Post-transcriptional Steps Research Articles

SAMD9L acts as an antiviral factor against HIV-1 and primate lentiviruses by restricting viral and cellular translation.

Sterile alpha motif domain-containing proteins 9 and 9-like (SAMD9/9L) are associated with life-threatening genetic diseases in humans and are restriction factors of poxviruses. Yet, their cellular function and the extent of their antiviral role are poorly known. Here, we found that interferon-stimulated human SAMD9L restricts HIV-1 in the late phases of replication, at the posttranscriptional and prematuration steps, impacting viral translation and, possibly, endosomal trafficking. Surprisingly, the paralog SAMD9 exerted an opposite effect, enhancing HIV-1. More broadly, we showed that SAMD9L restricts primate lentiviruses, but not a gammaretrovirus (MLV), nor 2 RNA viruses (arenavirus MOPV and rhabdovirus VSV). Using structural modeling and mutagenesis of SAMD9L, we identified a conserved Schlafen-like active site necessary for HIV-1 restriction by human and a rodent SAMD9L. By testing a gain-of-function constitutively active variant from patients with SAMD9L-associated autoinflammatory disease, we determined that SAMD9L pathogenic functions also depend on the Schlafen-like active site. Finally, we found that the constitutively active SAMD9L strongly inhibited HIV, MLV, and, to a lesser extent, MOPV. This suggests that the virus-specific effect of SAMD9L may involve its differential activation/sensing and the virus ability to evade from SAMD9L restriction. Overall, our study identifies SAMD9L as an HIV-1 antiviral factor from the cell autonomous immunity and deciphers host determinants underlying the translational repression. This provides novel links and therapeutic avenues against viral infections and genetic diseases.

In vivo Polycystin-1 interactome using a novel Pkd1 knock-in mouse model.

PKD1 is the most commonly mutated gene causing autosomal dominant polycystic kidney disease (ADPKD). It encodes Polycystin-1 (PC1), a putative membrane protein that undergoes a set of incompletely characterized post-transcriptional cleavage steps and has been reported to localize in multiple subcellular locations, including the primary cilium and mitochondria. However, direct visualization of PC1 and detailed characterization of its binding partners remain challenging. We now report a new mouse model with HA epitopes and eGFP knocked-in frame into the endogenous mouse Pkd1 gene by CRISPR/Cas9. Using this model, we sought to visualize endogenous PC1-eGFP and performed affinity-purification mass spectrometry (AP-MS) and network analyses. We show that the modified Pkd1 allele is fully functional but the eGFP-tagged protein cannot be detected without signal amplification by secondary antibodies. Using nanobody-coupled beads and large quantities of tissue, AP-MS identified an in vivo PC1 interactome, which is enriched for mitochondrial proteins and components of metabolic pathways. These studies suggest this mouse model and interactome data will be useful to understand PC1 function, but that new methods and brighter tags will be required to track endogenous PC1.

Tip60's Novel RNA-Binding Function Modulates Alternative Splicing of Pre-mRNA Targets Implicated in Alzheimer's Disease.

The severity of Alzheimer's disease (AD) progression involves a complex interplay of genetics, age, and environmental factors orchestrated by histone acetyltransferase (HAT)-mediated neuroepigenetic mechanisms. While disruption of Tip60 HAT action in neural gene control is implicated in AD, alternative mechanisms underlying Tip60 function remain unexplored. Here, we report a novel RNA binding function for Tip60 in addition to its HAT function. We show that Tip60 preferentially interacts with pre-mRNAs emanating from its chromatin neural gene targets in the Drosophila brain and this RNA binding function is conserved in human hippocampus and disrupted in Drosophila brains that model AD pathology and in AD patient hippocampus of either sex. Since RNA splicing occurs co-transcriptionally and alternative splicing (AS) defects are implicated in AD, we investigated whether Tip60-RNA targeting modulates splicing decisions and whether this function is altered in AD. Replicate multivariate analysis of transcript splicing (rMATS) analysis of RNA-Seq datasets from wild-type and AD fly brains revealed a multitude of mammalian-like AS defects. Strikingly, over half of these altered RNAs are identified as bona-fide Tip60-RNA targets that are enriched for in the AD-gene curated database, with some of these AS alterations prevented against by increasing Tip60 in the fly brain. Further, human orthologs of several Tip60-modulated splicing genes in Drosophila are well characterized aberrantly spliced genes in human AD brains, implicating disruption of Tip60's splicing function in AD pathogenesis. Our results support a novel RNA interaction and splicing regulatory function for Tip60 that may underly AS impairments that hallmark AD etiology.SIGNIFICANCE STATEMENT Alzheimer's disease (AD) has recently emerged as a hotbed for RNA alternative splicing (AS) defects that alter protein function in the brain yet causes remain unclear. Although recent findings suggest convergence of epigenetics with co-transcriptional AS, whether epigenetic dysregulation in AD pathology underlies AS defects remains unknown. Here, we identify a novel RNA interaction and splicing regulatory function for Tip60 histone acetyltransferase (HAT) that is disrupted in Drosophila brains modeling AD pathology and in human AD hippocampus. Importantly, mammalian orthologs of several Tip60-modulated splicing genes in Drosophila are well characterized aberrantly spliced genes in human AD brain. We propose that Tip60-mediated AS modulation is a conserved critical posttranscriptional step that may underlie AS defects now characterized as hallmarks of AD.

MicroRNA Biogenesis in Cell Senescence Induced by Chronic Endoplasmic Reticulum Stress

MicroRNAs are small noncoding RNAs that regulate gene expression; stabilize the cell phenotype; and play an important role in cell differentiation, development, and apoptosis. A canonical microRNA biogenesis pathway includes several posttranscriptional steps of processing and transport and ends with cytoplasmic cleavage of pre-miRNA by type III ribonuclease DICER to form a mature duplex, which is included in RISC. MicroRNA biogenesis and role in cell stress are still poorly understood. Using flow cytometry and high-throughput analysis of gene expression, we have shown that chronic endoplasmic reticulum (ER) stress, which is associated with improper protein folding in the ER, induce a cellular senescence phenotype in fibroblast-like FRSN cells. While acute ER stress can reduce miRNA biogenesis, chronic stress does not cause a significant drop in global microRNA expression and is accompanied by only a slight decrease in DICER1 mRNA expression. Heterogeneity with respect to lysosomal β-galactosidase activity was found to increase in the cell population exposed to ER stress. We do not exclude induced cell heterogeneity regarding expression of components of the microRNA biogenesis pathway.

Circulating microRNAs as emerging regulators of COVID-19.

Coronavirus disease 2019 (COVID-19), an infectious disease caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global pandemic that has high incidence rates, spreads rapidly, and has caused more than 6.5 million deaths globally to date. Currently, several drugs have been used in the clinical treatment of COVID-19, including antivirals (e.g., molnupiravir, baricitinib, and remdesivir), monoclonal antibodies (e.g., etesevimab and tocilizumab), protease inhibitors (e.g., paxlovid), and glucocorticoids (e.g., dexamethasone). Increasing evidence suggests that circulating microRNAs (miRNAs) are important regulators of viral infection and antiviral immune responses, including the biological processes involved in regulating COVID-19 infection and subsequent complications. During viral infection, both viral genes and host cytokines regulate transcriptional and posttranscriptional steps affecting viral replication. Virus-encoded miRNAs are a component of the immune evasion repertoire and function by directly targeting immune functions. Moreover, several host circulating miRNAs can contribute to viral immune escape and play an antiviral role by not only promoting nonstructural protein (nsp) 10 expression in SARS coronavirus, but among others inhibiting NOD-like receptor pyrin domain-containing (NLRP) 3 and IL-1β transcription. Consequently, understanding the expression and mechanism of action of circulating miRNAs during SARS-CoV-2 infection will provide unexpected insights into circulating miRNA-based studies. In this review, we examined the recent progress of circulating miRNAs in the regulation of severe inflammatory response, immune dysfunction, and thrombosis caused by SARS-CoV-2 infection, discussed the mechanisms of action, and highlighted the therapeutic challenges involving miRNA and future research directions in the treatment of COVID-19.

CNNSplice: Robust models for splice site prediction using convolutional neural networks

The identification of splice site, or segments of an RNA gene where noncoding and coding sequences are connected in the 5′ and 3′ directions, is an essential post-transcriptional step for the annotation of functional genes and is required for the study and analysis of biological function in eukaryotic organisms through protein production and gene expression. Splice site detection tools have been proposed for this purpose; however, the models of these tools have a specific use case and are inefficiently or typically untransferable between organisms. Here, we present CNNSplice, a set of deep convolutional neural network models for splice site prediction. Using the five-fold cross-validation model selection technique, we explore several models based on typical machine learning applications and propose five high-performing models to efficiently predict the true and false SS in balanced and imbalanced datasets. Our evaluation results indicate that CNNSplice’s models achieve a better performance compared with existing methods across five organisms’ datasets. In addition, our generality test shows CNNSplice’s model ability to predict and annotate splice sites in new or poorly trained genome datasets indicating a broad application spectrum. CNNSplice demonstrates improved model prediction, interpretability, and generalizability on genomic datasets compared to existing splice site prediction tools. We have developed a web server for the CNNSplice algorithm which can be publicly accessed here: http://www.cnnsplice.online

Inhibition of Aflatoxin Production by Citrinin and Non-Enzymatic Formation of a Novel Citrinin-Kojic Acid Adduct.

Screening for microorganisms that inhibit aflatoxin production from environments showed that Penicillium citrinum inhibited aflatoxin production by Aspergillus parasiticus. The inhibitory substance in the culture medium of P. citrinum was confirmed to be citrinin (CTN). RT-PCR analyses showed that CTN did not inhibit expressions of aflatoxin biosynthetic genes (aflR, pksL1, and fas-1) of A. parasiticus, whereas feeding experiments using A. parasiticus showed that CTN inhibited the in vivo conversion of dihydrosterigmatocystin to AFB2·AFG2. These results suggest that CTN inhibits a certain post-transcriptional step in aflatoxin biosynthesis. CTN in the culture medium of A. parasiticus was found to be decreased or lost with time, suggesting that a certain metabolite produced by A. parasiticus is the cause of the CTN decrease; we then purified, characterized, and then analyzed the substance. Physico-chemical analyses confirmed that the metabolite causing a decrease in CTN fluorescence was kojic acid (KA) and the resulting product was identified as a novel substance: (1R,3S,4R)-3,4-dihydro-6,8-dihydroxy-1-(3-hydroxy-6-(hydroxymethyl)-4-oxo-4H-pyran-2-yl)-3,4,5-trimethyl-1H-isochromene-7-carboxylic acid, which was named "CTN-KA adduct". Our examination of the metabolites' toxicities revealed that unlike CTN, the CTN-KA adduct did not inhibit aflatoxin production by A. parasiticus. These results indicate that CTN's toxicity was alleviated with KA by converting CTN to the CTN-KA adduct.

An Overview of Pentatricopeptide Repeat (PPR) Proteins in the Moss Physcomitrium patens and Their Role in Organellar Gene Expression.

Pentatricopeptide repeat (PPR) proteins are one type of helical repeat protein that are widespread in eukaryotes. In particular, there are several hundred PPR members in flowering plants. The majority of PPR proteins are localized in the plastids and mitochondria, where they play a crucial role in various aspects of RNA metabolism at the post-transcriptional and translational steps during gene expression. Among the early land plants, the moss Physcomitrium (formerly Physcomitrella) patens has at least 107 PPR protein-encoding genes, but most of their functions remain unclear. To elucidate the functions of PPR proteins, a reverse-genetics approach has been applied to P. patens. To date, the molecular functions of 22 PPR proteins were identified as essential factors required for either mRNA processing and stabilization, RNA splicing, or RNA editing. This review examines the P. patens PPR gene family and their current functional characterization. Similarities and a diversity of functions of PPR proteins between P. patens and flowering plants and their roles in the post-transcriptional regulation of organellar gene expression are discussed.

An Alternatively Spliced Variant of METTL3 Mediates Tumor Suppression in Hepatocellular Carcinoma.

Many post-transcriptional mRNA processing steps play crucial roles in tumorigenesis and the progression of cancers, such as N6-methyladenosine (m6A) modification and alternative splicing. Upregulation of methyltransferase-like 3 (METTL3), the catalytic core of the m6A methyltransferase complex, increases m6A levels and results in significant effects on the progression of hepatocellular carcinoma (HCC). However, alternative splicing of METTL3 has not been fully investigated, and the functions of its splice variants remain unclear. Here, we analyzed both our and online transcriptomic data, obtaining 13 splice variants of METTL3 in addition to canonical full-length METTL3-A in HCC cell lines and tissues. Validated by RT–qPCR and Western blotting, we found that METTL3-D, one of the splice variants expressing a truncated METTL3 protein, exhibits higher levels than METTL3-A in normal human livers but lower levels than METTL3-A in HCC tumor tissues and cell lines. Further functional assays demonstrated that METTL3-D expression decreased cellular m6A modification, inhibited the proliferation, migration, and invasion of HCC cells, and was negatively associated with the malignancy of patient tumors, exhibiting functions opposite to those of full-length METTL3-A. This study demonstrates that the METTL3-D splice variant is a tumor suppressor that could potentially be used as a target for HCC therapy.

Comprehensive analysis of plastid gene expression during fruit development and ripening of kiwifruit.

Global survey of plastid gene expression during fruit ripening in kiwifruit provides cis-elements for the futureengineering ofthe plastid genome of kiwifruit. A limitation in the application of plastid biotechnology for molecular farming is the low-level expression of transgenes in non-green plastids compared with photosynthetically active chloroplasts. Unlike other fruits, not all chloroplasts are transformed into chromoplasts during ripening of red-fleshed kiwifruit (Actinidia chinensis cv. Hongyang) fruits, which may make kiwifruit an ideal horticultural plant for recombinant protein production by plastid engineering. To identify cis-elements potentially triggering high-level transgene expression in edible tissues of the 'Hongyang' kiwifruit, here we report a comprehensive analysis of kiwifruit plastid gene transcription in green leaves and fruits at three different developmental stages. While transcripts of a few photosynthesis-related genes and most genetic system genes were substantially upregulated in green fruits compared with leaves, nearly all plastid genes were significantly downregulated at the RNA level during fruit development. Expression of a few genes remained unchanged, including psbA, the gene encoding the D1 polypeptide of photosystem II. However, PsbA protein accumulation decreased continuously during chloroplast-to-chromoplast differentiation. Analysis of post-transcriptional steps in mRNA maturation, including intron splicing and RNA editing, revealed that splicing and editing may contribute to regulation ofplastid gene expression. Altogether, 40 RNA editing sites were verified, and 5 of them were newly discovered. Taken together, this study has generated a valuable resource for the analysis of plastid gene expression and provides cis-elements for future efforts to engineer the plastid genome of kiwifruit.

Innovative Bioprocess Strategies Combining Physiological Control and Strain Engineering of Pichia pastoris to Improve Recombinant Protein Production.

The combination of strain and bioprocess engineering strategies should be considered to obtain the highest levels of recombinant protein production (RPP) while assuring product quality and process reproducibility of heterologous products. In this work, two complementary approaches were investigated to improve bioprocess efficiency based on the yeast P. pastoris. Firstly, the performance of two Candida rugosa lipase 1 producer clones with different gene dosage under the regulation of the constitutive P GAP were compared in chemostat cultures with different oxygen-limiting conditions. Secondly, hypoxic conditions in carbon-limited fed-batch cultures were applied by means of a physiological control based on the respiratory quotient (RQ). Stirring rate was selected to maintain RQ between 1.4 and 1.6, since it was found to be the most favorable in chemostat. As the major outcome, between 2-fold and 4-fold higher specific production rate (q P ) values were observed when comparing multicopy clone (MCC) and single-copy clone (SCC), both in chemostat and fed-batch. Additionally, when applying oxygen limitation, between 1.5-fold and 3-fold higher q P values were obtained compared with normoxic conditions. Thus, notable increases of up to 9-fold in the production rates were reached. Furthermore, transcriptional analysis of certain key genes related to RPP and central carbon metabolism were performed. Results seem to indicate the presence of a limitation in post-transcriptional protein processing steps and a possible transcription attenuation of the target gene in the strains with high gene dosage. The entire approach, including both strain and bioprocess engineering, represents a relevant novelty involving physiological control in Pichia cell factory and is of crucial interest in bioprocess optimization, boosting RPP, allowing bioproducts to be economically competitive in the market, and helping develop the bioeconomy.

9-Aminoacridine Inhibits Ribosome Biogenesis by Targeting Both Transcription and Processing of Ribosomal RNA.

Aminoacridines, used for decades as antiseptic and antiparasitic agents, are prospective candidates for therapeutic repurposing and new drug development. Although the mechanisms behind their biological effects are not fully elucidated, they are most often attributed to the acridines’ ability to intercalate into DNA. Here, we characterized the effects of 9-aminoacridine (9AA) on pre-rRNA metabolism in cultured mammalian cells. Our results demonstrate that 9AA inhibits both transcription of the ribosomal RNA precursors (pre-rRNA) and processing of the already synthesized pre-rRNAs, thereby rapidly abolishing ribosome biogenesis. Using a fluorescent intercalator displacement assay, we further show that 9AA can bind to RNA in vitro, which likely contributes to its ability to inhibit post-transcriptional steps in pre-rRNA maturation. These findings extend the arsenal of small-molecule compounds that can be used to block ribosome biogenesis in mammalian cells and have implications for the pharmacological development of new ribosome biogenesis inhibitors.

Measuring mRNA Decay with Roadblock-qPCR.

The control of mRNA stability is fundamental to gene regulation, and a deeper understanding of this post-transcriptional regulatory step can provide key insights into gene function. Measuring mRNA half-lives directly, however, is challenging. The most common strategies for evaluating mRNA stability and decay involve blocking general transcription and then measuring the decline in mRNA levels over time. The downside of these approaches, however, is that they severely impact cell function and viability, indirectly perturbing gene expression. Here, we describe Roadblock-qPCR, a simple method for measuring mRNA decay kinetics in living cells that is both economical and quick. Cells are first incubated with the nucleoside analog 4-thiouridine (4sU), which is readily incorporated into nascent mRNAs during transcription. RNA is then extracted and treated with N-ethylmaleimide (NEM), a sulfhydryl alkylating agent that selectively modifies 4sU, before proceeding to cDNA synthesis. Because the NEM-modified 4sU creates a chemical "roadblock" that interferes with reverse transcription, this treatment ultimately results in the depletion of the nascent 4sU-containing transcripts from the cDNA pool. As such, the decay rate of the non-4sU-labeled pre-existing mRNAs can be monitored by quantitative PCR (qPCR). In combination with spike-in standards, this approach can be used to efficiently and accurately measure the half-lives of endogenous mRNAs with a wide range of stabilities, while avoiding the artifacts of transcription shutoff strategies. © 2022 Wiley Periodicals LLC. Basic Protocol: Roadblock-qPCR Support Protocol: Synthesis of spike-in mRNA.

Toward a Disease-Modifying Therapy of Alpha-Synucleinopathies: New Molecules and New Approaches Came into the Limelight.

The accumulation of the various products of alpha-synuclein aggregation has been associated with the etiology and pathogenesis of several neurodegenerative conditions, including both familial and sporadic forms of Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). It is now well established that the aggregation and spread of alpha-synuclein aggregation pathology activate numerous pathogenic mechanisms that contribute to neurodegeneration and, ultimately, to disease progression. Therefore, the development of a safe and effective disease-modifying therapy that limits or prevents the accumulation of the toxic intermediate products of alpha-synuclein aggregation and the spread of alpha-synuclein aggregation pathology could provide significant positive clinical outcomes in PD/DLB cohorts. It has been suggested that this goal can be achieved by reducing the intracellular and/or extracellular levels of monomeric and already aggregated alpha-synuclein. The principal aim of this review is to critically evaluate the potential of therapeutic strategies that target the post-transcriptional steps of alpha-synuclein production and immunotherapy-based approaches to alpha-synuclein degradation in PD/DLB patients. Strategies aimed at the downregulation of alpha-synuclein production are at an early preclinical stage of drug development and, although they have shown promise in animal models of alpha-synuclein aggregation, many limitations need to be resolved before in-human clinical trials can be seriously considered. In contrast, many strategies aimed at the degradation of alpha-synuclein using immunotherapeutic approaches are at a more advanced stage of development, with some in-human Phase II clinical trials currently in progress. Translational barriers for both strategies include the limitations of alpha-synuclein aggregation models, poor understanding of the therapeutic window for the alpha-synuclein knockdown, and variability in alpha-synuclein pathology across patient cohorts. Overcoming such barriers should be the main focus of further studies. However, it is already clear that these strategies do have the potential to achieve a disease-modifying effect in PD and DLB.

Investigation of the effect of Staufen1 overexpression on the HIV-1 virus production

In this study, we investigated how Staufen1 influences the HIV-1 production. The overexpression of Staufen1 increased virus production without any negative affect on the viral infectivity. This increase was not caused by transcriptional activation; but by influencing post-transcriptional steps. Using multiple Gag protein derivatives, we confirmed that the zinc-finger domains of the HIV-1 nucleocapsid (NC) are important for its interaction with Staufen1. We also found that Staufen1 colocalized in stress granules with the mature form of the HIV-1 NC protein.

Full Length Transcriptome Highlights the Coordination of Plastid Transcript Processing

Plastid gene expression involves many post-transcriptional maturation steps resulting in a complex transcriptome composed of multiple isoforms. Although short-read RNA-Seq has considerably improved our understanding of the molecular mechanisms controlling these processes, it is unable to sequence full-length transcripts. This information is crucial, however, when it comes to understanding the interplay between the various steps of plastid gene expression. Here, we describe a protocol to study the plastid transcriptome using nanopore sequencing. In the leaf of Arabidopsis thaliana, with about 1.5 million strand-specific reads mapped to the chloroplast genome, we could recapitulate most of the complexity of the plastid transcriptome (polygenic transcripts, multiple isoforms associated with post-transcriptional processing) using virtual Northern blots. Even if the transcripts longer than about 2500 nucleotides were missing, the study of the co-occurrence of editing and splicing events identified 42 pairs of events that were not occurring independently. This study also highlighted a preferential chronology of maturation events with splicing happening after most sites were edited.

Engineering and standardization of posttranscriptional biocircuitry in Saccharomyces cerevisiae.

This short review considers to what extent posttranscriptional steps of gene expression can provide the basis for novel control mechanisms and procedures in synthetic biology and biotechnology. The term biocircuitry is used here to refer to functionally connected components comprising DNA, RNA or proteins. The review begins with an overview of the diversity of devices being developed and then considers the challenges presented by trying to engineer more scaled-up systems. While the engineering of RNA-based and protein-based circuitry poses new challenges, the resulting 'toolsets' of components and novel mechanisms of operation will open up multiple new opportunities for synthetic biology. However, agreed procedures for standardization will need to be placed at the heart of this expanding field if the full potential benefits are to be realized.

ECOA-3. MLL4, UTX, and EZH2 regulate the expression of transcription factors associated with the epithelial-to-mesenchymal transition of lung cancer during metastasis to the brain at posttranscriptional steps

PurposeThe objective of this study was to investigate the epigenetic role of histone lysine methylation/demethylation on the expression of epithelial-to-mesenchymal transition (EMT) associated transcriptional factors (TFs) during the metastasis of lung adenocarcinoma to the brain.MethodsPaired samples of lung adenocarcinoma and brain metastasis (BM) were analyzed in 46 individual patients. Both samples were obtained by surgical resection or biopsy of the lung and brain. The paraffin-fixed formalin-embedded samples were obtained from the pathology archives in our institute. In samples of lung adenocarcinoma and BM, immunohistochemical staining was performed for epithelial markers, mesenchymal markers, EMT-TFs, histone lysine methyltransferase and demethylase. And the verification of the present result was performed by qRT-PCR.ResultsThe immunoreactivity of EMT-TFs such as Slug (15.6% vs. 42.6%, p = 0.005), Twist (23.6% vs. 45.9%, p = 0.010) and ZEB1 (15.0% vs. 55.9%, p = 0.002) was increased in BM compared with that in lung adenocarcinoma. Epigenetic inducers such as H3K4 methyltransferase (MLL4, p = 0.018) and H3K36me3 demethylase (UTX, p = 0.003) were statistically increased, and epigenetic repressors such as EZH2 (H3K27 methyltransferase, p = 0.046) were significantly decreased in BM compared with those in lung adenocarcinoma. The expression of UTX-ZEB1 (R2 linear = 1.204) and MLL4-Slug (R2 linear = 0.987) was increased in direct proportion, and EZH2-Twist (R2 linear = - 2.723) decreased in reverse proportion. The qRT-PCR showed the same results.ConclusionThe results suggest that certain histone lysine methyltransferase/demethylase, such as MLL4, UTX, and EZH2, regulate the expression of EMT-TFs such as Slug, ZEB1, and Twist epigenetically, which may thereby influence cancer metastasis from the lung to the brain.

Making sense of mRNA landscapes: Translation control in neurodevelopment.

Like all other parts of the central nervous system, the mammalian neocortex undergoes temporally ordered set of developmental events, including proliferation, differentiation, migration, cellular identity, synaptogenesis, connectivity formation, and plasticity changes. These neurodevelopmental mechanisms have been characterized by studies focused on transcriptional control. Recent findings, however, have shown that the spatiotemporal regulation of post-transcriptional steps like alternative splicing, mRNA traffic/localization, mRNA stability/decay, and finally repression/derepression of protein synthesis (mRNA translation) have become just as central to the neurodevelopment as transcriptional control. A number of dynamic players act post-transcriptionally in the neocortex to regulate these steps, as RNA binding proteins (RBPs), ribosomal proteins (RPs), long non-coding RNAs, and/or microRNA. Remarkably, mutations in these post-transcriptional regulators have been associated with neurodevelopmental, neurodegenerative, inherited, or often co-morbid disorders, such as microcephaly, autism, epilepsy, intellectual disability, white matter diseases, Rett-syndrome like phenotype, spinocerebellar ataxia, and amyotrophic lateral sclerosis. Here, we focus on the current state, advanced methodologies and pitfalls of this exciting and upcoming field of RNA metabolism with vast potential in understanding fundamental neurodevelopmental processes and pathologies. This article is categorized under: Translation > Translation Regulation RNA in Disease and Development > RNA in Disease RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Schizosaccharomyces pombe Ppr10 and Mpa1 together mediate mitochondrial translational initiation

Pentatricopeptide repeat (PPR) proteins are a large family of proteins that act primarily at different posttranscriptional steps of organellar gene expression. We have previously found that the Schizosaccharomyces pombe PPR protein mpal10 interacts with mitochondrial translational activator Mpa1, and both are essential for mitochondrial protein synthesis. However, it is unclear how these two proteins function in mitochondrial protein synthesis in S. pombe. In this study, we further investigated the role of Ppr10 and Mpa1 in mitochondrial protein synthesis. Mitochondrial translational initiation requires two initiation factors, Mti2 and Mti3, which bind to the small subunit of the mitochondrial ribosome (mt-SSU) during the formation of the mitochondrial translational initiation complex. Using sucrose gradient sedimentation analysis, we found that disruption of ppr10, mpa1, or the PPR motifs in Ppr10 impairs the association of Mti2 and Mti3 with the mt-SSU, suggesting that both Ppr10 and Mpa1 may be required for the interaction of Mti2 and Mti3 with the mt-SSU during the assembly of mitochondrial translational initiation complex. Loss of Ppr10 perturbs the association of mitochondrially encoded cytochrome b (cob1) and cytochrome c oxidase subunit 1 (cox1) mRNAs with assembled mitochondrial ribosomes. Proteomic analysis revealed that a fraction of Ppr10 and Mpa1 copurified with a subset of mitoribosomal proteins. The PPR motifs of Ppr10 are necessary for its interaction with Mpa1 and that disruption of these PPR motifs impairs mitochondrial protein synthesis. Our results suggest that Ppr10 and Mpa1 function together to mediate mitochondrial translational initiation.