Recognition sites for microRNAs (miRNAs) have been reported to be located in the 3 untranslated regions of transcripts. In a computational screen for highly conserved motifs within coding regions, we found an excess of sequences conserved at the nucleotide level within coding regions in the human genome, the highest scoring of which are enriched for miRNA target sequences. To validate our results, we experimentally demonstrated that the let-7 miRNA directly targets the miRNA-processing enzyme Dicer within its coding sequence, thus establishing a mechanism for a miRNA/Dicer autoregulatory negative feedback loop. We also found computational evidence to suggest that miRNA target sites in coding regions and 3 UTRs may differ in mechanism. This work demonstrates that miRNAs can directly target transcripts within their coding region in animals, and it suggests that a complete search for the regulatory targets of miRNAs should be expanded to include genes with recognition sites within their coding regions. As more genomes are sequenced, the methodological approach that we used for identifying motifs with high sequence conservation will be increasingly valuable for detecting functional sequence motifs within coding regions.computational biology ͉ posttranscriptional regulation ͉ comparative genomics ͉ multiple-sequence alignment ͉ evolutionary conservation M icroRNAs (miRNAs) are endogenously encoded, singlestranded regulatory RNAs that bind to and inhibit the translation of transcripts with complementary sequence (1). Computational evidence suggests that miRNAs regulate at least 20% of human genes and have been implicated in the regulation of a wide range of biological systems (2). In plants, miRNA targets can be predicted with relatively high confidence because of the extensive base pairing between plant miRNAs and their target mRNAs (1). In animals, in contrast, miRNAs typically bind to their targets with significantly less complementarity, and the short target sequences are, therefore, difficult to identify on the basis of sequence alone. As a result, most computational approaches to predict miRNA-target interactions rely on conservation of target sites (3-6).Although early studies reported some evidence for the targeting of miRNAs to sites within protein coding regions (4, 6), subsequent research has reported that there is minimal functionality for sites in ORFs or 5Ј UTRs (7). A focus on miRNAs present within 3Ј UTRs is supported by evidence suggesting that the G-cap/poly(A) tail interface (which connects the two ends of eukaryotic mRNAs during translation) is important for miRNA function (8) and that miRNAs tend to be more effective when localized at the end of the 3Ј UTR rather than the middle (7, 9). Indeed, the protein translation machinery might be expected to displace an miRNA complex present within a gene's coding sequence. However, exogenously added siRNAs that target coding sequences, including siRNAs with imperfect base pairing, are effective at silencing (10). More recent reports have also shown that, c...

Metabolomics technology reveals that fibroblast that have exited the proliferative cell cycle nevertheless utilize glucose throughout central carbon metabolism and rely on the pentose phosphate pathway for viability.

Cell polarity, as reflected by polarized growth and organelle segregation during cell division in yeast, appears to follow a simple hierarchy. On the basis of physical cues from previous cell cycles or stochastic processes, yeast cells select a site for bud emergence that also defines the axis of cell division. Once polarity is established, rho protein-based signal pathways set up a polarized cytoskeleton by activating localized formins to nucleate and assemble polarized actin cables. These serve as tracks for the transport of secretory vesicles, the segregation of the trans Golgi network, the vacuole, peroxisomes, endoplasmic reticulum, mRNAs for cell fate determination, and microtubules that orient the nucleus in preparation for mitosis, all by myosin-Vs encoded by the MYO2 and MYO4 genes. Most of the proteins participating in these processes in yeast are conserved throughout the kingdoms of life, so the emerging models are likely to be generally applicable. Indeed, several parallels to cellular organization in animals are evident.

overcome the energy barrier required for membrane fusion (Fasshauer et al., 1997;Hanson et al., 1997). Furthermore, Membrane proteins located on vesicles (v-SNAREs) using liposomes reconstituted with t-or v-SNAREs, and on the target membrane (t-SNAREs) mediateRothman and co-workers showed that the v-SNAREspecific recognition and, possibly, fusion between a t-SNARE complex per se fulfills the minimal requirement transport vesicle and its target membrane. The activity for fusion between two membranes (Weber et al., 1998; of SNARE molecules is regulated by several soluble Nickel et al., 1999;Parlati et al., 1999). Yet, based on an cytosolic proteins. We have cloned a bovine brain in vitro system that reconstitutes homotypic fusion of yeast cDNA encoding a conserved 117 amino acid polypepvacuoles, Ungermann and co-workers (1998) deduced tide, denoted Golgi-associated ATPase Enhancer of that the formation of the SNARE complex is only an 16 kDa (GATE-16), that functions as a soluble transport intermediate step in the overall fusion reaction. According factor. GATE-16 interacts with N-ethylmaleimideto this view, SNARE molecules are involved in docking sensitive factor (NSF) and significantly stimulates its between donor and acceptor membranes, while another ATPase activity. It also interacts with the Golgi set of proteins participates in subsequent stages of the v-SNARE GOS-28 in an NSF-dependent manner. We fusion process. This notion is supported by Peters and propose that GATE-16 modulates intra-Golgi transport Mayer (1998), who suggested that calmodulin and other through coupling between NSF activity and SNAREs as yet unidentified factors are involved in mediating late activation.stages of vacuolar fusion.

Mitochondria undergo cycles of fusion and fission crucial for organelle homeostasis. Fission is regulated partially by recruitment of the large GTPase Dnm1p to the outer mitochondrial membrane. Using three-dimensional time-lapse fluorescence imaging of Saccharomyces cerevisiae cells, we found that Dnm1p-EGFP appears and disappears at "hot spots" along mitochondrial tubes. It forms patches that convert rapidly into different shapes regardless of whether mitochondrial fission ensues or not. Moreover, the thickness of the mitochondrial matrix displays frequent temporal fluctuations apparently unrelated to fission or to recruitment of Dnm1p-EGFP. These results suggest that mitochondrial fission requires coordination of at least two distinct processes.