Peptidomic discovery of short open reading frame-encoded peptides in human cells

The amount of the transcriptome that is translated into polypeptides is of fundamental importance. We developed a peptidomic strategy to detect short ORF (sORF)-encoded polypeptides (SEPs) in human cells. We identified 90 SEPs, 86 of which are novel, the largest number of human SEPs ever reported. SEP abundances range from 10-1000 molecules per cell, identical to known proteins. SEPs arise from sORFs in non-coding RNAs as well as multi-cistronic mRNAs, and many SEPs initiate with non-AUG start codons, indicating that non-canonical translation may be more widespread in mammals than previously thought. In addition, coding sORFs are present in a small fraction (8/1866) of long intergenic non-coding RNAs (lincRNAs). Together, these results provide the strongest evidence to date that the human proteome is more complex than previously appreciated

Phylogenomic analyses of the genus Drosophila reveals genomic signals of climate adaptation

Many Drosophila species differ widely in their distributions and climate niches, making them excellent subjects for evolutionary genomic studies. Here, we have developed a database of high-quality assemblies for 46 Drosophila species and one closely related Zaprionus. Fifteen of the genomes were newly sequenced, and 20 were improved with additional sequencing. New or improved annotations were generated for all 47 species, assisted by new transcriptomes for 19. Phylogenomic analyses of these data resolved several previously ambiguous relationships, especially in the melanogaster species group. However, it also revealed significant phylogenetic incongruence among genes, mainly in the form of incomplete lineage sorting in the subgenus Sophophora but also including asymmetric introgression in the subgenus Drosophila. Using the phylogeny as a framework and taking into account these incongruences, we then screened the data for genome-wide signals of adaptation to different climatic niches. First, phylostratigraphy revealed relatively high rates of recent novel gene gain in three temperate pseudoobscura and five desert-adapted cactophilic mulleri subgroup species. Second, we found differing ratios of nonsynonymous to synonymous substitutions in several hundred orthologues between climate generalists and specialists, with trends for significantly higher ratios for those in tropical and lower ratios for those in temperate-continental specialists respectively than those in the climate generalists. Finally, resequencing natural populations of 13 species revealed tropics-restricted species generally had smaller population sizes, lower genome diversity and more deleterious mutations than the more widespread species. We conclude that adaptation to different climates in the genus Drosophila has been associated with large-scale and multifaceted genomic changes

Deep Annotation of Populus trichocarpa microRNAs from Diverse Tissue Sets

Populus trichocarpa is an important woody model organism whose entire genome has been sequenced. This resource has facilitated the annotation of microRNAs (miRNAs), which are short non-coding RNAs with critical regulatory functions. However, despite their developmental importance, P. trichocarpa miRNAs have yet to be annotated from numerous important tissues. Here we significantly expand the breadth of tissue sampling and sequencing depth for miRNA annotation in P. trichocarpa using high-throughput smallRNA (sRNA) sequencing. miRNA annotation was performed using three individual next-generation sRNA sequencing runs from separate leaves, xylem, and mechanically treated xylem, as well as a fourth run using a pooled sample containing vegetative apices, male flowers, female flowers, female apical buds, and male apical and lateral buds. A total of 276 miRNAs were identified from these datasets, including 155 previously unannotated miRNAs, most of which are P. trichocarpa specific. Importantly, we identified several xylem-enriched miRNAs predicted to target genes known to be important in secondary growth, including the critical reaction wood enzyme xyloglucan endo-transglycosylase/hydrolase and vascular-related transcription factors. This study provides a thorough genome-wide annotation of miRNAs in P. trichocarpa through deep sRNA sequencing from diverse tissue sets. Our data significantly expands the P. trichocarpa miRNA repertoire, which will facilitate a broad range of research in this major model system

Non-equilibrium statistical mechanics: From a paradigmatic model to biological transport

Unlike equilibrium statistical mechanics, with its well-established foundations, a similar widely-accepted framework for non-equilibrium statistical mechanics (NESM) remains elusive. Here, we review some of the many recent activities on NESM, focusing on some of the fundamental issues and general aspects. Using the language of stochastic Markov processes, we emphasize general properties of the evolution of configurational probabilities, as described by master equations. Of particular interest are systems in which the dynamics violate detailed balance, since such systems serve to model a wide variety of phenomena in nature. We next review two distinct approaches for investigating such problems. One approach focuses on models sufficiently simple to allow us to find exact, analytic, non-trivial results. We provide detailed mathematical analyses of a one-dimensional continuous-time lattice gas, the totally asymmetric exclusion process (TASEP). It is regarded as a paradigmatic model for NESM, much like the role the Ising model played for equilibrium statistical mechanics. It is also the starting point for the second approach, which attempts to include more realistic ingredients in order to be more applicable to systems in nature. Restricting ourselves to the area of biophysics and cellular biology, we review a number of models that are relevant for transport phenomena. Successes and limitations of these simple models are also highlighted.Comment: 72 pages, 18 figures, Accepted to: Reports on Progress in Physic

A theoretical study of proton translocation in gramicidin A

Proton translocation (PTL) is studied in the ion channel gramicidin A (gA). Non-dissociable and fully charged dissociable models are found to be insufficient for studying this complex process. The partially charged and dissociable multi-state empirical valence bond (MS-EVB) model is modified to model the proton hopping process between water molecules in gA. A new and general method is developed for measuring the location of the excess charge in a protonated system. Molecular dynamics simulations are run for a system containing gA, a protonated water wire, a model membrane, and bulk water. Both the water turning defect and excess proton hopping defect contributions to PTL are explored. Proton transfers are observed in these simulations, but long range proton translocation is not, and the differences with experimental results are investigated. A large barrier for the hopping defect is observed, with a much smaller barrier being found for the turning defect. The hopping defect is therefore identified as the likely rate determining step for the overall process in this model. The structure, dynamics, and energetics of proton translocation in gA are found to be determined in large part by competition for hydrogen bonding between protonated and unprotonated waters inside the channel and the carbonyl oxygens lining the channel

A theoretical study of proton translocation in gramicidin A

Proton translocation (PTL) is studied in the ion channel gramicidin A (gA). Non-dissociable and fully charged dissociable models are found to be insufficient for studying this complex process. The partially charged and dissociable multi-state empirical valence bond (MS-EVB) model is modified to model the proton hopping process between water molecules in gA. A new and general method is developed for measuring the location of the excess charge in a protonated system. Molecular dynamics simulations are run for a system containing gA, a protonated water wire, a model membrane, and bulk water. Both the water turning defect and excess proton hopping defect contributions to PTL are explored. Proton transfers are observed in these simulations, but long range proton translocation is not, and the differences with experimental results are investigated. A large barrier for the hopping defect is observed, with a much smaller barrier being found for the turning defect. The hopping defect is therefore identified as the likely rate determining step for the overall process in this model. The structure, dynamics, and energetics of proton translocation in gA are found to be determined in large part by competition for hydrogen bonding between protonated and unprotonated waters inside the channel and the carbonyl oxygens lining the channel

miRNA expression clustergram.

<p>Clustering of smallRNA sequencing runs by miRNA expression. [miRNA expression = (Raw Abundance)/(Number of Mappable Reads)×1,000,000] reveals that xylem and mechanically treated xylem (MTX) are most similar. MATLAB <i>clustergram</i> algorithm was used for expression clustering.</p

Length distribution of smallRNA reads.

<p>Length distribution of smallRNAs of obtained from four individual sequencing runs after adaptor removal but prior to filtering.</p

Expression of miRNAs.

<p>miRNA expression is shown in normalized units according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033034#pone.0033034-Li1" target="_blank">[20]</a>. [miRNA expression = (Raw Abundance)/(Number of Mappable Reads)×1,000,000]. Conserved: miRNAs identified outside of <i>P. trichocarpa</i> in at least one other green plant species on miRBASE. <i>P. trichocarpa</i> specific: miRNAs only identified in <i>P. trichocarpa</i>.</p

Summary statistics of smallRNA sequencing.

<p>Summary statistics of smallRNA sequencing.</p