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The broadly conserved Class III HOMEODOMAIN LEUCINE ZIPPER (HD-ZIPIII) and KANADI transcription factors have opposing and transformational effects on polarity and growth in all tissues and stages of the plant's life. To obtain a comprehensive understanding of how these factors work, we have identified transcripts that change in response to induced HD-ZIPIII or KANADI function. Additional criteria used to identify high-confidence targets among this set were presence of an adjacent HD-ZIPIII binding site, expression enriched within a subdomain of the shoot apical meristem, mutant phenotype showing defect in polar leaf and/or meristem development, physical interaction between target gene product and HD-ZIPIII protein, opposite regulation by HD-ZIPIII and KANADI, and evolutionary conservation of the regulator-target relationship. We find that HD-ZIPIII and KANADI regulate tissue-specific transcription factors involved in subsidiary developmental decisions, nearly all major hormone pathways, and new actors (such as INDETERMINATE DOMAIN4) in the ad/abaxial regulatory network. Multiple feedback loops regulating HD-ZIPIII and KANADI are identified, as are mechanisms through which HD-ZIPIII and KANADI oppose each other. This work lays the foundation needed to understand the components, structure, and workings of the ad/abaxial regulatory network directing basic plant growth and development.

A Dicer and an Argonaute homolog are found in the genome of Schizosaccharomyces pombe, implying that siRNAs, miRNAs, or another class of small RNAs might play an important role in fission yeast. To investigate this possibility, Reinhart and Bartel cloned endogenous RNAs from exponentially growing S. pombe using a method designed to clone RNAs with the features of Dicer cleavage products.

MicroRNAs (miRNAs) are ∼22‐nucleotide noncoding RNAs that can regulate gene expression by directing mRNA degradation or inhibiting productive translation. Dominant mutations in PHABULOSA ( PHB ) and PHAVOLUTA ( PHV ) map to a miR165/166 complementary site and impair miRNA‐guided cleavage of these mRNAs in vitro . Here, we confirm that disrupted miRNA pairing, not changes in PHB protein sequence, causes the developmental defects in phb‐d mutants. In planta , disrupting miRNA pairing near the center of the miRNA complementary site had far milder developmental consequences than more distal mismatches. These differences correlated with differences in miRNA‐directed cleavage efficiency in vitro , where mismatch scanning revealed more tolerance for mismatches at the center and 3′ end of the miRNA compared to mismatches to the miRNA 5′ region. In this respect, miR165/166 resembles animal miRNAs in its pairing requirements. Pairing to the 5′ portion of the small silencing RNA appears crucial regardless of the mode of post‐transcriptional repression or whether it occurs in plants or animals, supporting a model in which this region of the silencing RNA nucleates pairing to its target.

The NASHmap model is a non-invasive tool using 14 variables (features) collected in standard clinical practice to classify patients as probable nonalcoholic steatohepatitis (NASH) or non-NASH, and here we have explored its performance and prediction accuracy. The National Institute of Diabetes and Digestive Kidney Diseases (NIDDK) NAFLD Adult Database and the Optum Electronic Health Record (EHR) were used for patient data. Model performance metrics were calculated from correct and incorrect classifications for 281 NIDDK (biopsy-confirmed NASH and non-NASH, with and without stratification by type 2 diabetes status) and 1,016 Optum (biopsy-confirmed NASH) patients. NASHmap sensitivity in NIDDK is 81%, with a slightly higher sensitivity in T2DM patients (86%) than non-T2DM patients (77%). NIDDK patients misclassified by NASHmap had mean feature values distinct from correctly predicted patients, particularly for aspartate transaminase (AST; 75.88 U/L true positive vs 34.94 U/L false negative), and alanine transaminase (ALT; 104.09 U/L vs 47.99 U/L). Sensitivity was slightly lower in Optum at 72%. In an undiagnosed Optum cohort at risk for NASH (n = 2.9 M), NASHmap predicted 31% of patients as NASH. This predicted NASH group had AST and ALT mean levels above normal range of 0–35 U/L, and 87% had HbA1C levels > 5.7%. Overall, NASHmap demonstrates good sensitivity in predicting NASH status in both datasets, and NASH patients misclassified as non-NASH by NASHmap have clinical profiles closer to non-NASH patients.

Two small RNAs regulate the timing of Caenorhabditis elegans development 1 , 2 . Transition from the first to the second larval stage fates requires the 22-nucleotide lin-4 RNA 1 , 3 , 4 , and transition from late larval to adult cell fates requires the 21-nucleotide let-7 RNA 2 . The lin-4 and let-7 RNA genes are not homologous to each other, but are each complementary to sequences in the 3′ untranslated regions of a set of protein-coding target genes that are normally negatively regulated by the RNAs 1 , 2 , 5 , 6 . Here we have detected let-7 RNAs of ∼21 nucleotides in samples from a wide range of animal species, including vertebrate, ascidian, hemichordate, mollusc, annelid and arthropod, but not in RNAs from several cnidarian and poriferan species, Saccharomyces cerevisiae , Escherichia coli or Arabidopsis . We did not detect lin-4 RNA in these species. We found that let-7 temporal regulation is also conserved: let-7 RNA expression is first detected at late larval stages in C. elegans and Drosophila , at 48 hours after fertilization in zebrafish, and in adult stages of annelids and molluscs. The let-7 regulatory RNA may control late temporal transitions during development across animal phylogeny.

The C. elegans heterochronic gene pathway consists of a cascade of regulatory genes that are temporally controlled to specify the timing of developmental events 1 . Mutations in heterochronic genes cause temporal transformations in cell fates in which stage-specific events are omitted or reiterated 2 . Here we show that let-7 is a heterochronic switch gene. Loss of let-7 gene activity causes reiteration of larval cell fates during the adult stage, whereas increased let-7 gene dosage causes precocious expression of adult fates during larval stages. let-7 encodes a temporally regulated 21-nucleotide RNA that is complementary to elements in the 3′ untranslated regions of the heterochronic genes lin-14 , lin-28 , lin-41 , lin-42 and daf-12 , indicating that expression of these genes may be directly controlled by let-7 . A reporter gene bearing the lin-41 3′ untranslated region is temporally regulated in a let-7 -dependent manner. A second regulatory RNA, lin-4 , negatively regulates lin-14 and lin-28 through RNA–RNA interactions with their 3′ untranslated regions 3 , 4 . We propose that the sequential stage-specific expression of the lin-4 and let-7 regulatory RNAs triggers transitions in the complement of heterochronic regulatory proteins to coordinate developmental timing.

Two major classes of small noncoding RNAs have emerged as important regulators of gene expression in eukaryotes, the short interfering RNAs (siRNAs) associated with RNA silencing and endogenous micro-RNAs (miRNAs) implicated in regulation of gene expression. Helper component-proteinase (HC-Pro) is a viral protein that blocks RNA silencing in plants. Here we examine the effect of HC-Pro on the accumulation of siRNAs and endogenous miRNAs. siRNAs were analyzed in transgenic tobacco plants silenced in response to three different classes of transgenes: sense-transgenes, inverted-repeat transgenes, and amplicon-transgenes. HC-Pro suppressed silencing in each line, blocking accumulation of the associated siRNAs and allowing accumulation of transcripts from the previously silenced loci. HC-Pro-suppression of silencing in the inverted-repeat- and amplicon-transgenic lines was accompanied by the apparent accumulation of long double-stranded RNAs and proportional amounts of small RNAs that are larger than the siRNAs that accumulate during silencing. Analysis of these results suggests that HC-Pro interferes with silencing either by inhibiting siRNA processing from double-stranded RNA precursors or by destabilizing siRNAs. In contrast to siRNAs, the accumulation of endogenous miRNAs was greatly enhanced in all of the HC-Pro-expressing lines. Thus, our results demonstrate that accumulation of siRNAs and miRNAs in plants can be differentially regulated by a viral protein. The fact that HC-Pro affects the miRNA pathway raises the possibility that this pathway is targeted by plant viruses as a means to control gene expression in the host.

The formation of leaves and other lateral organs in plants depends on the proper specification of adaxial-abaxial (upper-lower) polarity. KANADI1 (KAN1), a member of the GARP family of transcription factors, is a key regulator of abaxial identity, leaf growth, and meristem formation in Arabidopsis thaliana. Here, we demonstrate that the Myb-like domain in KAN1 binds the 6-bp motif GNATA(A/T) and that this motif alone is sufficient to squelch transcription of a linked reporter in vivo. In addition, we report that KAN1 acts as a transcriptional repressor. Among its targets are genes involved in auxin biosynthesis, auxin transport, and auxin response. Furthermore, we find that the adaxializing HD-ZIPIII transcription factor REVOLUTA has opposing effects on multiple components of the auxin pathway. We hypothesize that HD-ZIPIII and KANADI transcription factors pattern auxin accumulation and responsiveness in the embryo. Specifically, we propose the opposing actions of KANADI and HD-ZIPIII factors on cotyledon formation (KANADI represses and HD-ZIPIII promotes cotyledon formation) occur through their opposing actions on genes acting at multiple steps in the auxin pathway.

ceh-14, a LIM class homeobox gene from Caenorhabditis elegans, is the orthologue of the vertebrate Lhx3/Lhx4 genes. ceh-14 reporter constructs are expressed in several different cell types: head and tail neurons, spermatheca and hypodermis. An intriguing aspect of the hypodermal expression pattern is that it takes the form of a gradient which is strongest in the central body region in L4 to young adult hermaphrodites. Promoter deletion analyses revealed that important regulatory elements for hypodermal expression are located within the transcribed region of ceh-14. Since a large part of the hypodermis is a syncytium, we hypothesized that this expression is triggered in a non-cell-autonomous fashion, a possible source being the underlying gonad. In males, which have a different gonadal organisation, the ceh-14 reporter constructs are expressed in a gradient that is strongest in the tail. By laser ablation of the gonadal precursor cells we found that ceh-14 reporter construct expression is eliminated in the hermaphrodite hypodermis, suggesting that the gonad plays a role in the generation of the gradient. Several signaling pathways are known in the gonad and the vulva, thus we crossed the mutations lin-3, egl-17 and lin-12 with the ceh-14 reporter lines. However, the expression of the reporter constructs is not affected in these mutant backgrounds. This suggests that another, presently unknown, signal triggers the graded hypodermal expression.

The Caenorhabditis elegans heterochronic gene pathway consists of a regulatory hierarchy that coordinately controls the temporal identities of diverse types of postembryonic cells. Mutations in heterochronic genes cause temporal transformations in cell fate in which stage-specific events are either omitted with subsequent events occurring prematurely, or reiterated at the expense of later events. lin-14 plays a central role in the progression from early to late larval development. lin-14 directs two distinct temporal fates, L1 fate determination and L2 timing, and these two genetic activities not only act at different points in development but are independently mutable. The lin-14 locus produces three novel, nuclearly localized proteins, and molecular characterization of lin-14 alleles that cause the specific loss of L2 fates has shown that L2 fate determination is provided by the LIN-14B1/B2 isoforms. No temporal differences in isoform expression have been detected, making it unlikely that the stage-specific phenotypes are due to distinct temporal expression profiles of the isoforms. However, genetic experiments suggested that a single isoform can be sufficient for normal development. Expression of LIN-14 appears to be simply reduced in lin-14(b-) mutants, suggesting that the level of lin-14 activity is crucial for fate determination. At the end of the first larval stage, the lin-14 products are negatively regulated via their 3′ UTR by the 22 nt lin-4 RNA to allow progression to L2 and later stages of development. Few genes involved in the subsequent progression through late larval stages have been identified, but new candidates were isolated in a screen for suppressors of egl-35; lin-14. Mutations in one gene, let-7, cause animals to reiterate late larval fates and delay the onset of the adult stage. Positional cloning of let-7 identified a 21 nt untranslated RNA as the gene product. The 3′ UTRs of several heterochronic genes contain sequences complementary to the let-7 RNA, suggesting that let-7 physically interacts with these mRNAs. Genetic and molecular experiments support our theory that let-7 acts in the heterochronic pathway. A model is proposed whereby sequential stage-specific expression of the lin-4 and let-7 regulatory RNAs trigger transitions in the expression of heterochronic genes to allow developmental progression.