Performance and Photosynthesis Efficiency.

The regulation of gene expression is crucial for maintaining cellular activities and responding to environmental stimuli. RNA molecules are central to this regulatory network, influencing transcription, post-transcriptional processing, and translation. Recent advancements have expanded our understanding of RNA modifications beyond the nucleus, highlighting their impact on chloroplast function and photosynthesis efficiency. Chloroplasts, essential for photosynthesis, rely on precise genetic regulation to adapt to environmental changes. RNA modifications, such as methylation and pseudouridylation, are critical in regulating chloroplast RNA stability, processing, and translation. This review summarizes current knowledge of how RNA modifications affect chloroplast function and photosynthesis. It discusses the roles of specific RNA modifications occurring in chloroplast RNA, including N6-methyladenosine (m6A), 5-methylcytosine (m5C), and pseudouridylation, as well as the enzymes which are known to be involved in these processes. This review also explores extrachloroplastic RNA modifications that influence chloroplast function, emphasizing the importance of m6A and m5C modifications and their associated enzymes.

RNA editing-induced structural and functional adaptations of NAD9 in Triticum aestivum under drought stress.

Mitochondria are essential organelles in eukaryotic cells, producing ATP through the electron transport chain to supply energy for cellular activities. Beyond energy production, mitochondria play crucial roles in cellular signaling, stress responses, and the regulation of reactive oxygen species. In plants, mitochondria are one of the keys to responding to environmental stresses which can significantly affect crop productivity, particularly in crops like wheat. RNA editing, a post-transcriptional RNA modification process in mitochondria, is linked to regulating these stress responses. This study explores RNA editing patterns in the nad9 gene of wheat drought-tolerant (Giza168) and drought-sensitive (Gemmiza10) wheat cultivars under drought stress to understand plant adaptation mechanisms. RNA-seq data for these cultivars were analyzed using CLC Genomic Workbench to identify RNA editing sites in the nad9 gene, examining subsequent amino acid changes and predicting secondary structure modifications. These RNA editing sites were validated using qRT-PCR on drought-treated seedlings at 0, 2, and 12 hours post-treatment. Protein models were generated using AlphaFold, with functional predictions and structure verification conducted using various bioinformatics tools to investigate the effect of RNA editing on protein level. The results showed significant RNA editing events, especially C-to-T conversions, in the nad9 gene across different drought exposure times. Giza168 had 22 editing sites, while Gemmiza10 had 19, with several showing significant differences between control and stress conditions. RNA editing influenced the NAD9 protein's secondary structure, particularly beta sheets, and 3D modeling highlighted the structural impacts of these edits. The N-terminal region of NAD9 contained important regulatory motifs, suggesting a complex regulatory environment. This study reveals key editing sites that differ between drought-tolerant and sensitive wheat cultivars, impacting NAD9 protein structures and highlighting the role of RNA editing in enhancing drought resilience. Additionally, the study suggests potential regulatory mechanisms, including phosphorylation and ubiquitination that influence mitochondrial stability and function.