MicroRNAs (miRNAs) are important regulatory molecules in most eukaryotes and identification of their target mRNAs is essential for their functional analysis. Whereas conventional methods rely on computational prediction and subsequent experimental validation of target RNAs, we directly sequenced >28,000,000 signatures from the 5' ends of polyadenylated products of miRNA-mediated mRNA decay, isolated from inflorescence tissue of Arabidopsis thaliana, to discover novel miRNA-target RNA pairs. Within the set of approximately 27,000 transcripts included in the 8,000,000 nonredundant signatures, several previously predicted but nonvalidated targets of miRNAs were found. Like validated targets, most showed a single abundant signature at the miRNA cleavage site, particularly in libraries from a mutant deficient in the 5'-to-3' exonuclease AtXRN4. Although miRNAs in Arabidopsis have been extensively investigated, working in reverse from the cleaved targets resulted in the identification and validation of novel miRNAs. This versatile approach will affect the study of other aspects of RNA processing beyond miRNA-target RNA pairs.

Small RNAs (21-24 nt) are involved in gene regulation through translation inhibition, mRNA cleavage, or directing chromatin modifications. In rice, currently Ϸ240 microRNAs (miRNAs) have been annotated. We sequenced more than four million small RNAs from rice and identified another 24 miRNA genes. Among these, we found a unique class of miRNAs that derive from natural cisantisense transcript pairs. This configuration generates miRNAs that can perfectly match their targets. We provide evidence that the miRNAs function by inducing mRNA cleavage in the middle of their complementary site. Their production requires Dicer-like 1 (DCL1) activity, which is essential for canonical miRNA biogenesis. All of the natural antisense miRNAs (nat-miRNAs) identified in this study have large introns in their precursors that appear critical for nat-miRNA evolution and for the formation of functional miRNA loci. These findings suggest that other natural cis-antisense loci with similar exon-intron arrangements could be another source of miRNA genes.high-throughput sequencing ͉ siRNA ͉ small RNA ͉ massively parallel signature sequencing (MPSS) M ost eukaryotes possess small RNA-based gene silencing systems that can down-regulate genes at transcriptional and posttranscriptional levels (1, 2). At least five classes of these small regulatory RNAs (21-24 nt) have been characterized, including microRNAs (miRNAs), heterochromatic siRNAs, transacting siRNAs (ta-siRNAs), natural antisense siRNAs (nat-siRNAs), and, in metazoans, the Piwi-interacting RNAs (3-7). miRNAs are processed from self-complementary transcripts by the activity of Dicer ribonucleases. siRNAs originate from longer, doublestranded (ds)RNA molecules and usually represent both strands of the RNA, although they are similar in biochemical structure to miRNAs and have some functional similarities. In plants, siRNAs typically derive from transposons, repetitive sequences, and transgenes. These siRNAs could be involved in DNA methylation and histone modifications that silence target transcription (8). Although nat-siRNAs also have been identified (9, 10), natural antisense miRNAs (nat-miRNAs) have not been reported in any system.Plant miRNAs have near-perfect pairing to their targets and therefore generally cause mRNA cleavage. Numerous studies have demonstrated the critical role of miRNAs in controlling developmental processes and organ identity. As of April 2007, the miRNA Sanger database contained 916 plant miRNAs. The list is rapidly growing as a result of new deep-sequencing technologies for small RNA discovery. In Arabidopsis, small RNAs from various mutants, tissues, and developmental stages have been analyzed by highthroughput pyrosequencing (11-15). These efforts identified at least 184 miRNAs (Ϸ70 families) in Arabidopsis. Cloning of miRNAs from lower plants such as moss indicates that some miRNAs are conserved over a long evolutionary distance. In fact, most miRNAs identified in the early studies (21/28) are conserved in more than one plant species, although some miRNAs...

Histologically normal tissue adjacent to the tumor can provide insight of the microenvironmental alterations surrounding the cancerous lesion and affecting the progression of the disease. However, little is known about the molecular changes governing cancer initiation in cancer-free breast tissue. Here, we employed laser microdissection and whole-transcriptome profiling of the breast epithelium prior to and post tumor diagnosis to identify the earliest alterations in breast carcinogenesis. Furthermore, a comprehensive analysis of the three tissue compartments (microdissected epithelium, stroma, and adipose tissue) was performed on the breast donated by either healthy subjects or women prior to the clinical manifestation of cancer (labeled “susceptible normal tissue”). Although both susceptible and healthy breast tissues appeared histologically normal, the susceptible breast epithelium displayed a significant upregulation of genes involved in fatty acid uptake/transport (CD36 and AQP7), lipolysis (LIPE), and lipid peroxidation (AKR1C1). Upregulation of lipid metabolism- and fatty acid transport-related genes was observed also in the microdissected susceptible stromal and adipose tissue compartments, respectively, when compared with the matched healthy controls. Moreover, inter-compartmental co-expression analysis showed increased epithelium-adipose tissue crosstalk in the susceptible breasts as compared with healthy controls. Interestingly, reductions in natural killer (NK)-related gene signature and CD45+/CD20+ cell staining were also observed in the stromal compartment of susceptible breasts. Our study yields new insights into the cancer initiation process in the breast. The data suggest that in the early phase of cancer development, metabolic activation of the breast, together with increased epithelium-adipose tissue crosstalk may create a favorable environment for final cell transformation, proliferation, and survival.