Summary Eukaryotic cells make many types of primary and processed RNAs that are found either in specific sub-cellular compartments or throughout the cells. A complete catalogue of these RNAs is not yet available and their characteristic sub-cellular localizations are also poorly understood. Since RNA represents the direct output of the genetic information encoded by genomes and a significant proportion of a cell’s regulatory capabilities are focused on its synthesis, processing, transport, modifications and translation, the generation of such a catalogue is crucial for understanding genome function. Here we report evidence that three quarters of the human genome is capable of being transcribed, as well as observations about the range and levels of expression, localization, processing fates, regulatory regions and modifications of almost all currently annotated and thousands of previously unannotated RNAs. These observations taken together prompt to a redefinition of the concept of a gene.

The use of fluorescent proteins has revolutionized our understanding of biological processes. However, the requirement for external illumination precludes their universal application to the study of biological processes in all tissues. Although light can be created by chemiluminescence, light emission from existing chemiluminescent probes is too weak to use this imaging modality in situations when fluorescence cannot be used. Here we report the development of the brightest luminescent protein to date, Nano-lantern, which is a chimera of enhanced Renilla luciferase and Venus, a fluorescent protein with high bioluminescence resonance energy transfer efficiency. Nano-lantern allows real-time imaging of intracellular structures in living cells with spatial resolution equivalent to fluorescence and sensitive tumour detection in freely moving unshaved mice. We also create functional indicators based on Nano-lantern that can image Ca2+, cyclic adenosine monophosphate and adenosine 5′-triphosphate dynamics in environments where the use of fluorescent indicators is not feasible. These luminescent proteins allow visualization of biological phenomena at previously unseen single-cell, organ and whole-body level in animals and plants.

Data from the Encyclopedia of DNA Elements (ENCODE) project show over 9640 human genome loci classified as long noncoding RNAs (lncRNAs), yet only~100 have been deeply characterized to determine their role in the cell. To measure the protein-coding output from these RNAs, we jointly analyzed two recent data sets produced in the ENCODE project: tandem mass spectrometry (MS/MS) data mapping expressed peptides to their encoding genomic loci, and RNA-seq data generated by ENCODE in long polyA+ and polyA-fractions in the cell lines K562 and GM12878. We used the machinelearning algorithm RuleFit3 to regress the peptide data against RNA expression data. The most important covariate for predicting translation was, surprisingly, the Cytosol polyA-fraction in both cell lines. LncRNAs are~13-fold less likely to produce detectable peptides than similar mRNAs, indicating that~92% of GENCODE v7 lncRNAs are not translated in these two ENCODE cell lines. Intersecting 9640 lncRNA loci with 79,333 peptides yielded 85 unique peptides matching 69 lncRNAs. Most cases were due to a coding transcript misannotated as lncRNA. Two exceptions were an unprocessed pseudogene and a bona fide lncRNA gene, both with open reading frames (ORFs) compromised by upstream stop codons. All potentially translatable lncRNA ORFs had only a single peptide match, indicating low protein abundance and/or false-positive peptide matches. We conclude that with very few exceptions, ribosomes are able to distinguish coding from noncoding transcripts and, hence, that ectopic translation and cryptic mRNAs are rare in the human lncRNAome.

The success of shotgun proteomic analysis depends largely on how samples are prepared. Current approaches (such as those that are gel-, solution-, or filter-based), although being extensively employed in the field, are time-consuming and less effective with respect to the repetitive sample processing, recovery, and overall yield. As an alternative, the suspension trapping (S-Trap) filter has been commercially available very recently in the format of a single or 96-well filter plate. In contrast to the conventional filter-aided sample preparation (FASP) approach, which utilizes a molecular weight cut-off (MWCO) membrane as the filter and requires hours of processing before digestion-ready proteins can be obtained, the S-Trap employs a three-dimensional porous material as filter media and traps particulate protein suspensions with the subsequent depletion of interfering substances and in-filter digestion. Due to the large (submicron) pore size, each centrifugation cycle of the S-Trap filter only takes 1 min, which significantly reduces the total processing time from approximately 3 h by FASP to less than 15 min, suggesting an ultrafast sample-preparation approach for shotgun proteomics. Here, we comprehensively evaluate the performance of the individual S-Trap filter and 96-well filter plate in the context of global protein identification and quantitation using whole-cell lysate and clinically relevant sputum samples.

Over 100 mutations in Cu/Zn-superoxide dismutase (SOD1) result in familial amyotrophic lateral sclerosis. Dimer dissociation is the first step in SOD1 aggregation, and studies suggest nearly every amino acid residue in SOD1 is dynamically connected to the dimer interface. Post-translational modifications of SOD1 residues might be expected to have similar effects to mutations, but few modifications have been identified. Here we show, using SOD1 isolated from human erythrocytes, that human SOD1 is phosphorylated at threonine 2 and glutathionylated at cysteine 111. A second SOD1 phosphorylation was observed and mapped to either Thr-58 or Ser-59. Cysteine 111 glutathionylation promotes SOD1 monomer formation, a necessary initiating step in SOD1 aggregation, by causing a 2-fold increase in the K d . This change in the dimer stability is expected to result in a 67% increase in monomer concentration, 315 nM rather than 212 nM at physiological SOD1 concentrations. Because protein glutathionylation is associated with redox regulation, our finding that glutathionylation promotes SOD1 monomer formation supports a model in which increased oxidative stress promotes SOD1 aggregation. Familial amyotrophic lateral sclerosis (FALS)4 is the hereditary form of amyotrophic lateral sclerosis, a fatal disease characterized by progressive motor neuron loss (1). A subset of FALS is caused by mutations in the gene encoding homodimeric Cu/Zn-superoxide dismutase (SOD1), which forms intraneuronal aggregates (2). Although SOD1 aggregation is involved in SOD1-mediated FALS, it is generally believed that the functional properties of the enzyme are not related to the toxic gain of function imparted by mutations in SOD1 (3). However, the discovery of roles for SOD1 in the regulation of the cellular phosphorylation balance (4) and redox state (5) provides additional avenues for connecting the cellular role of SOD1 to FALS. The classical studies of SOD1 were generally performed using bovine erythrocyte SOD1 or recombinant human SOD1. Although recombinant methods are widely used to produce SOD1 mutants, a disadvantage of studying recombinant SOD1 is the absence of potentially important post-translational modifications present in human tissues. The initial SOD1 crystal structure was solved using bovine erythrocyte SOD1 (6) and no structure of human erythrocyte SOD1 is available. Here we report results using human erythrocyte SOD1 rather than the recombinant enzyme and find that the native enzyme features a consistent pattern of post-translational modifications. Using a combination of "bottom-up" and "top-down" mass spectrometry (MS) approaches, we show that SOD1 isolated from human erythrocytes is post-translationally phosphorylated and glutathionylated. These modifications occur near the SOD1 dimer interface. Because monomer formation is thought to be the first intermediate leading to SOD1 aggregation (7, 8), we tested the dimer stability of modified SOD1 found, as expected, that glutathionylation promotes the formation of SOD1 monomer. EXPERIMEN...