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Concept: Messenger RNA

167

The human selenoprotein thioredoxin reductase 1 (TrxR1), encoded by the TXNRD1 gene, is a key player in redox regulation. Alternative splicing generates several TrxR1 variants, one of which is v3 that carries an atypical N-terminal glutaredoxin domain. When overexpressed, v3 associates with membranes and triggers formation of filopodia. Here we found that membrane targeting of v3 is mediated by myristoylation and palmitoylation of its N-terminal MGC-motif, through which v3 specifically targets membrane rafts. This was suggested by its localization in cholera toxin subunit B-stained membrane areas and also shown using lipid fractionation experiments. Utilizing site-directed mutant variants we also found that v3-mediated generation of filopodia is independent of the Cys residues in its redox active site, but dependent upon its membrane raft targeting. These results identify v3 as an intricately regulated protein that expands TXNRD1-derived protein functions to the membrane raft compartment.

Concepts: Protein, Gene expression, Cell membrane, Messenger RNA, Posttranslational modification, Lipid raft, Selenocysteine, Thioredoxin reductase

167

Mizoribine monophosphate (MZP) is a specific inhibitor of the cellular inosine-5'-monophosphate dehydrogenase (IMPDH), the enzyme catalyzing the rate-limiting step of de novo guanine nucleotide biosynthesis. MZP is a highly potent antagonistic inhibitor of IMPDH that blocks the proliferation of T and B lymphocytes that use the de novo pathway of guanine nucleotide synthesis almost exclusively. In the present study, we investigated the ability of MZP to directly inhibit the human RNA capping enzyme (HCE), a protein harboring both RNA 5'-triphosphatase and RNA guanylyltransferase activities. HCE is involved in the synthesis of the cap structure found at the 5' end of eukaryotic mRNAs, which is critical for the splicing of the cap-proximal intron, the transport of mRNAs from the nucleus to the cytoplasm, and for both the stability and translation of mRNAs. Our biochemical studies provide the first insight that MZP can inhibit the formation of the RNA cap structure catalyzed by HCE. In the presence of MZP, the RNA 5'-triphosphatase activity appears to be relatively unaffected while the RNA guanylyltransferase activity is inhibited, indicating that the RNA guanylyltransferase activity is the main target of MZP inhibition. Kinetic studies reveal that MZP is a non-competitive inhibitor that likely targets an allosteric site on HCE. Mizoribine also impairs mRNA capping in living cells, which could account for the global mechanism of action of this therapeutic agent. Together, our study clearly demonstrates that mizoribine monophosphate inhibits the human RNA guanylyltransferase in vitro and impair mRNA capping in cellulo.

Concepts: DNA, Protein, Cell nucleus, Cell, RNA, Messenger RNA, Enzyme inhibitor, 5' cap

166

Ribosome profiling or ribo-seq is a new technique that provides genome-wide information on protein synthesis (GWIPS) in vivo. It is based on the deep sequencing of ribosome protected mRNA fragments allowing the measurement of ribosome density along all RNA molecules present in the cell. At the same time, the high resolution of this technique allows detailed analysis of ribosome density on individual RNAs. Since its invention, the ribosome profiling technique has been utilized in a range of studies in both prokaryotic and eukaryotic organisms. Several studies have adapted and refined the original ribosome profiling protocol for studying specific aspects of translation. Ribosome profiling of initiating ribosomes has been used to map sites of translation initiation. These studies revealed the surprisingly complex organization of translation initiation sites in eukaryotes. Multiple initiation sites are responsible for the generation of N-terminally extended and truncated isoforms of known proteins as well as for the translation of numerous open reading frames (ORFs), upstream of protein coding ORFs. Ribosome profiling of elongating ribosomes has been used for measuring differential gene expression at the level of translation, the identification of novel protein coding genes and ribosome pausing. It has also provided data for developing quantitative models of translation. Although only a dozen or so ribosome profiling datasets have been published so far, they have already dramatically changed our understanding of translational control and have led to new hypotheses regarding the origin of protein coding genes. WIREs RNA 2013. doi: 10.1002/wrna.1172 For further resources related to this article, please visit the WIREs website.

Concepts: DNA, Protein, Gene, Cell nucleus, Bacteria, RNA, Ribosome, Messenger RNA

164

TFIIIB and TFIIIC are multi-subunit factors required for transcription by RNA polymerase III. We present a genome-wide high-resolution footprint map of TFIIIB-TFIIIC complexes in Saccharomyces cerevisiae, obtained by paired-end sequencing of micrococcal nuclease-resistant DNA. On tRNA genes, TFIIIB and TFIIIC form stable complexes with the same distinctive occupancy pattern but in mirror image, termed ‘bootprints’. Global analysis reveals that the TFIIIB-TFIIIC transcription complex exhibits remarkable structural elasticity: tRNA genes vary significantly in length but remain protected by TFIIIC. Introns, when present, are markedly less protected. The RNA polymerase III transcription terminator is flexibly accommodated within the transcription complex and, unexpectedly, plays a major structural role by delimiting its 3'-boundary. The ETC sites, where TFIIIC binds without TFIIIB, exhibit different bootprints, suggesting that TFIIIC forms complexes involving other factors. We confirm six ETC sites and report a new site (ETC10). Surprisingly, TFIIIC, but not TFIIIB, interacts with some centromeric nucleosomes, suggesting that interactions between TFIIIC and the centromere may be important in the 3D organization of the nucleus.

Concepts: DNA, Gene, Genetics, Gene expression, Transcription, Molecular biology, RNA, Messenger RNA

162

The methyltransferase like 3 (METTL3)-containing methyltransferase complex catalyzes the N6-methyladenosine (m6A) formation, a novel epitranscriptomic marker; however, the nature of this complex remains largely unknown. Here we report two new components of the human m6A methyltransferase complex, Wilms' tumor 1-associating protein (WTAP) and methyltransferase like 14 (METTL14). WTAP interacts with METTL3 and METTL14, and is required for their localization into nuclear speckles enriched with pre-mRNA processing factors and for catalytic activity of the m6A methyltransferase in vivo. The majority of RNAs bound by WTAP and METTL3 in vivo represent mRNAs containing the consensus m6A motif. In the absence of WTAP, the RNA-binding capability of METTL3 is strongly reduced, suggesting that WTAP may function to regulate recruitment of the m6A methyltransferase complex to mRNA targets. Furthermore, transcriptomic analyses in combination with photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) illustrate that WTAP and METTL3 regulate expression and alternative splicing of genes involved in transcription and RNA processing. Morpholino-mediated knockdown targeting WTAP and/or METTL3 in zebrafish embryos caused tissue differentiation defects and increased apoptosis. These findings provide strong evidence that WTAP may function as a regulatory subunit in the m6A methyltransferase complex and play a critical role in epitranscriptomic regulation of RNA metabolism.Cell Research advance online publication 10 January 2014; doi:10.1038/cr.2014.3.

Concepts: DNA, Protein, Cell nucleus, Gene expression, Enzyme, RNA, Messenger RNA, Intron

157

Protein-coding genes with multiple alternative polyadenylation sites can generate mRNA 3'UTR sequences of different lengths, thereby causing the loss or gain of regulatory elements, which can affect stability, localization and translation efficiency. 3USS is a web-server developed with the aim of giving experimentalists the possibility to automatically identify alternative 3'UTRs (shorter or longer with respect to a reference transcriptome), an option that is not available in standard RNA-seq data analysis procedures. The tool reports as putative novel the 3'UTRs not annotated in available databases. Furthermore, if data from two related samples are uploaded, common and specific alternative 3'UTRs are identified and reported by the server. Availability: 3USS is freely available at http://www.biocomputing.it/3uss_server.

Concepts: DNA, Gene expression, RNA, Messenger RNA, Computer, Polyadenylation, Three prime untranslated region, Web server

143

Mitochondrial RNAs in the acellular slime mold Physarum polycephalum contain nucleotides that are not encoded in the mitochondrial genes from which they are transcribed. These site-specific changes are quite extensive, comprising ~4% of the residues within mRNAs and ~2% of rRNAs and tRNAs. These “extra” nucleotides are added co-transcriptionally, but the means by which this is accomplished have not been elucidated. The cox1 mRNA also contains four sites of C to U changes, which occur post-transcriptionally, most likely via targeted deamination. The currently available in vitro systems for studying P. polycephalum editing are limited in that the template is the entire ~63,000 bp mitochondrial genome. This presents a significant challenge when trying to define the signals that specify editing sites. In an attempt to overcome this issue, a method for introducing DNA into isolated P. polycephalum mitochondria via electroporation has been developed. Exogenous DNA is expressed, but the transcripts synthesized from these templates are not edited under the conditions tested. However, transcripts derived from the mitochondrial genome are accurately edited after electroporation, indicating that the editing machinery is still functional. These findings suggest that this method may ultimately provide a feasible approach to elucidating editing signals.

Concepts: DNA, Gene, Genetics, Gene expression, Bacteria, RNA, Messenger RNA, Physarum polycephalum

143

Osteopontin (OPN) promotes hepatic fibrosis, and developing therapies targeting OPN expression in settings of hepatic injury holds promise. The polyphenol epigallocatechin-3-gallate (EGCG), found in high concentrations in green tea, downregulates OPN expression through OPN mRNA degradation, but the mechanism is unknown. Previous work has shown that microRNAs can decrease OPN mRNA levels, and other studies have shown that EGCG modulates the expression of multiple microRNAs. In our study, we first demonstrated that OPN induces hepatic stellate cells to transform into an activated state. We then identified three microRNAs which target OPN mRNA: miR-181a, miR-10b, and miR-221. In vitro results show that EGCG upregulates all three microRNAs, and all three microRNAs are capable of down regulating OPN mRNA when administered alone. Interestingly, only miR-221 is necessary for EGCG-mediated OPN mRNA degradation and miR-221 inhibition reduces the effects of EGCG on cell function. In vivo experiments show that thioacetamide (TAA)-induced cell cytotoxicity upregulates OPN expression; treatment with EGCG blocks the effects of TAA. Furthermore, chronic treatment of EGCG in vivo upregulates all three microRNAs equally, suggesting that in more chronic treatment all three microRNAs are involved in modulating OPN expression. We conclude that in in vitro and in vivo models of TAA-induced hepatic fibrosis, EGCG inhibits OPN-dependent injury and fibrosis. EGCG works primarily by upregulating miR-221 to accelerate OPN degradation. EGCG may therefore have utility as a protective agent in settings of liver injury.

Concepts: Messenger RNA, In vivo, Enzyme inhibitor, In vitro, Inhibitor, Catechin, Green tea, Epigallocatechin gallate

119

Lateralization is a fundamental principle of nervous system organization but its molecular determinants are mostly unknown. In humans, asymmetric gene expression in the fetal cortex has been suggested as the molecular basis of handedness. However, human fetuses already show considerable asymmetries in arm movements before the motor cortex is functionally linked to the spinal cord, making it more likely that spinal gene expression asymmetries form the molecular basis of handedness. We analyzed genome-wide mRNA expression and DNA methylation in cervical and anterior thoracal spinal cord segments of five human fetuses and show development-dependent gene expression asymmetries. These gene expression asymmetries were epigenetically regulated by miRNA expression asymmetries in the TGF-β signaling pathway and lateralized methylation of CpG islands. Our findings suggest molecular mechanisms for epigenetic regulation within the spinal cord constitute the starting point for handedness, implying a fundamental shift in our understanding of the ontogenesis of hemispheric asymmetries in humans.

Concepts: DNA, Gene, Gene expression, Cell, Epigenetics, RNA, Messenger RNA, DNA methylation

107

Aging reduces skeletal muscle mass and strength, but the underlying molecular mechanisms remain elusive. Here, we used mouse models to investigate molecular mechanisms of age-related skeletal muscle weakness and atrophy, as well as new potential interventions for these conditions. We identified two small molecules that significantly reduce age-related deficits in skeletal muscle strength, quality and mass: ursolic acid (a pentacyclic triterpenoid found in apples) and tomatidine (a steroidal alkaloid derived from green tomatoes). Because small molecule inhibitors can sometimes provide mechanistic insight into disease processes, we used ursolic acid and tomatidine to investigate the pathogenesis of age-related muscle weakness and atrophy. We found that ursolic acid and tomatidine generate hundreds of small positive and negative changes in mRNA levels in aged skeletal muscle, and the mRNA expression signatures of the two compounds are remarkably similar. Interestingly, a subset of the mRNAs are repressed by ursolic acid and tomatidine in aged muscle are positively regulated by the transcription factor ATF4. Based on this finding, we investigated ATF4 as a potential mediator of age-related muscle weakness and atrophy. We found that a targeted reduction in skeletal muscle ATF4 expression reduces age-related deficits in skeletal muscle strength, quality and mass, similar to ursolic acid and tomatidine. These results elucidate ATF4 as a critical mediator of age-related muscle weakness and atrophy. In addition, these results identify ursolic acid and tomatidine as potential agents and/or lead compounds for reducing ATF4 activity, weakness, and atrophy in aged skeletal muscle.

Concepts: DNA, Protein, Energy, Molecule, Messenger RNA, Muscle, Enzyme inhibitor, Muscular system