Showing papers by "Phillip A. Sharp published in 2013"

Divergent transcription of long noncoding RNA/mRNA gene pairs in embryonic stem cells

Abstract: Many long noncoding RNA (lncRNA) species have been identified in mammalian cells, but the genomic origin and regulation of these molecules in individual cell types is poorly understood. We have generated catalogs of lncRNA species expressed in human and murine embryonic stem cells and mapped their genomic origin. A surprisingly large fraction of these transcripts (>60%) originate from divergent transcription at promoters of active protein-coding genes. The divergently transcribed lncRNA/mRNA gene pairs exhibit coordinated changes in transcription when embryonic stem cells are differentiated into endoderm. Our results reveal that transcription of most lncRNA genes is coordinated with transcription of protein-coding genes.

A Circuitous Route to Noncoding RNA

Abstract: Most genetic information is expressed as, and transacted by, proteins. Yet, less than 2% of the human genome actually codes for proteins, prompting a search for functions for the other 98% of the genome, once considered to be mostly “junk DNA.” Transcription is pervasive, however, and high-throughput sequencing has identified tens of thousands of distinct RNAs generated from the non—protein—coding portion of the genome ( 1 ). These so-called noncoding RNAs vary in length, but like protein-coding RNAs, appear to be linear molecules with 5′ and 3′ termini, reflecting the defined start and end points of RNA polymerase on the DNA template. But do all RNAs have to be linear?

Promoter directionality is controlled by U1 snRNP and polyadenylation signals

Abstract: Transcription of the mammalian genome is pervasive, but productive transcription outside of protein-coding genes is limited by unknown mechanisms. In particular, although RNA polymerase II (RNAPII) initiates divergently from most active gene promoters, productive elongation occurs primarily in the sense-coding direction. Here we show in mouse embryonic stem cells that asymmetric sequence determinants flanking gene transcription start sites control promoter directionality by regulating promoter-proximal cleavage and polyadenylation. We find that upstream antisense RNAs are cleaved and polyadenylated at poly(A) sites (PASs) shortly after initiation. De novo motif analysis shows PAS signals and U1 small nuclear ribonucleoprotein (snRNP) recognition sites to be the most depleted and enriched sequences, respectively, in the sense direction relative to the upstream antisense direction. These U1 snRNP sites and PAS sites are progressively gained and lost, respectively, at the 5' end of coding genes during vertebrate evolution. Functional disruption of U1 snRNP activity results in a dramatic increase in promoter-proximal cleavage events in the sense direction with slight increases in the antisense direction. These data suggest that a U1-PAS axis characterized by low U1 snRNP recognition and a high density of PASs in the upstream antisense region reinforces promoter directionality by promoting early termination in upstream antisense regions, whereas proximal sense PAS signals are suppressed by U1 snRNP. We propose that the U1-PAS axis limits pervasive transcription throughout the genome.

The Role of miRNAs in Regulating Gene Expression Networks

Abstract: MicroRNAs (miRNAs) are key regulators of gene expression. They are conserved across species, expressed across cell types, and active against a large proportion of the transcriptome. The sequence-complementary mechanism of miRNA activity exploits combinatorial diversity, a property conducive to network-wide regulation of gene expression, and functional evidence supporting this hypothesized systems-level role has steadily begun to accumulate. The emerging models are exciting and will yield deep insight into the regulatory architecture of biology. However, because of the technical challenges facing the network-based study of miRNAs, many gaps remain. Here, we review mammalian miRNAs by describing recent advances in understanding their molecular activity and network-wide function.

A Circuitous Route to Noncoding RNA

Abstract: Most genetic information is expressed as, and transacted by, proteins. Yet, less than 2% of the human genome actually codes for proteins, prompting a search for functions for the other 98% of the genome, once considered to be mostly “junk DNA.” Transcription is pervasive, however, and high-throughput sequencing has identified tens of thousands of distinct RNAs generated from the non—protein—coding portion of the genome ( 1 ). These so-called noncoding RNAs vary in length, but like protein-coding RNAs, appear to be linear molecules with 5′ and 3′ termini, reflecting the defined start and end points of RNA polymerase on the DNA template. But do all RNAs have to be linear?

Regulation of Glucose Transporter Expression in Human Intestinal Caco-2 Cells following Exposure to an Anthocyanin-Rich Berry Extract

Abstract: Polyphenols contained within plant tissues are consumed in significant amounts in the human diet and are known to influence a number of biological processes This study investigated the effects of an anthocyanin-rich berry-extract on glucose uptake by human intestinal Caco-2 cells Acute exposure (15 min) to berry extract (0125%, w/v) significantly decreased both sodium-dependent (Total uptake) and sodium-independent (facilitated uptake) 3H-D-glucose uptake In longer-term studies, SGLT1 mRNA and GLUT2 mRNA expression were reduced significantly Polyphenols are known to interact directly with glucose transporters to regulate the rate of glucose absorption Our in vitro data support this mechanism and also suggest that berry flavonoids may modulate post-prandial glycaemia by decreasing glucose transporter expression Further studies are warranted to investigate the longer term effects of berry flavonoids on the management of glycaemia in human volunteers

Copper deficiency leads to anemia, duodenal hypoxia, upregulation of HIF-2α and altered expression of iron absorption genes in mice.

Abstract: Iron and copper are essential trace metals, actively absorbed from the proximal gut in a regulated fashion. Depletion of either metal can lead to anemia. In the gut, copper deficiency can affect iron absorption through modulating the activity of hephaestin - a multi-copper oxidase required for optimal iron export from enterocytes. How systemic copper status regulates iron absorption is unknown. Mice were subjected to a nutritional copper deficiency-induced anemia regime from birth and injected with copper sulphate intraperitoneally to correct the anemia. Copper deficiency resulted in anemia, increased duodenal hypoxia and Hypoxia inducible factor 2α (HIF-2α) levels, a regulator of iron absorption. HIF-2α upregulation in copper deficiency appeared to be independent of duodenal iron or copper levels and correlated with the expression of iron transporters (Ferroportin - Fpn, Divalent Metal transporter – Dmt1) and ferric reductase – Dcytb. Alleviation of copper-dependent anemia with intraperitoneal copper injection resulted in down regulation of HIF-2α-regulated iron absorption genes in the gut. Our work identifies HIF-2α as an important regulator of iron transport machinery in copper deficiency.

Let-7 represses Nr6a1 and a mid-gestation developmental program in adult fibroblasts

Abstract: MicroRNAs (miRNAs) are critical to proliferation, differentiation, and development. Here, we characterize gene expression in murine Dicer-null adult mesenchymal stem cell lines, a fibroblast cell type. Loss of Dicer leads to derepression of let-7 targets at levels that exceed 10-fold to 100-fold with increases in transcription. Direct and indirect targets of this miRNA belong to a mid-gestation embryonic program that encompasses known oncofetal genes as well as oncogenes not previously associated with an embryonic state. Surprisingly, this mid-gestation program represents a distinct period that occurs between the pluripotent state of the inner cell mass at embryonic day 3.5 (E3.5) and the induction of let-7 upon differentiation at E10.5. Within this mid-gestation program, we characterize the let-7 target Nr6a1, an embryonic transcriptional repressor that regulates gene expression in adult fibroblasts following miRNA loss. In total, let-7 is required for the continual suppression of embryonic gene expression in adult cells, a mechanism that may underlie its tumor-suppressive function.

Quantifying Argonaute proteins in and out of GW/P-bodies: implications in microRNA activities.

Abstract: MicroRNAs (miRNAs) are a class of ∼22nt non-coding RNAs that regulate the translational potential and stability of mRNAs. Though constituting only 1–4% of human genes, miRNAs are predicted to regulate more than 60% of all mRNAs. The action of miRNAs is mediated through their associations with Argonaute proteins and mRNA targets. Previous studies indicated that though the majority of Argonaute proteins is diffusely distributed in the cytoplasm, a small fraction is consistently observed to be concentrated in a cytoplasmic compartment called GW/P-bodies. In this chapter, we will provide a quantitative and dynamic view of the subcellular localization of miRNA function, followed by a discussion on the possible roles of PBs in miRNA silencing.

Sugars increase non-heme iron bioavailability in human epithelial intestinal and liver cells.

Abstract: Previous studies have suggested that sugars enhance iron bioavailability, possibly through either chelation or altering the oxidation state of the metal, however, results have been inconclusive. Sugar intake in the last 20 years has increased dramatically, and iron status disorders are significant public health problems worldwide; therefore understanding the nutritional implications of iron-sugar interactions is particularly relevant. In this study we measured the effects of sugars on non-heme iron bioavailability in human intestinal Caco-2 cells and HepG2 hepatoma cells using ferritin formation as a surrogate marker for iron uptake. The effect of sugars on iron oxidation state was examined by measuring ferrous iron formation in different sugar-iron solutions with a ferrozine-based assay. Fructose significantly increased iron-induced ferritin formation in both Caco-2 and HepG2 cells. In addition, high-fructose corn syrup (HFCS-55) increased Caco-2 cell iron-induced ferritin; these effects were negated by the addition of either tannic acid or phytic acid. Fructose combined with FeCl3 increased ferrozine-chelatable ferrous iron levels by approximately 300%. In conclusion, fructose increases iron bioavailability in human intestinal Caco-2 and HepG2 cells. Given the large amount of simple and rapidly digestible sugars in the modern diet their effects on iron bioavailability may have important patho-physiological consequences. Further studies are warranted to characterize these interactions.

CD44 promotes Kras-dependent lung adenocarcinoma

Abstract: Kras-induced non-small-cell lung adenocarcinoma is the major subtype of lung cancers and is associated with poor prognosis. Using a lung cancer mouse model that expresses a cre-mediated KrasG12D mutant, we identified a critical role for the cell surface molecule CD44 in mediating cell proliferation downstream of oncogenic Kras signaling. The deletion of CD44 attenuates lung adenocarcinoma formation and prolongs the survival of these mice. Mechanistically, CD44 is required for the activation of Kras-mediated signaling through the mitogen-activated protein kinase (MAPK) pathway and thus promotes tumor cell proliferation. Together, these results reveal an unrecognized role for CD44 in oncogenic Kras-induced lung adenocarcinoma and suggest that targeting CD44 could be an effective strategy for halting Kras-dependent carcinomas.

Standing Up for GMOs

Abstract: On 8 August 2013, vandals destroyed a Philippine “Golden Rice” Field Trial. Officials and staff of the Philippine Department of Agriculture that conduct rice tests for the International Rice Research Institute (IRRI) and the Philippine Rice Research Institute (PhilRice) had gathered for a peaceful dialogue. They were taken by surprise when protesters invaded the compound, overwhelmed police and village security, and trampled the rice. Billed as an uprising of farmers, the destruction was actually carried out by protesters trucked in overnight in a dozen jeepneys.

Production of stable non-polyadenylated RNAs

Abstract: The invention relates in aspects to hybrid RNAs lacking a poly-A tail and nucleic acid vectors for expressing the RNA. The hybrid RNAs in some instances have a 3' terminal stabilizing triple helical structure. Related methods for expressing said RNAs in vivo and in vitro are also disclosed.

Duodenal cytochrome b plays a role in copper uptake

Abstract: Introduction: Iron and copper are absorbed in their reduced forms (ferrous and cuprous, respectively). Duodenal cytochrome b (Dcytb) functions as an intestinal ferric reductase; however, the nature of the putative intestinal cupric reductase remains unclear. We have shown previously that cells over-expressing Dcytb show not only enhanced ferric reductase activity but also display significant cupric reductase activity, suggesting that Dcytb may function as a dual metallo-reductase in intestinal enterocytes (Wyman et al FEBS Lett 2008, 582:1901-6). The aims of this study were to investigate whether intestinal Dcytb expression was regulated by copper, and whether Dcytb might play a role in cellular copper uptake. Methods and Materials: Human intestinal epithelial cells (Caco-2 and HuTu-80) were exposed to either the copper chelator TETA (0.5 mM) or copper (50 µM) for 24 h. Cells were harvested for RNA and expression of Dcytb and the copper transporter Ctr1 determined by qPCR. To determine whether Dcytb plays a role in copper uptake we employed a MDCK cell line which over-expresses Dcytb under the control of a tetracycline-repressible promoter (TET-Off MDCK, Clontech; Wyman et al 2008). Copper uptake was measured by ICP-OES. Results: In Caco-2 cells, Ctr1 expression was significantly increased by TETA treatment (p< 0.01) and significantly decreased by exposure to high copper medium (p<0.05). Basal Dcytb expression in Caco-2 cells was below the detection threshold in our studies so we used HuTu-80 cells as an alternative intestinal model. Dcytb expression was significantly increased in TETA-treated cells (p<0.01). Copper treatment did not significantly alter Dcytb mRNA compared with untreated controls. Copper uptake in Dcytb-overexpressing MDCK cells was significantly increased compared with untransfected control cells (+6.7-fold; p<0.01). Treatment with doxycycline (20ng/ml) significantly reduced copper uptake to levels seen in untransfected cells (p<0.01). Conclusion: These data demonstrate that Dcytb is a copper-regulated gene and that it plays a role in copper uptake. Taken together with our previous work we hypothesize that Dcytb functions as an intestinal cupric reductase providing Cu+ to Ctr1 for cellular uptake. This work forges a further link between mammalian iron and copper metabolism.

Divergent Transcription: A Driving Force for New Gene Origination?

Abstract: The mammalian genome is extensively transcribed, a large fraction of which is divergent transcription from promoters and enhancers that is tightly coupled with active gene transcription. Here, we propose that divergent transcription may shape the evolution of the genome by new gene origination.

Promoter directionality is controlled by U1 snRNP and polyadenylation signals

Abstract: Transcription of the mammalian genome is pervasive, but productive transcription outside of protein-coding genes is limited by unknown mechanisms. In particular, although RNA polymerase II (RNAPII) initiates divergently from most active gene promoters, productive elongation occurs primarily in the sense-coding direction. Here we show in mouse embryonic stem cells that asymmetric sequence determinants flanking gene transcription start sites control promoter directionality by regulating promoter-proximal cleavage and polyadenylation. We find that upstream antisense RNAs are cleaved and polyadenylated at poly(A) sites (PASs) shortly after initiation. De novo motif analysis shows PAS signals and U1 small nuclear ribonucleoprotein (snRNP) recognition sites to be the most depleted and enriched sequences, respectively, in the sense direction relative to the upstream antisense direction. These U1 snRNP sites and PAS sites are progressively gained and lost, respectively, at the 5' end of coding genes during vertebrate evolution. Functional disruption of U1 snRNP activity results in a dramatic increase in promoter-proximal cleavage events in the sense direction with slight increases in the antisense direction. These data suggest that a U1-PAS axis characterized by low U1 snRNP recognition and a high density of PASs in the upstream antisense region reinforces promoter directionality by promoting early termination in upstream antisense regions, whereas proximal sense PAS signals are suppressed by U1 snRNP. We propose that the U1-PAS axis limits pervasive transcription throughout the genome.