RNA interference is thought to require near-identity between the small interfering RNA (siRNA) and its cognate mRNA. Here, we used gene expression profiling to characterize the specificity of gene silencing by siRNAs in cultured human cells. Transcript profiles revealed siRNA-specific rather than target-specific signatures, including direct silencing of nontargeted genes containing as few as eleven contiguous nucleotides of identity to the siRNA. These results demonstrate that siRNAs may cross-react with targets of limited sequence similarity.

microRNAs (miRNAs) are abundant, ϳ21-nucleotide, noncoding regulatory RNAs. Each miRNA may regulate hundreds of mRNA targets, but the identities of these targets and the processes they regulate are poorly understood. Here we have explored the use of microarray profiling and functional screening to identify targets and biological processes triggered by the transfection of human cells with miRNAs. We demonstrate that a family of miRNAs sharing sequence identity with miRNA-16 (miR-16) negatively regulates cellular growth and cell cycle progression. miR-16-down-regulated transcripts were enriched with genes whose silencing by small interfering RNAs causes an accumulation of cells in G 0 /G 1 . Simultaneous silencing of these genes was more effective at blocking cell cycle progression than disruption of the individual genes. Thus, miR-16 coordinately regulates targets that may act in concert to control cell cycle progression.

The hypoxia-inducible factor (HIF) pathway is essential for cell survival under low oxygen and plays an important role in tumor cell homeostasis. We investigated the function of miR-210, the most prominent microRNA upregulated by hypoxia and a direct transcriptional target of HIFs. miR-210 expression was elevated in multiple cancer types and correlated with metastasis of breast and melanoma tumors. miR-210 overexpression in cancer cell lines bypassed hypoxia-induced cell cycle arrest and partially reversed the hypoxic gene expression signature. We identified MNT, a known MYC antagonist, as a miR-210 target. MNT mRNA contains multiple miR-210 binding sites in the 3' UTR and its knockdown phenocopied miR-210 overexpression. Furthermore, loss of MYC abolished miR-210-mediated override of hypoxia-induced cell cycle arrest. Comparison of miR-210 and MYC overexpression with MNT knockdown signatures also indicated that miR-210 triggered a "MYC-like" transcriptional response. Thus, miR-210 influences the hypoxia response in tumor cells through targeting a key transcriptional repressor of the MYC-MAX network.

CCR5 and CXCR4 are the two major coreceptors that have been identified for human immunodeficiency virus (HIV) entry. We have modified several beta-galactosidase-based HIV indicator cell lines to express CCR5 and/or CXCR4. Using these new reagents, we have been able to detect all primary isolates tested using one or both of these cell lines. However, there is large variation in the absolute viral infectivity among primary strains. Furthermore, all HIV strains are capable of causing syncytia in the indicator cells when the coreceptor is present regardless of whether they had previously been characterized as "syncytia-inducing" or "non-syncytium-inducing."

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor, consisting of an alpha subunit and a beta subunit, that controls cellular responses to hypoxia. HIF␣ contains two transcriptional activation domains called the N-terminal transactivation domain (NTAD) and the C-terminal transactivation domain (CTAD). HIF␣ is destabilized by prolyl hydroxylation catalyzed by EglN family members. In addition, CTAD function is inhibited by asparagine hydroxylation catalyzed by FIH1. Both hydroxylation reactions are linked to oxygen availability. The von Hippel-Lindau tumor suppressor protein (pVHL) is frequently mutated in kidney cancer and is part of the ubiquitin ligase complex that targets prolyl hydroxylated HIF␣ for destruction. Recent studies suggest that HIF2␣ plays an especially important role in promoting tumor formation by pVHL-defective renal carcinoma cells among the three HIF␣ paralogs. Here we dissected the relative contribution of the two HIF2␣ transactivation domains to hypoxic gene activation and renal carcinogenesis and investigated the regulation of the HIF2␣ CTAD by FIH1. We found that the HIF2␣ NTAD is capable of activating both artificial and naturally occurring HIF-responsive promoters in the absence of the CTAD. Moreover, we found that the HIF2␣ CTAD, in contrast to the HIF1␣ CTAD, is relatively resistant to the inhibitory effects of FIH1 under normoxic conditions and that, perhaps as a result, both the NTAD and CTAD cooperate to promote renal carcinogenesis in vivo.HIF (hypoxia-inducible factor) is a master transcriptional regulator of hypoxia-inducible genes and is composed of the ␣ subunit HIF1␣, or one of its paralogs HIF2␣ or HIF3␣, and a HIF␤ subunit, such as HIF1␤ (also called the aryl hydrocarbon receptor nuclear translocator [ARNT]) (12,14,17,20,50,53). Whereas HIF1␤ is constitutively present, the HIF␣ members are highly unstable except under low-oxygen conditions (hypoxia). Under hypoxic conditions, the HIF␣ subunits accumulate, bind to a HIF␤ subunit, and transcriptionally activate hypoxia-inducible genes bearing canonical HIF DNA-binding sites called hypoxia-responsive elements (HRE). These genes include genes that control angiogenesis (such as vascular endothelial growth factor [VEGF]), energy metabolism (for example, the glucose transporter 1 [GLUT1] and carbonic anhydrase IX [CAIX]), erythropoiesis (such as erythropoietin [EPO]), and mitogenesis (for example, transforming growth factor ␣ and platelet-derived growth factor B) (18, 49). In the presence of oxygen, HIF␣ proteins are hydroxylated on conserved prolyl residues by members of the egg-laying-defective nine (EglN) prolyl hydroxylases (also called PHD or HPH hydroxylases) (2,13,(26)(27)(28)55). Prolyl hydroxylation of HIF␣ creates a binding site for the von Hippel-Lindau (VHL) ubiquitin ligase complex, which then targets HIF␣ for polyubiquitination and proteasomal degradation (30). In cells lacking functional pVHL, HIF␣ is not degraded properly, leading to activation of HIF target genes. More than half of clear cell r...