Significant fractions of eukaryotic genomes give rise to RNA, much of which is unannotated and has reduced protein-coding potential. The genomic origins and the associations of human nuclear and cytosolic polyadenylated RNAs longer than 200 nucleotides (nt) and whole-cell RNAs less than 200 nt were investigated in this genome-wide study. Subcellular addresses for nucleotides present in detected RNAs were assigned, and their potential processing into short RNAs was investigated. Taken together, these observations suggest a novel role for some unannotated RNAs as primary transcripts for the production of short RNAs. Three potentially functional classes of RNAs have been identified, two of which are syntenically conserved and correlate with the expression state of protein-coding genes. These data support a highly interleaved organization of the human transcriptome.

Drosophila melanogaster is one of the most well studied genetic model organisms, nonetheless its genome still contains unannotated coding and non-coding genes, transcripts, exons, and RNA editing sites. Full discovery and annotation are prerequisites for understanding how the regulation of transcription, splicing, and RNA editing directs development of this complex organism. We used RNA-Seq, tiling microarrays, and cDNA sequencing to explore the transcriptome in 30 distinct developmental stages. We identified 111,195 new elements, including thousands of genes, coding and non-coding transcripts, exons, splicing and editing events and inferred protein isoforms that previously eluded discovery using established experimental, prediction and conservation-based approaches. Together, these data substantially expand the number of known transcribed elements in the Drosophila genome and provide a high-resolution view of transcriptome dynamics throughout development.

Mechanosensory transduction underlies a wide range of senses, including proprioception, touch, balance, and hearing. The pivotal element of these senses is a mechanically gated ion channel that transduces sound, pressure, or movement into changes in excitability of specialized sensory cells. Despite the prevalence of mechanosensory systems, little is known about the molecular nature of the transduction channels. To identify such a channel, we analyzed Drosophila melanogaster mechanoreceptive mutants for defects in mechanosensory physiology. Loss-of-function mutations in the no mechanoreceptor potential C (nompC) gene virtually abolished mechanosensory signaling. nompC encodes a new ion channel that is essential for mechanosensory transduction. As expected for a transduction channel, D. melanogaster NOMPC and a Caenorhabditis elegans homolog were selectively expressed in mechanosensory organs.

Noncoding RNA molecules (ncRNAs) have been implicated in numerous biological processes including transcriptional regulation and the modulation of protein function. Yet, in spite of the apparent abundance of ncRNA, little is known about the biological role of the projected thousands of ncRNA genes present in the human genome. To facilitate functional analysis of these RNAs, we have created an arrayed library of short hairpin RNAs (shRNAs) directed against 512 evolutionarily conserved putative ncRNAs and, via cell-based assays, we have begun to determine their roles in cellular pathways. Using this system, we have identified an ncRNA repressor of the nuclear factor of activated T cells (NFAT), which interacts with multiple proteins including members of the importin-beta superfamily and likely functions as a specific regulator of NFAT nuclear trafficking.

Recent evidence of genome-wide transcription in several species indicates that the amount of transcription that occurs cannot be entirely accounted for by current sets of genome-wide annotations. Evidence indicates that most of both strands of the human genome might be transcribed, implying extensive overlap of transcriptional units and regulatory elements. These observations suggest that genomic architecture is not colinear, but is instead interleaved and modular, and that the same genomic sequences are multifunctional: that is, used for multiple independently regulated transcripts and as regulatory regions. What are the implications and consequences of such an interleaved genomic architecture in terms of increased information content, transcriptional complexity, evolution and disease states?

Drosophila melanogaster cell lines are important resources for cell biologists. Here, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, often distant, genes. Sixty-four percent of genes are expressed detectably in at least one line, but only 21% are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signaling pathways are well represented: most differentiation pathways are “off” and survival/growth pathways “on.” Roughly 50% of the genes expressed by each line are not part of the common set, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most remarkable is that imaginal disc-derived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common “cell line“ gene expression pattern.

The widespread occurrence of noncoding (nc) RNAs--unannotated eukaryotic transcripts with reduced protein coding potential--suggests that they are functionally important. Study of ncRNAs is increasing our understanding of the organization and regulation of genomes.

Small interfering RNAs (siRNAs) are short, double-stranded RNAs that use the endogenous RNAi pathway to mediate gene silencing. Phosphorylation facilitates loading of a siRNA into the Ago2 complex and subsequent cleavage of the target mRNA. In this study, 2′, 3′ seco nucleoside modifications, which contain an acylic ribose ring and are commonly called unlocked nucleic acids (UNAs), were evaluated at all positions along the guide strand of a siRNA targeting apolipoprotein B (ApoB). UNA modifications at positions 1, 2 and 3 were detrimental to siRNA activity. UNAs at positions 1 and 2 prevented phosphorylation by Clp1 kinase, abrogated binding to Ago2, and impaired Ago2-mediated cleavage of the mRNA target. The addition of a 5′-terminal phosphate to siRNA containing a position 1 UNA restored ApoB mRNA silencing, Ago2 binding, and Ago2 mediated cleavage activity. Position 1 UNA modified siRNA containing a 5′-terminal phosphate exhibited a partial restoration of siRNA silencing activity in vivo. These data reveal the complexity of interpreting the effects of chemical modification on siRNA activity, and exemplify the importance of using multiple biochemical, cell-based and in vivo assays to rationally design chemically modified siRNA destined for therapeutic use.