New types of small RNAs distinct from microRNAs (miRNAs) are progressively being discovered in various organisms. In order to discover such novel small RNAs, a library of 17-to 26-base-long RNAs was created from prostate cancer cell lines and sequenced by ultra-high-throughput sequencing. A significant number of the sequences are derived from precise processing at the 59 or 39 end of mature or precursor tRNAs to form three series of tRFs (tRNA-derived RNA fragments): the tRF-5, tRF-3, and tRF-1 series. These sequences constitute a class of short RNAs that are second most abundant to miRNAs. Northern hybridization, quantitative RT-PCR, and splinted ligation assays independently measured the levels of at least 17 tRFs. To demonstrate the biological importance of tRFs, we further investigated tRF-1001, derived from the 39 end of a Ser-TGA tRNA precursor transcript that is not retained in the mature tRNA. tRF-1001 is expressed highly in a wide range of cancer cell lines but much less in tissues, and its expression in cell lines was tightly correlated with cell proliferation. siRNAmediated knockdown of tRF-1001 impaired cell proliferation with the specific accumulation of cells in G2, phenotypes that were reversed specifically by cointroducing a synthetic 29-O-methyl tRF-1001 oligoribonucleotide resistant to the siRNA. tRF-1001 is generated in the cytoplasm by tRNA 39-endonuclease ELAC2, a prostate cancer susceptibility gene. Our data suggest that tRFs are not random by-products of tRNA degradation or biogenesis, but an abundant and novel class of short RNAs with precise sequence structure that have specific expression patterns and specific biological roles.[Keywords: Small RNA; tRNA; deep sequencing; cancer cell proliferation] Supplemental material is available at http://www.genesdev.org.

During the last few years, studies on microRNA (miRNA) and cancer have burst onto the scene. Profiling of the miRNome (global miRNA expression levels) has become prevalent and abundant miRNome data are currently available from various cancers. The pattern of miRNA expression can be correlated with cancer type, stage, and other clinical variables, so that miRNA profiling can be used as a tool for cancer diagnosis and prognosis. miRNA expression analyses also suggested oncogenic (or tumor suppressive) roles of miRNAs. miRNAs play roles in almost all aspects of cancer biology such as proliferation, apoptosis, invasion/metastasis, and angiogenesis. Given that many miRNAs are deregulated in cancers but have not yet been further studied, it is expected that more miRNAs will emerge as players in the etiology and progression of cancer. miRNAs will be also discussed as a tool for cancer therapy. SYNOPSIS During the last decade, a major discovery in biology was the discovery of small RNAs, including miRNA (microRNA) and siRNA (small interfering RNA), as highlighted by the 2002 December issue of Science magazine (1). Since RNA interference (RNAi) phenomenon was discovered in nematodes (2), siRNA has provided a technical breakthrough for short term genetics in mammalian systems. The big impact of small RNAs was well celebrated by the 2006 Nobel prize awarded to the two scientists who discovered RNAi. On the other side, miRNAs shed new insight on the post-transcriptional regulation of gene expression. miRNAs were also first discovered in worms (3, 4), and later in a number of animals, plants, and viruses. During the last couple of years, the miRNA field has been expanding with many recent publications implicating miRNAs in diverse cellular processes. Cancer is a major cause of death in the United States (“Cancer Facts & Figures 2007” from American Cancer Society; http://www.cancer.org/docroot/stt/stt_0.asp). Cancer is a complex genetic disease caused by the accumulation of mutations that lead to deregulation of gene expression and uncontrolled cell proliferation. Given the wide impact of miRNAs on gene expression, it is not surprising that a number of miRNAs have been implicated in cancer. In this review, the links between miRNA and cancer will be comprehensively described and discussed.

HMGA2, a high-mobility group protein, is oncogenic in a variety of tumors, including benign mesenchymal tumors and lung cancers. Knockdown of Dicer in HeLa cells revealed that the HMGA2 gene is transcriptionally active, but its mRNA is destabilized in the cytoplasm through the microRNA (miRNA) pathway. HMGA2 was derepressed upon inhibition of let-7 in cells with high levels of the miRNA. Ectopic expression of let-7 reduced HMGA2 and cell proliferation in a lung cancer cell. The effect of let-7 on HMGA2 was dependent on multiple target sites in the 3 untranslated region (UTR), and the growth-suppressive effect of let-7 on lung cancer cells was rescued by overexpression of the HMGA2 ORF without a 3UTR. Our results provide a novel example of suppression of an oncogene by a tumor-suppressive miRNA and suggest that some tumors activate the oncogene through chromosomal translocations that eliminate the oncogene's 3UTR with the let-7 target sites.Supplemental material is available at http://www.genesdev.org.Received February 12, 2007; revised version accepted March 8, 2007. HMGA2 (also called HMGI-C), a member of the highmobility group AT-hook (HMGA) family of nonhistone chromatin proteins, is an architectural transcription factor (for review, see Reeves 2001). HMGA2 protein plays a critical role in growth during embryonic development, as evidenced by the pigmy phenotype of mutant mice deficient in hmga2 and by its high expression in embryos (Zhou et al. 1995;Rogalla et al. 1996). It is normally expressed at low levels in adult tissues, but disruption of the gene by chromosomal rearrangements at chr12q13-15 and attendant overexpression of the protein result in benign mesenchymal tumors such as lipoma, uterine leiomyoma, pulmonary chondroid hamartoma (for review, see Fedele et al. 2001), pleomorphic salivary gland adenoma (Geurts et al. 1997), and endometrial polyps (Bol et al. 1996). The chromosomal break separates the ORF of HMGA2 from the 3Ј untranslated region (UTR) and leads to overexpression of HMGA2 protein (Fig. 3A, below). As most of the breakpoints are within the 140-kb third intron, the rearrangement results in the expression of a chimeric HMGA2 protein that lacks a C-terminal acidic domain but is fused to another protein from the translocated region. Although the deletion and/or fusion partners were initially thought to be important for tumorigenesis, overexpression of full-length HMGA2 protein was found to be sufficient to cause a neoplastic transformation of mesenchymal cells (Zaidi et al. 2006). Consistent with this, at least in a couple of cases the translocation site was mapped in the 3ЈUTR, leaving the ORF intact (Schoenmakers et al. 1995;Geurts et al. 1997;Quade et al. 2003).HMGA2 protein is also elevated in several mouse and human neoplasias such as non-small-cell carcinoma of the lung (Giancotti et al. 1989;Sarhadi et al. 2006). HMGA2 was overexpressed in 90% of lung cancers, and the presence of the protein in the nucleus correlated with high cell proliferation and poor survival. The HMGA2 gene...

Three muscle-specific microRNAs, miR-206, -1, and -133, are induced during differentiation of C2C12 myoblasts in vitro. Transfection of miR-206 promotes differentiation despite the presence of serum, whereas inhibition of the microRNA by antisense oligonucleotide inhibits cell cycle withdrawal and differentiation, which are normally induced by serum deprivation. Among the many mRNAs that are down-regulated by miR-206, the p180 subunit of DNA polymerase α and three other genes are shown to be direct targets. Down-regulation of the polymerase inhibits DNA synthesis, an important component of the differentiation program. The direct targets are decreased by mRNA cleavage that is dependent on predicted microRNA target sites. Unlike small interfering RNA–directed cleavage, however, the 5′ ends of the cleavage fragments are distributed and not confined to the target sites, suggesting involvement of exonucleases in the degradation process. In addition, inhibitors of myogenic transcription factors, Id1-3 and MyoR, are decreased upon miR-206 introduction, suggesting the presence of additional mechanisms by which microRNAs enforce the differentiation program.

The discovery of small noncoding RNAs (sncRNAs) with regulatory functions is a recent breakthrough in biology. Among sncRNAs, microRNA (miRNA), derived from host or virus, has emerged as elements with high importance in control of viral replication and host responses. However, the expression pattern and functional aspects of other types of sncRNAs, following viral infection, are unexplored. In order to define expression patterns of sncRNAs, as well as to discover novel regulatory sncRNAs in response to viral infection, we applied deep sequencing to cells infected with human respiratory syncytial virus (RSV), the most common cause of bronchiolitis and pneumonia in babies. RSV infection leads to abundant production of transfer RNA (tRNA)-derived RNA Fragments (tRFs) that are ~30 nucleotides (nts) long and correspond to the 5'-half of mature tRNAs. At least one tRF, which is derived from tRNA-Glu-CTC, represses target mRNA in the cytoplasm and promotes RSV replication. This demonstrates that this tRF is not a random by-product of tRNA degradation but a functional molecule. The biogenesis of this tRF is also specific, as it is mediated by the endonuclease angiogenin (ANG), not by other nucleases. In summary, our study presents novel information on the induction of a functional tRF by viral infection.