Project description:Recent efforts towards the comprehensive identification of RNA-bound proteomes have revealed a large, surprisingly diverse family of candidate RNA-binding proteins (RBPs). Quantitative metrics for characterization and validation of protein-RNA interactions and their dynamic interactions have, however, proven to be analytically challenging and prone to error. Here we report a novel method termed LEAP-RBP for the selective, quantitative recovery of UV-crosslinked RNA-protein complexes. By virtue of its high specificity and yield, LEAP-RBP distinguishes RNA-bound and RNA-free protein levels and reveals common sources of experimental noise in RNA-centric RBP enrichment methods. We introduce new strategies for accurate RBP identification and signal-based metrics for quantifying protein-RNA complex enrichment, relative RNA occupancy, and method specificity. The utility of our approach is validated by comprehensive identification of RBPs whose association with mRNA is modulated in response to global mRNA translation state changes and through in-depth benchmark comparisons with current methodologies.
Project description:Seamounts, often rising hundreds of metres above the surrounding seafloor, obstruct the flow of deep-ocean water. While the resultant entrainment of deep-water by seamounts is predicted from ocean circulation models, its empirical validation has been hampered by the large scale and slow rate of the interaction. To overcome these limitations we use the growth of planktonic bacteria to assess the interaction rate. The selected study site, Tropic Seamount, in the North-Eastern Atlantic represents the majority of isolated seamounts, which do not affect the surface ocean waters. We prove deep-water is entrained by the seamount by measuring 2.3 times higher bacterial concentrations in the seamount-associated or ‘sheath’ water than in deep-ocean water unaffected by seamounts. Genomic analyses of the dominant sheath-water bacteria confirm their planktonic origin, whilst proteomic analyses indicate their slow growth. According to our radiotracer experiments, the doubling time of sheath-water bacterioplankton is 1.5 years. Therefore, for bacterioplankton concentration to reach 2.3 times higher in the ambient seawater, the seamount would need to retain deep-ocean water for more than 3.5 years. We propose that turbulent mixing of the retained sheath-water could stimulate bacterioplankton growth by increasing the cell encounter rate with the ambient dissolved organic molecules. If some of these molecules chelate hydroxides of iron and manganese, bacterioplankton consumption of the organic chelators would result in precipitation of insoluble hydroxides. Hence precipitated hydroxides would form ferromanganese deposits as a result of the bacterioplankton-mediated deep-water seamount interaction.
Project description:Polyamines, such as putrescine and spermidine, are aliphatic organic compounds with multiple amino groups. They are found ubiquitously in marine systems. However, compared with the extensive studies on the concentration and fate of other dissolved organic nitrogen compounds in seawater, such as dissolved free amino acids (DFAA), investigations of bacterially-mediated polyamine transformations have been rare. Bioinformatic analysis identified genes encoding polyamine transporters in 74 of 109 marine bacterial genomes surveyed, a surprising frequency for a class of organic nitrogen compounds not generally recognized as an important source of carbon and nitrogen for marine bacterioplankton. The genome sequence of marine model bacterium Silicibacter pomeroyi DSS-3 contains a number of genes putatively involved in polyamine use, including six four-gene ATP-binding cassette transport systems. In the present study, polyamine uptake and metabolism by S. pomeroyi was examined to confirm the role of putative polyamine-related genes, and to investigate how well current gene annotations reflect function. A comparative whole-genome microarray approach (Bürgmann et al., 2007) allowed us to identify key genes for transport and metabolism of spermidine in this bacterium, and specify candidate genes for in situ monitoring of polyamine transformations in marine bacterioplankton communities.
Project description:CEH-60 binding profile by comparison of DNA methylation of a C. elegans strain expression ceh-60::dam to a control strain expressing gfp::dam. Sequencing libraries prepared using NEBNext Singleplex oligos for Illumina®. Data analysis performed with GeneDamIDseq (Sharma, Ritler, and Meister 2016).
Project description:DEEPEND: Microbiome and bacterioplankton rRNA gene sequence data collected from Gulf of Mexico seawater samples. Cruises DP03 and DP04 from Jan 2016 - December 2016