MicroRNAs (miRNAs) are small regulatory RNAs that derive from distinctive hairpin transcripts. To learn more about the miRNAs of mammals, we sequenced 60 million small RNAs from mouse brain, ovary, testes, embryonic stem cells, three embryonic stages, and whole newborns. Analysis of these sequences confirmed 398 annotated miRNA genes and identified 108 novel miRNA genes. More than 150 previously annotated miRNAs and hundreds of candidates failed to yield sequenced RNAs with miRNA-like features. Ectopically expressing these previously proposed miRNA hairpins also did not yield small RNAs, whereas ectopically expressing the confirmed and newly identified hairpins usually did yield small RNAs with the classical miRNA features, including dependence on the Drosha endonuclease for processing. These experiments, which suggest that previous estimates of conserved mammalian miRNAs were inflated, provide a substantially revised list of confidently identified murine miRNAs from which to infer the general features of mammalian miRNAs. Our analyses also revealed new aspects of miRNA biogenesis and modification, including tissue-specific strand preferences, sequential Dicer cleavage of a metazoan precursor miRNA (pre-miRNA), consequential 59 heterogeneity, newly identified instances of miRNA editing, and evidence for widespread pre-miRNA uridylation reminiscent of miRNA regulation by Lin28.[Keywords: MicroRNA; miRNA biogenesis; noncoding RNA genes; high-throughput sequencing] Supplemental material is available at http://www.genesdev.org.

SUMMARY To use microRNAs to down-regulate mRNA targets, cells must first process these ~22 nt RNAs from primary transcripts (pri-miRNAs). These transcripts form RNA hairpins important for processing, but additional determinants must distinguish pri-miRNAs from the many other hairpin-containing transcripts expressed in each cell. Illustrating the complexity of this recognition, we show that most Caenorhabditis elegans pri-miRNAs lack determinants required for processing in human cells. To find these determinants, we generated >1011 variants of four human pri-miRNAs, sequenced millions that retained function and compared them with the starting variants. Our results confirmed the importance of pairing in the stem and revealed three primary-sequence determinants, including an SRp20-binding motif (CNNC) found downstream of most pri-miRNA hairpins in bilaterian animals but not in nematodes. Adding this and other determinants to C. elegans pri-miRNAs imparted efficient processing in human cells, thereby confirming the importance of primary-sequence determinants for distinguishing pri-miRNAs from other hairpin-containing transcripts.

FoxP3 ؉ regulatory T cells (Tregs) protect against autoimmunity, type 1 diabetes (T1D) in particular, prompting the hypothesis that a deficiency in Tregs is a critical determinant of diabetes susceptibility in NOD mice. However, tests of this hypothesis have yielded contradictory results. We confirmed that NOD mice, compared with reference strains, do not have a primary deficit in Treg numbers in the lymphoid organs, whether in prediabetic mice of any age or in animals with recent-onset diabetes. NOD Tregs did show a defect in standard in vitro T cell suppression assays, particularly at low suppressor/effector ratios. Gene expression profiling revealed the vast majority of transcripts constituting the ''Treg signature'' to be normally distributed in NOD Tregs versus CD4 ؉ T conventional (Tconv) cells, although there were a few differences affecting one or the other population. According to results from criss-cross experiments, the functional inefficacy was not rooted in NOD Tregs, which suppressed as well as their C57BL/6 (B6) counterparts, but rather in NOD Tconv, which were less prone to suppression than were B6 Tconv cells. They also responded more effectively to anti-CD3/28 monoclonal antibody (mAb) stimulation in vitro or to a natural pancreatic antigen in vivo. This difference was independent of autoimmune inflammation, did not map to the idd3 region, and was not due to the overproduction of interleukin-21 in NOD mice. That the immune dysregulation in this T1D model is rooted in the ability of effector T cells to be regulated, rather than in Tregs themselves, has implications for proposed therapeutic interventions.conventional T cells ͉ regulatory T cells ͉ type 1 diabetes

Specific, potent, and sustained short hairpin RNA (shRNA)-mediated gene silencing is crucial for the successful application of RNA interference technology to therapeutic interventions. We examined the effects of shRNA expression in primary human lymphocytes (PBLs) using lentiviral vectors bearing different RNA polymerase III promoters. We found that the U6 promoter is more efficient than the H1 promoter for shRNA expression and for reducing expression of CCR5 in PBLs. However, shRNA expression from the U6 promoter resulted in a gradual decline of the transduced cell populations. With one CCR5 shRNA this decline could be attributed to elevated apoptosis but another CCR5 shRNA that caused cytotoxicity did not show evidence of apoptosis, suggesting sequence-specific mechanisms for cytotoxicity. In contrast to the U6 promoter, PBLs transduced by vectors expressing shRNAs from the H1 promoter could be maintained without major cytotoxic effects. Since a lower level of shRNA expression appears to be advantageous to maintaining the shRNA-transduced population, lentiviral vectors bearing the H1 promoter are more suitable for stable transduction and expression of shRNA in primary human T lymphocytes. Our results suggest that functional shRNA screens should include tests for both potency and adverse metabolic effects upon primary cells.

To study conformational transitions at the muscle nicotinic acetylcholine (ACh) receptor (nAChR), a rhodamine fluorophore was tethered to a Cys side chain introduced at the ␤19 position in the M2 region of the nAChR expressed in Xenopus oocytes. This procedure led to only minor changes in receptor function. During agonist application, fluorescence increased by (⌬F͞F) Ϸ10%, and the emission peak shifted to lower wavelengths, indicating a more hydrophobic environment for the fluorophore. The dose-response relations for ⌬F agreed well with those for epibatidine-induced currents, but were shifted Ϸ100-fold to the left of those for ACh-induced currents. Because (i) epibatidine binds more tightly to the ␣␥-binding site than to the ␣␦ site and (ii) ACh binds with reverse-site selectivity, these data suggest that ⌬F monitors an event linked to binding specifically at the ␣␦-subunit interface. In experiments with flash-applied agonists, the earliest detectable ⌬F occurs within milliseconds, i.e., during activation. T he muscle nicotinic acetylcholine receptor (nAChR) is a well studied member of the Cys-loop family of neurotransmittergated ion channels. A 4.6-Å structure (1) shows five Ϸ160-Å-long rod-shaped subunits surrounding a central channel. From the extracellular side the subunits have a counterclockwise order of ␣␥␣␦␤ (2, 3). Each subunit has a large extracellular Nterminal or ligand-binding domain followed by four transmembrane regions, M1-M4 (4). The structure of the extracellular ligand-binding sites, at the ␣␥ and ␣␦ interfaces, resembles that of a homologous molluscan ACh-binding protein (3). Numerous biochemical and electrophysiological experiments indicate that M2 lines the channel (5).The nAChR exists in at least four distinct, interconvertible conformational states: resting, open, fast-onset-desensitized, and slow-onset-desensitized (6). The open and the fast-onsetdesensitized states presumably have moderate affinity for ACh, are metastable (on millisecond time scales), and are present in low concentrations at equilibrium. The supralinear doseresponse relation (Hill coefficient Ͼ1) suggests that the open state of the channel is much more likely to be associated with the presence of two bound agonist molecules than with a single bound agonist (7). In the prevailing kinetic scheme, receptors in the resting state bind two agonist molecules, isomerize to the open state, and in the continued presence of agonist, desensitize.After removal of agonist, the agonist-receptor complex dissociates and the channel closes within milliseconds; but desensitized receptors isomerize more slowly to the resting state (tens of milliseconds to hundreds of seconds). Thermodynamic considerations suggest that the resting state has low affinity for agonist, whereas the slow-onset-desensitized state is the most stable state in the presence of agonist because of its high affinity.Kinetic analyses of single-channel and macroscopic function suggests that in the resting state, the affinity of ACh for the two sites differs by a factor of...