A comprehensive survey of C. elegans argonaute proteins reveals organism-wide gene regulatory networks and functions.

Argonaute (AGO) proteins associate with small RNAs to direct their effector function on complementary transcripts. The nematode Caenorhabditis elegans contains an expanded family of 19 functional AGO proteins, many of which have not been fully characterized. In this work, we systematically analyzed every C. elegans AGO using CRISPR-Cas9 genome editing to introduce GFP::3xFLAG tags. We have characterized the expression patterns of each AGO throughout development, identified small RNA binding complements, and determined the effects of ago loss on small RNA populations and developmental phenotypes. Our analysis indicates stratification of subsets of AGOs into distinct regulatory modules, and integration of our data led us to uncover novel stress-induced fertility and pathogen response phenotypes due to ago loss.

DRUID: a pipeline for transcriptome-wide measurements of mRNA stability.

Control of messenger RNA (mRNA) stability is an important aspect of gene regulation. The gold standard for measuring mRNA stability transcriptome-wide uses metabolic labeling, biochemical isolation of labeled RNA populations, and high-throughput sequencing. However, difficult normalization procedures have inhibited widespread adoption of this approach. Here, we present DRUID (for determination of rates using intron dynamics), a new computational pipeline that is robust, easy to use, and freely available. Our pipeline uses endogenous introns to normalize time course data and yields reproducible half-lives, even with data sets that were otherwise unusable. DRUID can handle data sets from a variety of organisms, spanning yeast to humans, and we even applied it retroactively on published data sets. We anticipate that DRUID will allow broad application of metabolic labeling for studies of transcript stability.

Determining mRNA half-lives on a transcriptome-wide scale.

Every step in the life cycle of an RNA transcript provides opportunity for regulation. One important aspect of post-transcriptional control is the regulation of RNA stability. Of the many strategies for determining mRNA stability, transcription inhibition and metabolic labeling have proved the most amenable to high-throughput analysis and have opened the door to dissecting mRNA decay transcriptome-wide. Here, we describe experimental and computational methods to determine transcriptome-wide RNA stabilities using both pharmacological inhibition of transcription and metabolic labeling. To aid in the analysis of these experiments, we discuss key characteristics of high-quality experiments and address other experimental and computational considerations for the analysis of mRNA stability. Broader application of these approaches will further our understanding of mRNA decay and illuminate its contribution to different biological processes.

The influence of microRNAs and poly(A) tail length on endogenous mRNA-protein complexes.

BACKGROUND: All mRNAs are bound in vivo by proteins to form mRNA-protein complexes (mRNPs), but changes in the composition of mRNPs during posttranscriptional regulation remain largely unexplored. Here, we have analyzed, on a transcriptome-wide scale, how microRNA-mediated repression modulates the associations of the core mRNP components eIF4E, eIF4G, and PABP and of the decay factor DDX6 in human cells. RESULTS: Despite the transient nature of repressed intermediates, we detect significant changes in mRNP composition, marked by dissociation of eIF4G and PABP, and by recruitment of DDX6. Furthermore, although poly(A)-tail length has been considered critical in post-transcriptional regulation, differences in steady-state tail length explain little of the variation in either PABP association or mRNP organization more generally. Instead, relative occupancy of core components correlates best with gene expression. CONCLUSIONS: These results indicate that posttranscriptional regulatory factors, such as microRNAs, influence the associations of PABP and other core factors, and do so without substantially affecting steady-state tail length.

ME31B globally represses maternal mRNAs by two distinct mechanisms during the Drosophila maternal-to-zygotic transition.

In animal embryos, control of development is passed from exclusively maternal gene products to those encoded by the embryonic genome in a process referred to as the maternal-to-zygotic transition (MZT). We show that the RNA-binding protein, ME31B, binds to and represses the expression of thousands of maternal mRNAs during the Drosophila MZT. However, ME31B carries out repression in different ways during different phases of the MZT. Early, it represses translation while, later, its binding leads to mRNA destruction, most likely as a consequence of translational repression in the context of robust mRNA decay. In a process dependent on the PNG kinase, levels of ME31B and its partners, Cup and Trailer Hitch (TRAL), decrease by over 10-fold during the MZT, leading to a change in the composition of mRNA-protein complexes. We propose that ME31B is a global repressor whose regulatory impact changes based on its biological context.

Phosphoproteomic network analysis in the sea urchin Strongylocentrotus purpuratus reveals new candidates in egg activation.

Fertilization triggers a dynamic symphony of molecular transformations induced by a rapid rise in intracellular calcium. Most prominent are surface alterations, metabolic activation, cytoskeletal reorganization, and cell-cycle reentry. While the activation process appears to be broadly evolutionarily conserved, and protein phosphorylation is known to play a key role, the signaling networks mediating the response to fertilization are not well described. To address this gap, we performed a time course phosphoproteomic analysis of egg activation in the sea urchin Strongylocentrotus purpuratus, a system that offers biochemical tractability coupled with exquisite synchronicity. By coupling large-scale phosphopeptide enrichment with unbiased quantitative MS, we identified striking changes in global phosphoprotein patterns at 2- and 5-min postfertilization as compared to unfertilized eggs. Overall, we mapped 8796 distinct phosphosite modifications on 2833 phosphoproteins, of which 15% were differentially regulated in early egg activation. Activated kinases were identified by phosphosite mapping, while enrichment analyses revealed conserved signaling cascades not previously associated with egg activation. This work represents the most comprehensive study of signaling associated with egg activation to date, suggesting novel mechanisms that can be experimentally tested and providing a valuable resource for the broader research community. All MS data have been deposited in the ProteomeXchange with identifier PXD002239 (http://proteomecentral.proteomexchange.org/dataset/PXD002239).

Large-scale label-free phosphoproteomics: from technology to data interpretation.

Protein phosphorylation plays a central role in the dynamic intracellular signaling and the control of biochemical pathways in all living cells. Recent advances in high-performance MS/MS-based technology make the large-scale identification and quantification of phosphorylation sites possible. Here, we review the full data generation pipeline, starting from sample preparation methods and LC-MS detection procedures, through to data processing and analysis software tools that facilitate the systematic comparative profiling of thousands of phosphoproteins in different biological specimens in a single experiment. We emphasize current challenges and promising avenues for the mechanistic interpretation and visualization of global phosphorylation networks and their relevance to human health and disease.

ComplexQuant: high-throughput computational pipeline for the global quantitative analysis of endogenous soluble protein complexes using high resolution protein HPLC and precision label-free LC/MS/MS.

The experimental isolation and characterization of stable multi-protein complexes are essential to understanding the molecular systems biology of a cell. To this end, we have developed a high-throughput proteomic platform for the systematic identification of native protein complexes based on extensive fractionation of soluble protein extracts by multi-bed ion exchange high performance liquid chromatography (IEX-HPLC) combined with exhaustive label-free LC/MS/MS shotgun profiling. To support these studies, we have built a companion data analysis software pipeline, termed ComplexQuant. Proteins present in the hundreds of fractions typically collected per experiment are first identified by exhaustively interrogating MS/MS spectra using multiple database search engines within an integrative probabilistic framework, while accounting for possible post-translation modifications. Protein abundance is then measured across the fractions based on normalized total spectral counts and precursor ion intensities using a dedicated tool, PepQuant. This analysis allows co-complex membership to be inferred based on the similarity of extracted protein co-elution profiles. Each computational step has been optimized for processing large-scale biochemical fractionation datasets, and the reliability of the integrated pipeline has been benchmarked extensively. This article is part of a Special Issue entitled: From protein structures to clinical applications.

Combined biological and membrane treatment of food-processing wastewater to achieve dry-ditch criteria: pilot and full-scale performance.

This study tested the applicability of a submerged vacuum ultrafiltration membrane technology in combination with the biological treatment system to achieve dry-ditch criteria stipulated as follows: BOD5, TSS, NH3-N, and total phosphorous (TP) concentration not exceeding 10, 10, 1, and 0.5 mg/L respectively for the treatment of high strength food-processing wastewater. During the study, the biological system, operated at average hydraulic retention time of 5-6 days, achieved 95-96.5% BOD removal and 96-99% COD removal. The external membrane system ensured the achievability of the BOD and TSS criteria, with BOD and TSS concentrations in the permeate of 1-2 and 1-8 mg/L respectively. Nitrate, and nitrite concentrations increased during membrane filtration, while ammonia concentrations decreased. The most salient finding of this study is that, contrary to common belief, for industrial wastewaters, the filterability of the mixed liquor is influenced by the soluble organics, and may be low, thus necessitating operation of bioreactors at low mixed liquor solids. This study demonstrated that bioreactors operated at low SRTs and in combination with ultrafiltration can still achieve superior effluent quality that may meet reuse criteria at reasonable cost.

Simultaneous nitrification-denitrification and clarification in a pseudoliquified activated sludge system.

This paper describes results from a pilot study of a novel wastewater treatment technology, which incorporates nutrient removal and solids separation to a single step. The pseudoliquified activated sludge process pilot system was tested on grit removal effluent at flowrates of 29.4 to 54.7 m3/d, three different solid residence times (SRT) (15, 37, and 57 days), and over a temperature range of 12 to 28 degrees C. Despite wide fluctuations in the influent characteristics, the system performed reliably and consistently with respect to organics and total suspended solids (TSS) removals, achieving biochemical oxygen demand (BOD) and TSS reductions of > 96% and approximately 90%, respectively, with BOD5 and TSS concentrations as low as 3 mg/L. Although the system achieved average effluent ammonia concentrations of 2.7 to 3.2 mg/L, nitrification efficiency appeared to be hampered at low temperatures (< 15 degrees C). The system achieved tertiary effluent quality with denitrification efficiencies of 90 and 91% total nitrogen removal efficiency at a total hydraulic retention time of 4.8 hours and an SRT of 12 to 17 days. With ferric chloride addition, effluent phosphorous concentrations of 0.5 to 0.8 mg/L were achieved. Furthermore, because of operation at high biomass concentrations and relatively long biological SRTs, sludge yields were over 50% below typical values for activated sludge plants. The process was modeled using activated sludge model No. 2, as a two-stage system comprised an aerobic activated sludge system followed by an anoxic system. Model predictions for soluble BOD, ammonia, nitrates, and orthophosphates agreed well with experimental data.