In this article, we discuss emerging frontiers in RNA biology from a historical perspective. The field is currently undergoing yet another transformative expansion. RNA-seq has revealed that splicing, and, more generally, RNA processing is far more complex than expected, and the mechanisms of regulation are correspondingly sophisticated. Our understanding of the molecular machines involved in RNA metabolism is incomplete and derives from small sample sizes. Even if we manage to complete a catalogue of molecular species, RNA isoforms and the ribonucleoprotein complexes that drive their genesis, the horizons of molecular dynamics and cell-type-specific processing mechanisms await. This is an exciting time to enter into the study of RNA biology; analytical tools, wet and dry, are advancing rapidly, and each new measurement modality brings into view another new function or activity of versatile RNA. Since the dawn of sequence-based RNA biology, we have come a long way.

The modENCODE (Model Organism Encyclopedia of DNA Elements) Consortium aimed to map functional elements-including transcripts, chromatin marks, regulatory factor binding sites, and origins of DNA replication-in the model organisms Drosophila melanogaster and Caenorhabditis elegans. During its five-year span, the consortium conducted more than 2,000 genome-wide assays in developmentally staged animals, dissected tissues, and homogeneous cell lines. Analysis of these data sets provided foundational insights into genome, epigenome, and transcriptome structure and the evolutionary turnover of regulatory pathways. These studies facilitated a comparative analysis with similar data types produced by the ENCODE Consortium for human cells. Genome organization differs drastically in these distant species, and yet quantitative relationships among chromatin state, transcription, and cotranscriptional RNA processing are deeply conserved. Of the many biological discoveries of the modENCODE Consortium, we highlight insights that emerged from integrative studies. We focus on operational and scientific lessons that may aid future projects of similar scale or aims in other, emerging model systems.

Distinct isoforms of spliceosomal RNAs may be involved in regulating pre-messenger RNA splicing in eukaryotic cells. During a large-scale effort to identify small noncoding RNAs in Drosophila, we isolated a U5 snRNA-like molecule containing a 5' segment identical to that of the canonical (major) U5 snRNA but with a variant Sm binding site and a distinct 3' hairpin sequence. Based on this finding, another six similar U5 snRNA-like sequences were identified within the Drosophila genome by sequence similarity to the invariant loop in the 5' half of U5. Interestingly, although all of these variants are expressed in vivo, each shows a distinct temporal expression profile during Drosophila development, and one is expressed primarily in fly heads. The presence of these U5 snRNA variants within RNP particles suggests their role in splicing and implies a possible connection to regulation of developmental and tissue-specific gene expression.

Acetobacter pomorum Oregon-R-modENCODE strain BDGP5 was isolated from Drosophila melanogaster for functional host-microbe interaction studies. The complete genome is composed of a single chromosomal circle of 2,848,089 bp, with a G+C content of 53% and three plasmids of 131,455 bp, 19,216 bp, and 9,160 bp.

Lactobacillus plantarum Oregon-R-modENCODE strain BDGP2 was isolated from the gut of Drosophila melanogaster for functional host microbial interaction studies. The complete genome comprised a single circular genome of 3,407,160 bp, with a G+C content of 44%, and four plasmids.

Spatial gene expression patterns enable the detection of local covariability and are extremely useful for identifying local gene interactions during normal development. The abundance of spatial expression data in recent years has led to the modeling and analysis of regulatory networks. The inherent complexity of such data makes it a challenge to extract biological information. We developed staNMF, a method that combines a scalable implementation of nonnegative matrix factorization (NMF) with a new stability-driven model selection criterion. When applied to a set ofDrosophilaearly embryonic spatial gene expression images, one of the largest datasets of its kind, staNMF identified 21 principal patterns (PP). Providing a compact yet biologically interpretable representation ofDrosophilaexpression patterns, PP are comparable to a fate map generated experimentally by laser ablation and show exceptional promise as a data-driven alternative to manual annotations. Our analysis mapped genes to cell-fate programs and assigned putative biological roles to uncharacterized genes. Finally, we used the PP to generate local transcription factor regulatory networks. Spatially local correlation networks were constructed for six PP that span along the embryonic anterior-posterior axis. Using a two-tail 5% cutoff on correlation, we reproduced 10 of the 11 links in the well-studied gap gene network. The performance of PP with theDrosophiladata suggests that staNMF provides informative decompositions and constitutes a useful computational lens through which to extract biological insight from complex and often noisy gene expression data.

Enterococcus durans Oregon-R-modENCODE strain BDGP3 was isolated from the Drosophila melanogaster gut for functional host-microbe interaction studies. The complete genome is composed of a single circular genome of 2,983,334 bp, with a G+C content of 38%, and a single plasmid of 5,594 bp.

Acetobacter tropicalis Oregon-R-modENCODE strain BDGP1 was isolated from Drosophila melanogaster for functional host-microbe interaction studies. The complete genome comprises a single chromosomal circle of 3,988,649 bp with a G+C content of 56% and a conjugative plasmid of 151,013 bp.