The canonical microRNA (miRNA) pathway converts primary hairpin precursor transcripts into approximately 22 nucleotide regulatory RNAs via consecutive cleavages by two RNase III enzymes, Drosha and Dicer. In this study, we characterize Drosophila small RNAs that derive from short intronic hairpins termed "mirtrons." Their nuclear biogenesis appears to bypass Drosha cleavage, which is essential for miRNA biogenesis. Instead, mirtron hairpins are defined by the action of the splicing machinery and lariat-debranching enzyme, which yield pre-miRNA-like hairpins. The mirtron pathway merges with the canonical miRNA pathway during hairpin export by Exportin-5, and both types of hairpins are subsequently processed by Dicer-1/loqs. This generates small RNAs that can repress perfectly matched and seed-matched targets, and we provide evidence that they function, at least in part, via the RNA-induced silencing complex effector Ago1. These findings reveal that mirtrons are an alternate source of miRNA-type regulatory RNAs.

During microRNA (miRNA) biogenesis, one strand of a ∼21-22-nucleotide RNA duplex is preferentially selected for entry into a silencing complex. The other strand, known as the miRNA* species, has typically been assumed to be a carrier strand. Here we show that, although Drosophila melanogaster miRNA* species are less abundant than their partners, they are often present at physiologically relevant levels and can associate with Argonaute proteins. Comparative genomic analyses revealed that >40% of miRNA* sequences resist nucleotide divergence across Drosophilid evolution, and at least half of these well-conserved miRNA* species select for conserved 3′ untranslated region seed matches well above background noise. Finally, we validated the inhibitory activity of miRNA* species in both cultured cells and transgenic animals. These data broaden the reach of the miRNA regulatory network and suggest an important mechanism that diversifies miRNA function during evolution.miRNAs are an abundant class of ∼21-22-nucleotide (nt) RNAs that typically function as posttranscriptional repressors of gene activity 1,2 . The biogenesis of animal miRNAs involves stepwise processing of precursor transcripts containing hairpin structures. Canonical primary miRNA transcripts are cleaved in the nucleus by the RNase III enzyme Drosha, releasing ∼60-80-nt pre-miRNA hairpins 3 . In addition, splicing and debranching of short hairpin introns termed 'mirtrons' can directly generate pre-miRNA-like hairpins 4-6 . In both cases, the hairpins are exported to the cytoplasm and cleaved by the RNase III enzyme Dicer, resulting in a ∼21-nt miRNA duplex 7-10 . Although both strands of miRNA duplexes are necessarily produced in equal amounts by transcription, their accumulation is asymmetric at steady state. The convention is to refer to the more abundant product of a pre-miRNA or mirtron hairpin as the miRNA and its rarer partner as a miRNA* species 11 .The function of miRNA strands is evident from the preferential conservation of 7-nt sequences in target transcripts with Watson-Crick complementarity to positions 2-8 of mature miRNAs (the 'seed' region). Although other features influence target-site efficacy, miRNA seed matches are often necessary and sufficient for target regulation 12-15 and are the basis of most genomewide predictions of miRNA regulatory sites 16-18 . Such studies conclude that most animal genes are either actively regulated by one or more miRNAs or actively avoid the acquisition © 2008 Nature Publishing Group Correspondence should be addressed to E.C.L. (E-mail: laie@mskcc.org 17,19 . The reach of the miRNA regulatory network may in fact be larger, depending on the extent to which additional miRNA genes remain to be discovered, the extent to which noncanonical target sites are functional, and the extent to which nonconserved sites are relevant in vivo 20 .The nonrandom nature of miRNA strand selection was posited to reflect an active process that minimizes the population of silencing complexes with illegitimate miRNA* speci...

The nucleotide 5-methylcytosine is involved in processes crucial in mammalian development, such as X-chromosome inactivation and gene imprinting. In addition, cytosine methylation has long been speculated to be involved in the establishment and maintenance of cell type specific expression of developmentally regulated genes; however, it has been difficult to identify clear examples of such genes, particularly in humans. Here we provide evidence that cytosine methylation of the maspin gene (SERPINB5) promoter controls, in part, normal cell type specific SERPINB5 expression. In normal cells expressing SERPINB5, the SERPINB5 promoter is unmethylated and the promoter region has acetylated histones and an accessible chromatin structure. By contrast, normal cells that do not express SERPINB5 have a completely methylated SERPINB5 promoter with hypoacetylated histones, an inaccessible chromatin structure and a transcriptional repression that is relieved by inhibition of DNA methylation. These findings indicate that cytosine methylation is important in the establishment and maintenance of cell type restricted gene expression.

How multifunctional signals combine to specify unique cell fates during pattern formation is not well understood. Here, we demonstrate that together with the transcription factor Lozenge, the nuclear effectors of the EGFR and Notch signaling pathways directly regulate D-Pax2 transcription in cone cells of the Drosophila eye disc. Moreover, the specificity of D-Pax2 expression can be altered upon genetic manipulation of these inputs. Thus, a relatively small number of temporally and spatially controlled signals received by a set of pluripotent cells can create the unique combinations of activated transcription factors required to regulate target genes and ultimately specify distinct cell fates within this group. We expect that similar mechanisms may specify pattern formation in vertebrate developmental systems that involve intercellular communication.

Histone deacetylase (HDAC) inhibitors are promising antitumor agents, but they have not been extensively explored in B-cell lymphomas. Many of these lymphomas have the t(14;18) translocation, which results in increased bcl-2 expression and resistance to apoptosis. In this study, we examined the effects of two structurally different HDAC inhibitors, trichostatin A (TSA) and sodium butyrate (NaB), on the cell cycle, apoptosis, and bcl-2 expression in t(14;18) lymphoma cells. We found that in addition to potent cell cycle arrest, TSA and NaB also dramatically induced apoptosis and down-regulated bcl-2 expression, and overexpression of bcl-2 inhibited TSA-induced apoptosis. The repression of bcl-2 by TSA occurred at the transcriptional level. Western blot analysis and quantitative chromatin immunoprecipitation (ChIP) assay showed that even though HDAC inhibitors increased overall acetylation of histones, localized histone H3 deacetylation occurred at both bcl-2 promoters. TSA treatment increased the acetylation of the transcription factors Sp1 and C/EBP␣ and decreased their binding as well as the binding of CBP and HDAC2 to the bcl-2 promoters. Mutation of Sp1 and C/EBP␣ binding sites reduced the TSA-induced repression of bcl-2 promoter activity. This study provides a mechanistic rationale for the use of HDAC inhibitors in the treatment of human t(14;18) lymphomas.The cytogenetic hallmark of most follicular B-cell lymphomas is the chromosomal translocation of the antiapoptotic bcl-2 gene from 18q21 to the immunoglobulin heavy chain (IgH) locus at 14q32 (9, 54, 55). This t(14;18)(q32;q21) translocation constitutes the most common chromosomal translocation in human lymphoid malignancies. Approximately 85% of follicular and 20% of diffuse B-cell lymphomas possess this translocation. The t(14;18) translocation places bcl-2 in the same transcriptional orientation as IgH and results in deregulated overexpression of bcl-2 (15). Increased cell survival due to bcl-2 overexpression has been shown to contribute to the development of many B-cell lymphomas and confer resistance to a variety of anticancer therapies (12,26,43,50).Two promoters mediate transcriptional control of the bcl-2 gene (52). The 5Ј promoter (P1) is located 1,386 to 1,423 bp upstream of the bcl-2 translational start site, and it is GC-rich with multiple Sp1 sites. The start sites of the 3Ј promoter (P2) are located 1.3 kb downstream of the P1 promoter. P2 has a classic TATA and CAAT box and a simian virus 40 (SV40) decamer/Ig octamer motif. Important cis elements and associated trans-acting factors participating in the deregulation of bcl-2 have been characterized within the promoter regions. A major positive regulator of P1 activity is a cyclic AMP (cAMP) response element (CRE). CREB (CRE-binding protein) binds to this site and is essential for bcl-2 expression during B-cell development and for bcl-2 deregulation in t(14;18) lymphomas (27, 58). In addition, NF-B activates bcl-2 in t(14;18) lymphoma cells through interactions with the CRE and Sp1 binding sit...