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Richard W Padgett

Publications and source records attributed to Richard W Padgett.

15 recordsLinked to original sources
PubMed2006

Modulated microRNA expression during adult lifespan in Caenorhabditis elegans.

MicroRNAs (miRNAs) are small, abundant transcripts that can bind partially homologous target messages to inhibit their translation in animal cells. miRNAs have been shown to affect a broad spectrum of biological activities, including developmental fate determination, cell signaling and oncogenesis. Little is known, however, of miRNA contributions to aging. We examined the expression of 114 identified Caenorhabditis elegans miRNAs during the adult lifespan and find that 34 miRNAs exhibit changes in expression during adulthood (P<or= 0.05), 31 with more than a twofold level change. The majority of age-regulated miRNAs decline in relative abundance as animals grow older. Expression profiles of developmental timing regulators lin-4 and let-7 miRNAs, as well as conserved muscle miRNA miR-1, show regulation during adulthood. We also used bioinformatic approaches to predict miRNA targets encoded in the C. elegans genome and we highlight candidate miRNA-regulated genes among C. elegans genes previously shown to affect longevity, genes encoding insulin-like ligands, and genes preferentially expressed in C. elegans muscle. Our observations identify miRNAs as potential modulators of age-related decline and suggest a general reduction of message-specific translational inhibition during aging, a previously undescribed feature of C. elegans aging. Since many C. elegans age-regulated miRNAs are conserved across species, our observations identify candidate age-regulating miRNAs in both nematodes and humans.

Animals↗
PubMed2005

Pseudouridine modification in Caenorhabditis elegans spliceosomal snRNAs: unique modifications are found in regions involved in snRNA-snRNA interactions.

BACKGROUND: Pseudouridine (Psi) is an abundant modified nucleoside in RNA and a number of studies have shown that the presence of Psi affects RNA structure and function. The positions of Psi in spliceosomal small nuclear RNAs (snRNAs) have been determined for a number of species but not for the snRNAs from Caenorhabditis elegans (C. elegans), a popular experimental model system of development. RESULTS: As a prelude to determining the function of or requirement for this modification in snRNAs, we have mapped the positions of Psi in U1, U2, U4, U5, and U6 snRNAs from worms using a specific primer extension method. As with other species, C. elegans U2 snRNA has the greatest number of Psi residues, with nine, located in the 5' half of the U2 snRNA. U5 snRNA has three Psis, in or near the loop of the large stem-loop that dominates the structure of this RNA. U6 and U1 snRNAs each have one Psi, and two Psi residues were found in U4 snRNA. CONCLUSION: The total number of Psis found in the snRNAs of C. elegans is significantly higher than the minimal amount found in yeasts but it is lower than that seen in sequenced vertebrate snRNAs. When the actual sites of modification on C. elegans snRNAs are compared with other sequenced snRNAs most of the positions correspond to modifications found in other species. However, two of the positions modified on C. elegans snRNAs are unique, one at position 28 on U2 snRNA and one at position 62 on U4 snRNA. Both of these modifications are in regions of these snRNAs that interact with U6 snRNA either in the spliceosome or in the U4/U6 small nuclear ribonucleoprotein particle (snRNP) and the presence of Psi may be involved in strengthening the intermolecular association of the snRNAs.

Animals↗
PubMed2005

Rational probe optimization and enhanced detection strategy for microRNAs using microarrays.

MicroRNAs (miRNAs) are post-transcriptional regulators participating in biological processes ranging from differentiation to carcinogenesis. We developed a rational probe design algorithm and a sensitive labelling scheme for optimizing miRNA microarrays. Our microarray contains probes for all validated miRNAs from five species, with the potential for drawing on species conservation to identify novel miRNAs with homologous probes. These methods are useful for high-throughput analysis of micro RNAs from various sources, and allow analysis with limiting quantities of RNA. The system design can also be extended for use on Luminex beads or on 96-well plates in an ELISA-style assay. We optimized hybridization temperatures using sequence variations on 20 of the probes and determined that all probes distinguish wild-type from 2 nt mutations, and most probes distinguish a 1 nt mutation, producing good selectivity between closely-related small RNA sequences. Results of tissue comparisons on our microarrays reveal patterns of hybridization that agree with results from Northern blots and other methods.

Animals↗
PubMed2005

C. elegans serine-threonine kinase KIN-29 modulates TGFbeta signaling and regulates body size formation.

BACKGROUND: In C. elegans there are two well-defined TGFbeta-like signaling pathways. The Sma/Mab pathway affects body size morphogenesis, male tail development and spicule formation while the Daf pathway regulates entry into and exit out of the dauer state. To identify additional factors that modulate TGFbeta signaling in the Sma/Mab pathway, we have undertaken a genetic screen for small animals and have identified kin-29. RESULTS: kin-29 encodes a protein with a cytoplasmic serine-threonine kinase and a novel C-terminal domain. The kinase domain is a distantly related member of the EMK (ELKL motif kinase) family, which interacts with microtubules. We show that the serine-threonine kinase domain has in vitro activity. kin-29 mutations result in small animals, but do not affect male tail morphology as do several of the Sma/Mab signal transducers. Adult worms are smaller than the wild-type, but also develop more slowly. Rescue by kin-29 is achieved by expression in neurons or in the hypodermis. Interaction with the dauer pathway is observed in double mutant combinations, which have been seen with Sma/Mab pathway mutants. We show that kin-29 is epistatic to the ligand dbl-1, and lies upstream of the Sma/Mab pathway target gene, lon-1. CONCLUSION: kin-29 is a new modulator of the Sma/Mab pathway. It functions in neurons and in the hypodermis to regulate body size, but does not affect all TGFbeta outputs, such as tail morphogenesis.

Amino Acid Sequence↗
PubMed2005

Incorporating structure to predict microRNA targets.

MicroRNAs (miRNAs) are a recently discovered set of regulatory genes that constitute up to an estimated 1% of the total number of genes in animal genomes, including Caenorhabditis elegans, Drosophila, mouse, and humans [Lagos-Quintana, M., Rauhut, R., Lendeckel, W. & Tuschl, T. (2001) Science 294, 853-858; Lai, E. C., Tomancak, P., Williams, R. W. & Rubin, G.M. (2003) Genome Biol. 4, R42; Lau, N. C., Lim, L. P., Weinstein, E. G. & Bartel, D. P. (2001) Science 294, 858-862; Lee, R. C. & Ambros, V. (2001) Science 294, 862-8644; and Lee, R. C., Feinbaum, R. L. & Ambros, V. (1993) Cell 115, 787-798]. In animals, miRNAs regulate genes by attenuating protein translation through imperfect base pair binding to 3' UTR sequences of target genes. A major challenge in understanding the regulatory role of miRNAs is to accurately predict regulated targets. We have developed an algorithm for predicting targets that does not rely on evolutionary conservation. As one of the features of this algorithm, we incorporate the folded structure of mRNA. By using Drosophila miRNAs as a test case, we have validated our predictions in 10 of 15 genes tested. One of these validated genes is mad as a target for bantam. Furthermore, our computational and experimental data suggest that miRNAs have fewer targets than previously reported.

3' Untranslated Regions↗
PubMed2005

Methylation as a crucial step in plant microRNA biogenesis.

Methylation on the base or the ribose is prevalent in eukaryotic ribosomal RNAs (rRNAs) and is thought to be crucial for ribosome biogenesis and function. Artificially introduced 2'-O-methyl groups in small interfering RNAs (siRNAs) can stabilize siRNAs in serum without affecting their activities in RNA interference in mammalian cells. Here, we show that plant microRNAs (miRNAs) have a naturally occurring methyl group on the ribose of the last nucleotide. Whereas methylation of rRNAs depends on guide RNAs, the methyltransferase protein HEN1 is sufficient to methylate miRNA/miRNA* duplexes. Our studies uncover a new and crucial step in plant miRNA biogenesis and have profound implications in the function of miRNAs.

Arabidopsis↗
PubMed2005

BMP signaling is required for controlling somatic stem cell self-renewal in the Drosophila ovary.

BMP signaling is essential for promoting self-renewal of mouse embryonic stem cells and Drosophila germline stem cells and for repressing stem cell proliferation in the mouse intestine and skin. However, it remains unknown whether BMP signaling can promote self-renewal of adult somatic stem cells. In this study, we show that BMP signaling is necessary and sufficient for promoting self-renewal and proliferation of somatic stem cells (SSCs) in the Drosophila ovary. BMP signaling is required in SSCs to directly control their maintenance and division, but is dispensable for proliferation of their differentiated progeny. Furthermore, BMP signaling is required to control SSC self-renewal, but not survival. Moreover, constitutive BMP signaling prolongs the SSC lifespan. Therefore, our study clearly demonstrates that BMP signaling directly promotes SSC self-renewal and proliferation in the Drosophila ovary. Our work further suggests that BMP signaling could promote self-renewal of adult stem cells in other systems.

Animals↗
PubMed2004

Genome-wide microarray analysis of TGFbeta signaling in the Drosophila brain.

BACKGROUND: Members of TGFbeta superfamily are found to play important roles in many cellular processes, such as proliferation, differentiation, development, apoptosis, and cancer. In Drosophila, there are seven ligands that function through combinations of three type I receptors and two type II receptors. These signals can be roughly grouped into two major TGFbeta pathways, the dpp/BMP and activin pathways, which signal primarily through thick veins (tkv) and baboon (babo). Few downstream targets are known for either pathway, especially targets expressed in the Drosophila brain. RESULTS: tkv and babo both affect the growth of tissues, but have varying effects on patterning. We have identified targets for the tkv and babo pathways by employing microarray techniques using activated forms of the receptors expressed in the brain. In these experiments, we compare the similarities of target genes of these two pathways in the brain. About 500 of 13,500 examined genes changed expression at 95% confidence level (P < 0.05). Twenty-seven genes are co-regulated 1.5 fold by both the tkv and babo pathways. These regulated genes cluster into various functional groups such as DNA/RNA binding, signal transducers, enzymes, transcription regulators, and neuronal regulators. RNAi knockdown experiments of homologs of several of these genes show abnormal growth regulation, suggesting these genes may execute the growth properties of TGFbeta. CONCLUSIONS: Our genomic-wide microarray analysis has revealed common targets for the tkv and babo pathways and provided new insights into downstream effectors of two distinct TGFbeta like pathways. Many of these genes are novel and several genes are implicated in growth control. Among the genes regulated by both pathways is ultraspiracle, which further connects TGFbeta with neuronal remodeling.

Animals↗
PubMed2003

Caenorhabditis elegans pseudouridine synthase 1 activity in vivo: tRNA is a substrate, but not U2 small nuclear RNA.

The formation of pseudouridine (Psi) from uridine is post-transcriptional and catalysed by pseudouridine synthases, several of which have been characterized from eukaryotes. Pseudouridine synthase 1 (Pus1p) has been well characterized from yeast and mice. In yeast, Pus1p has been shown to have dual substrate specificity, modifying uridines in tRNAs and at position 44 in U2 small nuclear RNA (U2 snRNA). In order to study the in vivo activity of a metazoan Pus1p, a knockout of the gene coding for the homologue of Pus1p in Caenorhabditis elegans was obtained. The deletion encompasses the first two putative exons and includes the essential aspartate that is required for activity in truA pseudouridine synthases. The locations of most modified nucleotides on small RNAs in C. elegans are not known, and the positions of Psi were determined on four tRNAs and U2 snRNA. The uridine at position 27 of tRNA(Val) (AAC), a putative Pus1p-modification site, was converted into Psi in the wild-type worms, but the tRNA(Val) (AAC) from mutant worms lacked the modification. Psi formation at positions 13, 32, 38 and 39, all of which should be modified by other pseudouridine synthases, was not affected by the loss of Pus1p. The absence of Pus1p in C. elegans had no effect on the modification of U2 snRNA in vivo, even though worm U2 snRNA has a Psi at position 45 (the equivalent of yeast U2 snRNA position 44) and at four other positions. This result was unexpected, given the known dual specificity of yeast Pus1p.

Animals↗
PubMed2003

A small issue addressed.

Cell size is an important determinant of body size. While the genetic mechanisms of cell size regulation have been well studied in yeast, this process has only recently been addressed in multicellular organisms. One recent report by Wang et al. (2002) shows that in the nematode C. elegans, the TGFbeta-like pathway acts in the hypodermis to regulate cell size and consequently body size.1 This finding is an exciting step in discovering the molecular mechanisms that control cell and body size.

Animals↗
PubMed2003

Genetic screen for small body size mutants in C. elegans reveals many TGFbeta pathway components.

In the nematode Caenorhabditis elegans, a TGFbeta-related signaling pathway regulates body size and male tail morphogenesis. We sought to identify genes encoding components or modifiers of this pathway in a large-scale genetic screen. Remarkably, this screen was able to identify essentially all core components of the TGFbeta signaling pathway. Among 34 Small mutants, many mutations disrupt genes encoding recognizable components of the TGFbeta pathway: DBL-1 ligand, DAF-4 type II receptor, SMA-6 type I receptor, and SMA-2, SMA-3, and SMA-4 Smads. Moreover, we find that at least 11 additional complementation groups can mutate to the Small phenotype. Four of these 11 genes, sma-9, sma-14, sma-16, and sma-20 affect male tail morphogenesis as well as body size. Two genes, sma-11 and sma-20, also influence regulation of the developmentally arrested dauer larval stage, suggesting a role in a second characterized TGFbeta pathway in C. elegans. Other genes may represent tissue-specific factors or parallel pathways for body size control. Because of the conservation of TGFbeta signaling pathways, homologs of these genes may be involved in tissue specificity and/or crosstalk of TGFbeta pathways in other animals.

Animals↗
PubMed2002

lon-1 regulates Caenorhabditis elegans body size downstream of the dbl-1 TGF beta signaling pathway.

In Caenorhabditis elegans, two well-characterized TGF beta signaling cascades have been identified: the Small/Male tail abnormal (Sma/Mab) and Dauer formation (Daf) pathways. The Sma/Mab pathway regulates body size morphogenesis and male tail development. The ligand of the pathway, dbl-1, transmits its signal through two receptor serine threonine kinases, daf-4 and sma-6, which in turn regulate the activity of the Smads, sma-2, sma-3, and sma-4. In general, Smads have been shown to both positively and negatively regulate the transcriptional activity of downstream target genes in various organisms. In C. elegans, however, target genes have remained elusive. We have cloned and characterized lon-1, a gene with homology to the cysteine-rich secretory protein (CRISP) family of proteins. lon-1 regulates body size morphogenesis, but does not affect male tail development. lon-1 is expressed in hypodermal tissues, which is the focus of body size determination, similar to sma-2, sma-4, and sma-6. Using genetic methods, we show that lon-1 lies downstream of the Sma/Mab signaling cascade and demonstrate that lon-1 mRNA levels are up-regulated in sma-6-null mutant animals. This provides evidence that lon-1 is negatively regulated by Sma/Mab pathway signaling. Taken together, these data identify lon-1 as a novel downstream target gene of the dbl-1 TGF beta-like signaling pathway.

Amino Acid Sequence↗
PubMed2002

The other side of TGF-beta superfamily signal regulation: thinking outside the cell.

The transforming growth factor beta (TGF-beta) superfamily of paracrine and autocrine signaling molecules regulates a vast array of developmental and homeostatic processes and is itself exquisitely regulated. The misregulation of these molecules often results in cancer and other diseases. Here, we focus on new research that explores how TGF-beta superfamily signaling is controlled between the secreting cell and the target cell. Regulation can occur upon ligand secretion (in a latent protein complex) and in the creation of signaling gradients. Proteins in the extracellular milieu sequester ligand away from or facilitate ligand binding to receptor serine kinases. Ligands even positively regulate their own negative regulators. Studies of how TGF-beta signaling is regulated extracellularly have broadened our understanding of TGF-beta pathways, and could provide clues to our understanding and treatment of diseases resulting from misregulation of these pathways.

Animals↗
PubMedDate not supplied

MicroRNAs: Small regulators with a big impact.

MicroRNAs (miRNA) are non-coding small (approximately 22nt) RNAs that regulate diverse physiological and developmental processes. In animals, they regulate target genes by binding imperfectly to 3'UTR sequences in mRNAs and attenuate translation. There are hundreds of miRNA genes in animals, and current studies show they constitute a minimum of 1% of known genes. We are just beginning to understand the diverse roles they play in cellular processes, which include signaling pathways, developmental pathways, and possibly various types of cancers.

Animals↗