Post-transcriptional mRNA Processing Research Articles

Neuronal splicing of the unmethylated histone H3K4 reader, PHF21A, prevents excessive synaptogenesis

PHF21A is a histone-binding protein that recognizes unmethylated histone H3K4, the reaction product of LSD1 histone demethylase. PHF21A and LSD1 form a complex, and both undergo neuron-specific microexon splicing. The PHF21A neuronal microexon interferes with nucleosome binding, whereas the LSD1 neuronal microexon weakens H3K4 demethylation activity and can alter the substrate specificity to H3K9 or H4K20. However, the temporal expression patterns of PHF21A and LSD1 splicing isoforms during brain development and their biological roles remain unknown. In this work, we report that neuronal PHF21A isoform expression precedes neuronal LSD1 expression during human neuron differentiation and mouse brain development. The asynchronous splicing events resulted in stepwise deactivation of the LSD1-PHF21A complex in reversing H3K4 methylation. An unbiased proteomics survey revealed that the enzymatically inactive LSD1-PHF21A complex interacts with neuron-specific binding partners, including MYT1-family transcription factors and post-transcriptional mRNA processing proteins such as VIRMA. The interaction with the neuron-specific components, however, did not require the PHF21A microexon, indicating that the neuronal proteomic milieu, rather than the microexon-encoded PHF21A segment, is responsible for neuron-specific complex formation. Finally, by using two Phf21a mutant mouse models, we found that Phf21a neuronal splicing prevents excess synapse formation that otherwise would occur when canonical PHF21A is expressed in neurons. These results suggest that the role of the PHF21A microexon is to dampen LSD1-mediated H3K4 demethylation, thereby containing aberrant synaptogenesis.

Aberrant RNA polymerase initiation and processivity on the genome of a herpes simplex virus 1 mutant lacking ICP27.

Within the first 15 minutes of infection, herpes simplex virus 1 immediate early proteins repurpose cellular RNA polymerase (Pol II) for viral transcription. An important role of the viral-infected cell protein 27 (ICP27) is to facilitate viral pre-mRNA processing and export viral mRNA to the cytoplasm. Here, we use precision nuclear run-on followed by deep sequencing (PRO-seq) to characterize transcription of a viral ICP27 null mutant. At 1.5 and 3 hours post infection (hpi), we observed increased total levels of Pol II on the mutant viral genome and accumulation of Pol II downstream of poly A sites indicating increased levels of initiation and processivity. By 6 hpi, Pol II accumulation on specific mutant viral genes was higher than that on wild-type virus either at or upstream of poly A signals, depending on the gene. The PRO-seq profile of the ICP27 mutant on late genes at 6 hpi was similar but not identical to that caused by treatment with flavopiridol, a known inhibitor of RNA processivity. This pattern was different from PRO-seq profiles of other α gene mutants and upon inhibition of viral DNA replication with PAA. Together, these results indicate that ICP27 contributes to the repression of aberrant viral transcription at 1.5 and 3 hpi by inhibiting initiation and decreasing RNA processivity. However, ICP27 is needed to enhance processivity on most late genes by 6 hpi in a mechanism distinguishable from its role in viral DNA replication.IMPORTANCEWe developed and validated the use of a processivity index for precision nuclear run-on followed by deep sequencing data. The processivity index calculations confirm infected cell protein 27 (ICP27) induces downstream of transcription termination on certain host genes. The processivity indices and whole gene probe data implicate ICP27 in transient immediate early gene-mediated repression, a process that also requires ICP4, ICP22, and ICP0. The data indicate that ICP27 directly or indirectly regulates RNA polymerase (Pol II) initiation and processivity on specific genes at specific times post infection. These observations support specific and varied roles for ICP27 in regulating Pol II activity on viral genes in addition to its known roles in post transcriptional mRNA processing and export.

Stress-induced nuclear speckle reorganization is linked to activation of immediate early gene splicing.

Current models posit that nuclear speckles (NSs) serve as reservoirs of splicing factors and facilitate posttranscriptional mRNA processing. Here, we discovered that ribotoxic stress induces a profound reorganization of NSs with enhanced recruitment of factors required for splice-site recognition, including the RNA-binding protein TIAR, U1 snRNP proteins and U2-associated factor 65, as well as serine 2 phosphorylated RNA polymerase II. NS reorganization relies on the stress-activated p38 mitogen-activated protein kinase (MAPK) pathway and coincides with splicing activation of both pre-existing and newly synthesized pre-mRNAs. In particular, ribotoxic stress causes targeted excision of retained introns from pre-mRNAs of immediate early genes (IEGs), whose transcription is induced during the stress response. Importantly, enhanced splicing of the IEGs ZFP36 and FOS is accompanied by relocalization of the corresponding nuclear mRNA foci to NSs. Our study reveals NSs as a dynamic compartment that is remodeled under stress conditions, whereby NSs appear to become sites of IEG transcription and efficient cotranscriptional splicing.

Specific processing of meiosis-related transcript is linked to final maturation in human oocytes.

Human meiosis in oocytes entails an intricate regulation of the transcriptome to support late oocyte growth and early embryo development, both crucial to reproductive success. Currently, little is known about the co- and post-transcriptional mRNA processing mechanisms regulating the last meiotic phases, which contribute to transcriptome complexity and influence translation rates. We analyzed gene expression changes, splicing and pre-mRNA processing in an RNA sequencing set of 40 human oocytes at different meiotic maturation stages, matured both in vivo and in vitro. We found abundant untranslated region (UTR) processing, mostly at the 3' end, of meiosis-related genes between the germinal vesicle (GV) and metaphase II (MII) stages, supported by the differential expression of spliceosome and pre-mRNA processing related genes. Importantly, we found very few differences among GV oocytes across several durations of IVM, as long as they did not reach MII, suggesting an association of RNA processing and successful meiosis transit. Changes in protein isoforms are minor, although specific and consistent for genes involved in chromosome organization and spindle assembly. In conclusion, we reveal a dynamic transcript remodeling during human female meiosis, and show how pre-mRNA processing, specifically 3'UTR shortening, drives a selective translational regulation of transcripts necessary to reach final meiotic maturation.

Molecular basis of locus-specific H3K9 methylation catalyzed by SUVH6 in plants

Dimethylated histone H3 Lys9 (H3K9me2) is a conserved heterochromatic mark catalyzed by SUPPRESSOR OF VARIEGATION 3-9 HOMOLOG (SUVH) methyltransferases in plants. However, the mechanism underlying the locus specificity of SUVH enzymes has long been elusive. Here, we show that a conserved N-terminal motif is essential for SUVH6-mediated H3K9me2 deposition in planta. The SUVH6 N-terminal peptide can be recognized by the bromo-adjacent homology (BAH) domain of the RNA- and chromatin-binding protein ANTI-SILENCING 1 (ASI1), which has been shown to function in a complex to confer gene expression regulation. Structural data indicate that a classic aromatic cage of ASI1-BAH domain specifically recognizes an arginine residue of SUVH6 through extensive hydrogen bonding interactions. A classic aromatic cage of ASI1 specifically recognizes an arginine residue of SUVH6 through extensive cation-π interactions, playing a key role in recognition. The SUVH6-ASI1 module confers locus-specific H3K9me2 deposition at most SUVH6 target loci and gives rise to distinct regulation of gene expression depending on the target loci, either conferring transcriptional silencing or posttranscriptional processing of mRNA. More importantly, such mechanism is conserved in multiple plant species, indicating a coordinated evolutionary process between SUVH6 and ASI1. In summary, our findings uncover a conserved mechanism for the locus specificity of H3K9 methylation in planta. These findings provide mechanistic insights into the delicate regulation of H3K9 methylation homeostasis in plants.

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.

Stem cell-intrinsic mechanisms regulating adult hair follicle homeostasis.

Adult hair follicle stem cells (HFSCs) undergo dynamic and periodic molecular changes in their cellular states throughout the hair homeostatic cycle. These states are tightly regulated by cell-intrinsic mechanisms and by extrinsic signals from the microenvironment. HFSCs are essential not only for fuelling hair growth, but also for skin wound healing. Increasing evidence suggests an important role of HFSCs in organizing multiple skin components around the hair follicle, thus functioning as an organizing centre during adult skin homeostasis. Here, we focus on recent findings on cell-intrinsic mechanisms of HFSC homeostasis, which include transcription factors, histone modifications, DNA regulatory elements, non-coding RNAs, cell metabolism, cell polarity and post-transcriptional mRNA processing. Several transcription factors are now known to participate in well-known signalling pathways that control hair follicle homeostasis, as well as in super-enhancer activities to modulate HFSC and progenitor lineage progression. Interestingly, HFSCs have been shown to secrete molecules that are important in guiding the organization of several skin components around the hair follicle, including nerves, arrector pili muscle and vasculature. Finally, we discuss recent technological advances in the field such as single-cell RNA sequencing and live imaging, which revealed HFSC and progenitor heterogeneity and brought new light to understanding crosstalking between HFSCs and the microenvironment. The field is well on its way to generate a comprehensive map of molecular interactions that should serve as a solid theoretical platform for application in hair and skin disease and ageing.

Evolving Clinical Applications of Tissue Transcriptomics in Kidney Disease.

Nephrotic syndrome is classically categorized by the histopathology with examples including focal segmental glomerulosclerosis (FSGS) and minimal change disease. Pediatric patients are also classified by whether their nephrotic syndrome is sensitive to, dependent on, or resistant to steroids. However, this traditional classification system overlooks the frequent clinical conundrum when, for example, one patient with FSGS responds briskly to steroids, and another quickly progresses to end stage kidney disease despite therapy. Two patients may have similar histopathologic appearances on kidney biopsy but entirely different clinical characteristics, rates of progression, and treatment responses. Transcriptional regulation of gene activation and posttranscriptional processing of mRNA may drive the unique and heterogeneous phenotypes which are incompletely understood in kidney disease and are a recent focus of research. Gene expression profiles provide insight on active transcriptional programs in tissues, are being used to understand biologic mechanisms of progressive chronic kidney disease, and may help to identify patients with shared mechanisms of kidney damage. This mini-review discusses clinically relevant techniques of bulk tissue and single cell transcriptomics, as well as strengths and limitations of each methodology. Further, we summarize recent examples in kidney research achieved through transcriptomics. This review offers an outlook on the role of transcriptomics in an integrative systems biology model with the goal of defining unique disease subgroups, finding targets for drug development, and aligning the right drug with the right patient.

Post-transcriptional Processing of mRNA in Neurons: The Vestiges of the RNA World Drive Transcriptome Diversity.

Neurons are morphologically complex cells that rely on the compartmentalization of protein expression to develop and maintain their extraordinary cytoarchitecture. This formidable task is achieved, at least in part, by targeting mRNA to subcellular compartments where they are rapidly translated. mRNA transcripts are the conveyor of genetic information from DNA to the translational machinery, however, they are also endowed with additional functions linked to both the coding sequence (open reading frame, or ORF) and the flanking 5′ and 3′ untranslated regions (UTRs), that may harbor coding-independent functions. In this review, we will highlight recent evidences supporting new coding-dependent and -independent functions of mRNA and discuss how nuclear and cytoplasmic post-transcriptional modifications of mRNA contribute to localization and translation in mammalian cells with specific emphasis on neurons. We also describe recently developed techniques that can be employed to study RNA dynamics at subcellular level in eukaryotic cells in developing and regenerating neurons.

The nuclear GSK-3β regulated post-transcriptional processing of mRNA through phosphorylation of SC35.

Glycogen synthase kinase-3β (GSK-3β) is a multifunctional serine/threonine kinase and regulates a variety of biological processes. Recent studies show GSK-3β can regulate pre-mRNA processing and transcription through phosphorylation of multiple splicing factors, but the detailed mechanism is still undetermined. In this study, we further proved that GSK-3β could specifically co-localize with SC35 in nuclear speckles depending on its kinase activity. Immunofluorescence and FISH studies showed the activity of nuclear GSK-3β regulated the assembly of nuclear speckles and consequently modulated the post-transcriptional processing of mRNA. In addition, GSK-3β phosphorylated SC35 and promoted its hyperphosphorylation, in which the unique C-terminal sequences were particularly important to efficiently sequential multiple phosphorylation of SC35. Hyperphosphorylated SC35 converged into cluster and lost its ability to perform splicing in nuclear speckles. More importantly, the nuclear GSK-3β activity could be a part of Wnt/β-catenin signaling activation by TCF4 and might take part in embryonic or tumorigenesis of cells.

Influence of amino acids that come from the diet in the expression of genes

Objective: to make a descriptive review about the influence of the dietary amino acids in gene expression. Materials and method: the bibliographic research included the following written sources: Scielo, PubMed, Medline, NCBI, Springer, Scopus, Science Direct and Elsevier, retrieved until May 2018 from critical reviews of scientific articles. One hundred and five records were found after the combination of key words. Fundamental selection criteria were taken into account (title, authors, summary and results) using Maeda's reasoned reduction and Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) as a systematic review methodology. Conclusion: there are genes that are regulated in various stages including transcription, post-transcriptional processing, nuclear export and translation of mature mRNA. Amino acids can influence these processes through the activation of transcription factors. In terms of translation, amino acids can regulate protein synthesis through changes in eIF2B, phosphorylation of 4E-BP1 and S6 proteins. In addition, amino acids affect the regulation of the growth factor expression (insulin like grow factor: IGF-I) in humans.

Abstract 2352: Effect of inhibition of cell cycle versus transcription cyclin-dependent kinases (CDKs) in ovarian cancer cells

Abstract Background. Cell cycle is regulated by cyclin-dependent kinases (CDKs) activity, whose deregulation can lead to uncontrolled proliferation and cancer. Other CDKs are engaged in the regulation of transcription and post-transcriptional mRNA processing through the phosphorylation of the C-terminal domain of RNA polymerase II, such as CDK9 and CDK12. Inhibitors of cell cycle CDKs have been developed as anticancer agents and some of them are under clinical validation (e.g. palbociclib). While recent data would suggest that inhibition of CDK9 is feasible and has antitumor effect, the data on the therapeutic role of CDK12 inhibition are very scanty. Methods. Ovarian cancer cell lines were maintained in RPMI medium supplemented with 5% glutamine and 10% FBS. Cells were treated with different drug concentrations and after 72 hours cell survival was evaluated by MTS assay (Promega). IC50 values were calculated by interpolation method. Cell cycle analysis and apoptosis were performed with standard flow cytometric methods. A2780 and SKOV3 ovarian cancer cell lines knocked out for CDK12 were generated with CRISPR/CAS9 genome editing tool. Results. The cytotoxicity of palbociclib (a CDK4/6 inhibitor) and LDC000067 (a CDK9 inhibitor) was tested in a panel of ovarian cancer cell lines (A2780, SKOV3, OVCAR3, OVCAR5, OVCAR8, OVCA432, OVCA433, IGROV1, EFO27). Sensitivity of cells was similar for palbociclib and LDC000067, ranging from 10 to 33 and from 8 to 60 μM, respectively. A preferential G1 block was observed with palbociclib, while LDC000067 caused a S-G2 block. A higher induction of apoptosis was observed after LDC000067 than after palbociclib treatment in both A2780 and SKOV3. The palbociclib-induced G1 block was associated with decreased Rb phosphorylation, while no modulation of the Ser2 in the carboxyterminal domain of RNA polymerase II was observed after LDC000067 treatment. We generated CDK12 knocked out cells transfecting CRISPR/CAS9 engineered plasmid in both A2780 and SKOV3 ovarian cancer lines. The biological and pharmacological characterization of these clones is under study. Conclusions. Palbociclib and LDC000067 showed a dose dependent cytotoxic effect in the panel of ovarian cancer cell lines tested and were active in the μM range. Preliminary data of treatment induced cell cycle perturbation and apoptosis suggest that the two drugs behave in a different manner and have distinct molecular effects on cells. Citation Format: Rosaria Chilà, Nicolò Panini, Eugenio Erba, Giovanna Damia, Massimo Broggini. Effect of inhibition of cell cycle versus transcription cyclin-dependent kinases (CDKs) in ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2352. doi:10.1158/1538-7445.AM2017-2352

Immunogenicity of mammary tumor cells can be induced by shikonin via direct binding-interference with hnRNPA1.

Immunogenic cell death (ICD) of tumor cells occurs via various pathways that activate immune cell systems against cancer. Previous studies have demonstrated that shikonin (SK), a plant secondary metabolite, can confer strong pharmacological activities that activate ICD and strong immunogenicity of tumor cells. However, the exact hierarchical regulatory mechanisms including the molecular targets of SK-activated immunogenicity are still unknown. Here, the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) was revealed to serve as a specific protein target for SK. This binding plays a key role in SK-stimulated ICD activity and the suppression of post-transcriptional mRNA processing, including nuclear export activity of newly synthesized mRNAs in mammary carcinoma cells in vitro. Moreover, it also mechanistically mediates the anti-metastatic effect of a tumor cell lysate (TCL) vaccine, which can be readily generated from SK-treated 4T1 tumor cells (SK-TCL), and the derived tumor-immunogenicity of SK-TCL-treated dendritic cells in vivo. Together, the identification of hnRNPA1 as the intracellular molecular target provides compelling pharmacology-based knowledge for the potential clinical use of SK-induced immunogenicity. In addition, SK may also serve as a potent suppressor that interferes with specific post-transcriptional activities, a mechanism which may be useful for exploitation in cancer therapeutics.

Abstract 2212: Super-enhancer landscapes specify molecular subtypes and novel targets in acute myeloid leukemia

Abstract AML is a highly genotypically and phenotypically heterogeneous cancer with many subtypes of the disease currently lacking clear targeted therapies. While several genetic alterations currently serve to stratify patients into prognostic subgroups and guide treatment decisions, the classification and treatment options for AML patients remain unsatisfactory. Numerous genome-wide analyses have provided evidence linking recurring genetic mutations in de novo AML to perturbations in epigenomic signaling, transcriptional regulation, and post-transcriptional mRNA processing, indicating that misregulation of gene expression is a key molecular feature of AML pathogenesis yet the diagnostic value and clinical utility of these findings remain unclear. Recently, a novel class of densely clustered cis-regulatory elements termed super-enhancers have emerged as key effectors initiating and maintaining cell type-specific gene expression in a variety of physiological and disease settings, including cancer. Tumor-specific super-enhancers regulate expression of key oncogenes and other cancer-essential genes, providing a novel strategy for detecting both known and unrecognized cancer dependencies of high diagnostic and therapeutic value. Here we describe the discovery and characterization of super-enhancer domains across a large cohort of AML patients and relevant normal hematopoietic stem and progenitor cell controls. To this end, we profiled over 60 primary samples at both the level of epigenomic activity and transcript abundance and identify transcriptionally hyperactive regions in AML patients compared to normal healthy donors. By combining patient-specific super-enhancer landscapes with gene expression profiles, we elucidate the transcription factor repertoires controlling chromatin dynamics and lineage specification in cellular derivatives of normal hematopoietic development and purified AML samples. Further analyses yield a striking delineation of disease-unique transcriptional subtypes which are specified by distinct patterns of super-enhancers at well-known AML-related genes such as HOXA9, MEIS1, ETV6, and HOXB3. In addition, we identify clusters of AML cases with shared super-enhancer domains suggesting convergence on common key drivers of AML. Together, our results provide the biological and disease relevance of super-enhancers in the context of normal and tumor cell states and demonstrate how the comprehensive mapping of gene regulatory elements elucidate clinically relevant subtypes and new therapeutic targets in AML. Citation Format: Matthew Eaton, Ryan Corces-Zimmerman, Jeremy Lopez, Christian Fritz, Eric Olson, Ravindra Majeti, Jakob Loven. Super-enhancer landscapes specify molecular subtypes and novel targets in acute myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2212. doi:10.1158/1538-7445.AM2015-2212

Quantitative proteomic analysis identifies targets and pathways of a 2-aminobenzamide HDAC inhibitor in Friedreich's ataxia patient iPSC-derived neural stem cells.

Members of the 2-aminobenzamide class of histone deacetylase (HDAC) inhibitors show promise as therapeutics for the neurodegenerative diseases Friedreich’s ataxia (FRDA) and Huntington’s disease (HD). While it is clear that HDAC3 is one of the important targets of the 2-aminobenzamide HDAC inhibitors, inhibition of other class I HDACs (HDACs 1 and 2) may also be involved in the beneficial effects of these compounds in FRDA and HD, and other HDAC interacting proteins may be impacted by the compound. To this end, we synthesized activity-based profiling probe (ABPP) versions of one of our HDAC inhibitors (compound 106), and in the present study we used a quantitative proteomic method coupled with multidimensional protein identification technology (MudPIT) to identify the proteins captured by the ABPP 106 probe. Nuclear proteins were extracted from FRDA patient iPSC-derived neural stem cells, and then were reacted with control and ABPP 106 probe. After reaction, the bound proteins were digested on the beads, and the peptides were modified using stable isotope-labeled formaldehyde to form dimethyl amine. The selectively bound proteins determined by mass spectrometry were subjected to functional and pathway analysis. Our findings suggest that the targets of compound 106 are involved not only in transcriptional regulation but also in posttranscriptional processing of mRNA.

True and false alternative transcripts of calcium-dependent protein kinase CPK9 and CPK3a genes in Vitis amurensis

Calcium-dependent protein kinases (CDPKs) are major Ca2+ sensors in plants playing important roles in plant development and stress responses. In the present study, RT-PCR analysis revealed that VaCPK3a and VaCPK9 cDNAs lacking extensive regions in the kinase, autoinhibitory, and Ca2+-binding domains were numerous in probes derived from wild grapevine Vitis amurensis. Most of the VaCPK3a and VaCPK9-modified transcripts lacked canonical splice sites and possessed short direct repeated sequences (SDRs) instead. Three VaCPK9 transcript variants were generated using canonical 5′GT and 3′AG splice sites and lacked several subdomains in the kinase domain, including ATP-binding site, which is known to be indispensable for kinase activities. These observations indicate that post-transcriptional mRNA processing might lead to production of CDPKs with abolished phosphorylation activities. Recombinant proteins VaCPK3aSF2, lacking autoinhibitory and Ca2+-binding domain, and VaCPK3aSF3, lacking VIII–XI kinase subdomains and autoinhibitory domain, phosphorylated exogenous substrate in a Ca2+-independent manner. However, reverse transcription at +65 °C using heat-stable reverse transcriptase (RT) markedly lowered abundance of the unusual VaCPK3a and VaCPK9 cDNAs with SDRs, while it did not eliminate VaCPK9 cDNAs generated using canonical splice sites. The results show that VaCPK9 gene undergoes unproductive alternative splicing mediated by canonical splice sites to generate three mRNA isoforms lacking important catalytic kinase subdomains. The unusual VaCPK3a and VaCPK9 transcripts with SDRs are likely to be false alternative transcripts generated by RT template switching in vitro. The data demonstrate that using non-thermostable RTs for studying alternative splicing are not appropriate.

Transcriptomic response to prolonged ethanol production in the cyanobacterium Synechocystis sp. PCC6803

BackgroundThe production of biofuels in photosynthetic microalgae and cyanobacteria is a promising alternative to the generation of fuels from fossil resources. To be economically competitive, producer strains need to be established that synthesize the targeted product at high yield and over a long time. Engineering cyanobacteria into forced fuel producers should considerably interfere with overall cell homeostasis, which in turn might counteract productivity and sustainability of the process. Therefore, in-depth characterization of the cellular response upon long-term production is of high interest for the targeted improvement of a desired strain.ResultsThe transcriptome-wide response to continuous ethanol production was examined in Synechocystis sp. PCC6803 using high resolution microarrays. In two independent experiments, ethanol production rates of 0.0338% (v/v) ethanol d-1 and 0.0303% (v/v) ethanol d-1 were obtained over 18 consecutive days, measuring two sets of biological triplicates in fully automated photobioreactors. Ethanol production caused a significant (~40%) delay in biomass accumulation, the development of a bleaching phenotype and a down-regulation of light harvesting capacity. However, microarray analyses performed at day 4, 7, 11 and 18 of the experiment revealed only three mRNAs with a strongly modified accumulation level throughout the course of the experiment. In addition to the overexpressed adhA (slr1192) gene, this was an approximately 4 fold reduction in cpcB (sll1577) and 3 to 6 fold increase in rps8 (sll1809) mRNA levels. Much weaker modifications of expression level or modifications restricted to day 18 of the experiment were observed for genes involved in carbon assimilation (Ribulose bisphosphate carboxylase and Glutamate decarboxylase). Molecular analysis of the reduced cpcB levels revealed a post-transcriptional processing of the cpcBA operon mRNA leaving a truncated mRNA cpcA* likely not competent for translation. Moreover, western blots and zinc-enhanced bilin fluorescence blots confirmed a severe reduction in the amounts of both phycocyanin subunits, explaining the cause of the bleaching phenotype.ConclusionsChanges in gene expression upon induction of long-term ethanol production in Synechocystis sp. PCC6803 are highly specific. In particular, we did not observe a comprehensive stress response as might have been expected.

Abstract W P206: Analysis of SUMO3-Modified Proteome in Post-Ischemic Mouse Brain

Background and Purpose: Small ubiquitin-like modifier (SUMO) conjugation modulates many key cellular processes. Transient cerebral ischemia dramatically activates SUMO2/3 conjugation, and this is believed to be a protective stress response. It is, therefore, of tremendous clinical interest to characterize the SUMO-modified proteome regulated by transient ischemia. We generated a novel SUMO transgenic mouse and performed the first SUMO proteomics study using post-ischemic brain samples. Methods: CAG-loxP-STOP-loxP-SUMO (CAG-SUMO) mice were generated in which His-SUMO1, HA-SUMO2, and FLAG-SUMO3 were expressed from a single multicistronic transgene in a Cre-dependent manner. CAG-SUMO mice were mated with Emx1 Cre/Cre mice to generate double transgenic CAG-SUMO/Emx1-Cre mice as experimental mice and Emx1 Cre/+ mice as control mice. Double transgenic mice were subjected to 10 min global cerebral ischemia followed by 1 h reperfusion or sham operation. FLAG-SUMO3-conjugated proteins were enriched from cortical tissues and analyzed. Results: Characterization of double transgenic mice demonstrated that exogenous expressed tagged SUMO paralogues were functionally intact and did not perturb the endogenous SUMOylation machinery in the brain. FLAG pulldown of cortical samples from sham and ischemia mice followed by GeLC-MS/MS analysis identified 91 candidates whose SUMOylation states were up-regulated in ischemic samples. Data analysis revealed several potentially important processes in which SUMO3 conjugation may play a key role during ischemia/reperfusion, including the cross-talk between SUMOylation and ubiquitination, glucocorticoid receptor signaling, and modulation of posttranscriptional mRNA processing. Conclusions: SUMO proteomic analysis identified important processes and pathways modulated by SUMOylation in the post-ischemic brain that warrant future investigations, since they could be the key to understand the overall impact of SUMOylation on the fate and functions of post-ischemic neurons. The conditional SUMO transgenic mouse will be an invaluable tool for in-depth in vivo analysis of the SUMO-modified proteome in various pathological states.

Novel Mutations in the SCNN1A Gene Causing Pseudohypoaldosteronism Type 1

Pseudohypoaldosteronism type 1 (PHA1) is a rare inherited disease characterized by resistance to the actions of aldosterone. Mutations in the subunit genes (SCNN1A, SCNN1B, SCNN1G) of the epithelial sodium channel (ENaC) and the NR3C2 gene encoding the mineralocorticoid receptor, result in systemic PHA1 and renal PHA1 respectively. Common clinical manifestations of PHA1 include salt wasting, hyperkalaemia, metabolic acidosis and elevated plasma aldosterone levels in the neonatal period. In this study, we describe the clinical and biochemical manifestations in two Chinese patients with systemic PHA1. Sequence analysis of the SCNN1A gene revealed a compound heterozygous mutation (c.1311delG and c.1439+1G>C) in one patient and a homozygous mutation (c.814_815insG) in another patient, all three variants are novel. Further analysis of the splicing pattern in a minigene construct showed that the c.1439+1G>C mutation can lead to the retainment of intron 9 as the 5′-donor splice site disappears during post-transcriptional processing of mRNA. In conclusion, our study identified three novel SCNN1A gene mutations in two Chinese patients with systemic PHA1.

Short-Term Chromium-Stress-Induced Alterations in the Maize Leaf Proteome

Soil contamination by chromium (Cr) has become an increasing problem worldwide as a result of extensive industrial activities. Chromium, especially hexavalent Cr, impairs the growth and productivity of plants. Although it has been proposed that plants could modify their metabolism to adapt to Cr stress by reprogramming the expression of genes, especially those related to the antioxidant system, damage response, and electron transport chain, evidence at the protein expression level is lacking. To better understand the precise mechanisms underlying Cr phytoxicity and the plant response to Cr exposure, the time-course of changes in the protein expression profile induced by short-term hexavalent Cr exposure (1, 6 and 24 h) were analyzed in maize leaves. Among the over 1200 protein spots detected reproducibly by two-dimensional electrophoresis (2-DE), 60 were found to be differentially accumulated during Cr stress treatment. Of the Cr-regulated proteins, 58 were identified using tandem mass spectrometry (MS/MS). The Cr-regulated proteins identified were mainly involved in ROS detoxification and defense responses (26%), photosynthesis and chloroplast organization (22%), post-transcriptional processing of mRNA and rRNA (12%), protein synthesis and folding (10%), the DNA damage response (5%), and the cytoskeleton (3%). The possible involvement of these Cr stress-responsive proteins in Cr phytoxicity and the plant response to Cr exposure in maize is discussed, taking into consideration the information available from other plant models. Our results provide preliminary evidence that will facilitate understanding the molecular mechanisms underlying Cr toxicity in maize.