In Drosophila, repeat-associated small interfering RNAs (rasiRNAs) are produced in the germ line by a Dicer-independent pathway and function through the PIWI subfamily of Argonautes to ensure silencing of retrotransposons. We sequenced small RNAs associated with the PIWI subfamily member AGO3. Although other members of PIWI, Aubergine (Aub) and Piwi, associated with rasiRNAs derived mainly from the antisense strand of retrotransposons, AGO3-associated rasiRNAs arose mainly from the sense strand. Aub- and Piwi-associated rasiRNAs showed a strong preference for uracil at their 5' ends, and AGO3-associated rasiRNAs showed a strong preference for adenine at nucleotide 10. Comparisons between AGO3- and Aub-associated rasiRNAs revealed pairs of rasiRNAs showing complementarities in their first 10 nucleotides. Aub and AGO3 exhibited Slicer activity in vitro. These data support a model in which formation of a 5' terminus within rasiRNA precursors is guided by rasiRNAs originating from transcripts of the other strand in concert with the Slicer activity of PIWI.

The ubiquitous presence of long noncoding RNAs (lncRNAs) in eukaryotes points to the importance of understanding how their sequences impact function. As many lncRNAs regulate nuclear events and thus must localize to nuclei, we analyzed the sequence requirements for nuclear localization in an intergenic lncRNA named BORG (BMP2-OP1-responsive gene), which is both spliced and polyadenylated but is strictly localized in nuclei. Subcellular localization of BORG was not dependent on the context or level of its expression or decay but rather depended on the sequence of the mature, spliced transcript. Mutational analyses indicated that nuclear localization of BORG was mediated through a novel RNA motif consisting of the pentamer sequence AGCCC with sequence restrictions at positions ؊8 (T or A) and ؊3 (G or C) relative to the first nucleotide of the pentamer. Mutation of the motif to a scrambled sequence resulted in complete loss of nuclear localization, while addition of even a single copy of the motif to a cytoplasmically localized RNA was sufficient to impart nuclear localization. Further, the presence of this motif in other cellular RNAs showed a direct correlation with nuclear localization, suggesting that the motif may act as a general nuclear localization signal for cellular RNAs.

Recent genome-wide studies have revealed the presence of thousands of long non-protein-coding RNAs (lncRNAs), some of which may play critical roles in the cell. We have previously shown that a large number of lncRNAs show differential expression in response to interferon (IFN)α stimulation in primary human cells. Here, we show that a subset of IFN-induced lncRNAs are positioned in proximity of protein-coding IFN-stimulated genes (ISGs). The majority of gene pairs originated from bidirectional promoters and showed positively correlated expression. We focused our analysis on a pair consisting of the known protein-coding ISG, BST2, and an un-studied putative lncRNA originating from the promoter region of BST2 in a divergent orientation. We showed that this transcript was a multi-exonic, polyadenylated long RNA that lacked protein-coding capacity. BST2 and the lncRNA were both induced in response to IFNα in diverse cell types. The induction of both genes was mediated through the JAK–STAT pathway, suggesting that IFN-stimulated response elements within the shared promoter activated the transcription of both genes. RNAi-mediated knock-down of the lncRNA resulted in down-regulation of BST2, and we could show that this down-regulation occurred at the level of transcription. Forced overexpression of this lncRNA, which we named BST2 IFN-Stimulated Positive Regulator (BISPR), resulted in up-regulation of BST2, indicating that the regulation of expression of BST2 by BISPR is mediated through interactions involving BISPR RNA itself, rather than the impact of its transcription from an adjacent locus. Importantly, upon IFN stimulation, transcriptional activation of BISPR preceded the induction of BST2, suggesting that expression of BISPR facilitated the initiation of transcription in its paired protein-coding gene. The lncRNA-mediated transcriptional regulation described in this study may help govern the expression of additional protein-coding RNAs involved in IFN response and other cellular processes.