A large-scale binding and functional map of human RNA-binding proteins.

Many proteins regulate the expression of genes by binding to specific regions encoded in the genome1. Here we introduce a new data set of RNA elements in the human genome that are recognized by RNA-binding proteins (RBPs), generated as part of the Encyclopedia of DNA Elements (ENCODE) project phase III. This class of regulatory elements functions only when transcribed into RNA, as they serve as the binding sites for RBPs that control post-transcriptional processes such as splicing, cleavage and polyadenylation, and the editing, localization, stability and translation of mRNAs. We describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. Integrative analyses using five assays identify RBP binding sites on RNA and chromatin in vivo, the in vitro binding preferences of RBPs, the function of RBP binding sites and the subcellular localization of RBPs, producing 1,223 replicated data sets for 356 RBPs. We describe the spectrum of RBP binding throughout the transcriptome and the connections between these interactions and various aspects of RNA biology, including RNA stability, splicing regulation and RNA localization. These data expand the catalogue of functional elements encoded in the human genome by the addition of a large set of elements that function at the RNA level by interacting with RBPs.

Sequence, Structure, and Context Preferences of Human RNA Binding Proteins.

RNA binding proteins (RBPs) orchestrate the production, processing, and function of mRNAs. Here, we present the affinity landscapes of 78 human RBPs using an unbiased assay that determines the sequence, structure, and context preferences of these proteins in vitro by deep sequencing of bound RNAs. These data enable construction of "RNA maps" of RBP activity without requiring crosslinking-based assays. We found an unexpectedly low diversity of RNA motifs, implying frequent convergence of binding specificity toward a relatively small set of RNA motifs, many with low compositional complexity. Offsetting this trend, however, we observed extensive preferences for contextual features distinct from short linear RNA motifs, including spaced "bipartite" motifs, biased flanking nucleotide composition, and bias away from or toward RNA structure. Our results emphasize the importance of contextual features in RNA recognition, which likely enable targeting of distinct subsets of transcripts by different RBPs that recognize the same linear motif.

Phylogenetic analyses of ribosomal DNA-containing bacterioplankton genome fragments from a 4000 m vertical profile in the North Pacific Subtropical Gyre.

High-throughput identification of rRNA gene-containing clones in large insert metagenomic libraries is difficult, because of the high background of host ribosomal RNA (rRNA) and rRNA genes. To address this challenge, a membrane hybridization method was developed to identify all bacterial small subunit rRNA-containing fosmid clones of microbial community DNA from seven different depths in the North Pacific Subtropical Gyre. Out of 101,376 clones screened, 751 rDNA-containing clones were identified that grouped in approximately 60 different clades. Several rare sequences only remotely related to known groups were detected, including a Wolbachia-related sequence containing a putative intron or intervening sequence, as well as seven sequences from Order Myxococcales not previously detected in pelagic habitats. Stratified, depth-specific population structure was evident within both cultured and uncultured lineages. Conversely, some eurybathyal members of the genera Alcanivorax and Rhizobium shared identical small subunit ribosomal DNA sequences that were distributed from surface waters to the 4000 m depth. Comparison with similar analyses in Monterey Bay microbial communities revealed previously recognized, as well as some distinctive, depth-stratified partitioning that distinguished coastal from open ocean bacterioplankton populations. While some bias was evident in fosmid clone recovery in a few particular lineages, the overall phylogenetic group recovery and distributions were consistent with previous studies, as well as with direct shotgun sequence data from the same source DNA.

Large-scale screens of metagenomic libraries.

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.