The bacteriophage T4 AsiA protein contacts the beta-flap domain of RNA polymerase.
Proc Natl Acad Sci U S A. 106:6597-602. Abstract
To initiate transcription from specific promoters, the bacterial RNA polymerase (RNAP) core enzyme must associate with the initiation factor sigma, which contains determinants that allow sequence-specific interactions with promoter DNA. Most bacteria contain several sigma factors, each of which directs recognition of a distinct set of promoters. A large and diverse family of proteins known as "anti-sigma factors" regulates promoter utilization by targeting specific sigma factors. The founding member of this family is the AsiA protein of bacteriophage T4. AsiA specifically targets the primary sigma factor in Escherichia coli, sigma(70), and inhibits transcription from the major class of sigma(70)-dependent promoters. AsiA-dependent transcription inhibition has been attributed to a well-documented interaction between AsiA and conserved region 4 of sigma(70). Here, we establish that efficient AsiA-dependent transcription inhibition also requires direct protein-protein contact between AsiA and the RNAP core. In particular, we demonstrate that AsiA contacts the flap domain of the RNAP beta-subunit (the beta-flap). Our findings support the emerging view that the beta-flap is a target site for regulatory proteins that affect RNAP function during all stages of the transcription cycle.
Methylation as a crucial step in plant microRNA biogenesis.
Science (New York, NY). 307:932-5. Abstract
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.
The Drosophila decapentaplegic and short gastrulation genes function antagonistically during adult wing vein development.
Development (Cambridge, England). 122:4033-44. Abstract
TGF-beta-related signaling pathways play diverse roles during vertebrate and invertebrate development. A common mechanism for regulating the activity of TGF-beta family members is inhibition by extracellular antagonists. Recently, the Drosophila short gastrulation (sog) gene was shown to encode a predicted diffusible factor which antagonizes signaling mediated by the TGF-beta-like Decapentaplegic (Dpp) pathway in the early blastoderm embryo. sog and dpp, which are among the earliest zygotic genes to be activated, are expressed in complementary dorsal-ventral domains. The opposing actions of sog and dpp in the early embryo have been highly conserved during evolution as their vertebrate counterparts, chordin and BMP-4, function homologously to define neural versus non-neural ectoderm in Xenopus. Here we exploit the genetically sensitive adult wing vein pattern to investigate the generality of the antagonistic relationship between sog and dpp. We show that dpp is expressed in vein primordia during pupal wing development and functions to promote vein formation. In contrast, sog is expressed in complementary intervein cells and suppresses vein formation. sog and dpp function during the same phenocritical periods (i.e. 16-28 hours after pupariation) to influence the vein versus intervein cell fate choice. The conflicting activities of dpp and sog are also revealed by antagonistic dosage-sensitive interactions between these two genes during vein development. Analysis of vein and intervein marker expression in dpp and sog mutant wings suggests that dpp promotes vein fates indirectly by activating the vein gene rhomboid (rho), and that sog functions by blocking an autoactivating Dpp feedback loop. These data support the view that Sog is a dedicated Dpp antagonist.
Mammalian dwarfins are phosphorylated in response to transforming growth factor-β and are implicated in control of cell growth.
Proceedings of the National Academy of Sciences of the United States of America. 93:8940-4. Abstract
The dwarfin protein family has been genetically implicated in transforming growth factor beta (TGF-beta)-like signaling pathways in Drosophila and Caenorhabditis elegans. To investigate the role of these proteins in mammalian signaling pathways, we have isolated and studied two murine dwarfins, dwarfin-A and dwarfin-C. Using antibodies against dwarfin-A and dwarfin-C, we show that these two dwarfins and an immunogenically related protein, presumably also a dwarfin, are phosphorylated in a time- and dose-dependent manner in response to TGF-beta. Bone morphogenetic protein 2, a TGF-beta superfamily ligand, induces phosphorylation of only the related dwarfin protein. Thus, TGF-beta superfamily members may use overlapping yet distinct dwarfins to mediate their intracellular signals. Furthermore, transient overexpression of either dwarfin-A or dwarfin-C causes growth arrest, implicating the dwarfins in growth regulation. This work provides strong biochemical and preliminary functional evidence that dwarfin-A and dwarfin-C represent prototypic members of a family of mammalian proteins that may serve as mediators of signaling pathways for TGF-beta superfamily members.
Genome-wide microarray analysis of TGFβ signaling in the Drosophila brain.
BMC developmental biology. 4:14. Abstract
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.
Circadian regulation of a limited set of conserved microRNAs in Drosophila.
BMC Genomics. 9:83. Abstract
BACKGROUND: MicroRNAs (miRNAs) are short non-coding RNA molecules that target mRNAs to control gene expression by attenuating the translational efficiency and stability of transcripts. They are found in a wide variety of organisms, from plants to insects and humans. Here, we use Drosophila to investigate the possibility that circadian clocks regulate the expression of miRNAs. RESULTS: We used a microarray platform to survey the daily levels of D. melanogaster miRNAs in adult heads of wildtype flies and the arrhythmic clock mutant cyc01. We find two miRNAs (dme-miR-263a and -263b) that exhibit robust daily changes in abundance in wildtype flies that are abolished in the cyc01 mutant. dme-miR-263a and -263b reach trough levels during the daytime, peak during the night and their levels are constitutively elevated in cyc01 flies. A similar pattern of cycling is also observed in complete darkness, further supporting circadian regulation. In addition, we identified several miRNAs that appear to be constitutively expressed but nevertheless differ in overall daily levels between control and cyc01 flies. CONCLUSION: The circadian clock regulates miRNA expression in Drosophila, although this appears to be highly restricted to a small number of miRNAs. A common mechanism likely underlies daily changes in the levels of dme-miR-263a and -263b. Our results suggest that cycling miRNAs contribute to daily changes in mRNA and/or protein levels in Drosophila. Intriguingly, the mature forms of dme-miR-263a and -263b are very similar in sequence to several miRNAs recently shown to be under circadian regulation in the mouse retina, suggesting conserved functions.
MicroRNAs: Small regulators with a big impact.
Cytokine & growth factor reviews. 16:387-93. Abstract
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.