RNA steady-state defects in myotonic dystrophy are linked to nuclear exclusion of SHARP

We describe a new mechanism by which CTG tract expansion affects myotonic dystrophy (DM1). Changes to the levels of a panel of RNAs involved in muscle development and function that are downregulated in DM1 are due to aberrant localization of the transcription factor SHARP (SMART/HDAC1-associated repressor protein). Mislocalization of SHARP in DM1 is consistent with increased CRM1-mediated export of SHARP to the cytoplasm. A direct link between CTG repeat expression and SHARP mislocalization is demonstrated as expression of expanded CTG repeats in normal cells recapitulates cytoplasmic SHARP localization. These results demonstrate a role for the inactivation of SHARP transcription in DM1 biology

Synthesis of highly water-soluble fluorescent conjugated glycopoly(p-phenylene)s for lectin and Escherichia coli

Two facile, convenient, and versatile synthetic approaches are used to covalently attach carbohydrate residues to conjugated poly(p-phenylene)s (PPPs) for highly water-soluble PPPs bearing α-mannopyranosyl and β-glucopyranosyl pendants (polymers A and B), which highly fluoresce in phosphate buffer (pH 7.0). The post-polymerization functionalization approach is to treat bromo-bearing PPP (polymer 1) with 1-thiolethyl-α-D-mannose tetraacetate or 1-thiol-β-D-glucose tetraacetate in THF solution in the presence of K2CO3 at room temperature through formation of thioether bridges, affording polymer 2a or 2b. The prepolymerization functionalization approach is to polymerize a well-defined sugar-carrying monomer, affording polymer 2a. Polymers 2a and 2b were deacetylated under Zemplén conditions in methanol and methylene chloride containing sodium methoxide, affording polymers A and B, respectively. The multivalent display of carbohydrates on the fluorescent conjugated glycopolymer overcomes the characteristic low binding affinity of the individual carbohydrates to their receptor proteins. Titration of concanavalin A (Con A) to α-mannose-bearing polymer A resulted in significant fluorescent quenching of the polymer with Stern-Volmer quenching constant of 4.5 × 107. Incubation of polymer A with Escherichia coli (E. coli) lead to formation of fluorescently stained bacterial clusters. β-Glucose-bearing polymer B displayed no response to Con A and E. coli. © 2006 American Chemical Society

WRN Controls Formation of Extrachromosomal Telomeric Circles and Is Required for TRF2ΔB-Mediated Telomere Shortening▿ †

Telomere dysfunction has been proposed to contribute to the pathogenesis of Werner syndrome (WS), a premature-aging disorder. The WS protein WRN binds TRF2, a telomere-specific factor that protects chromosome ends. TRF2 possesses an amino-terminal domain that plays an essential role in preventing telomere shortening, as expression of TRF2ΔB, which lacks this domain, leads to the formation of telomeric circles, telomere shortening, and cell senescence. Our data show that the TRF2ΔB-induced telomeric-loop homologous-recombination pathway requires WRN helicase. In addition, we show that WRN represses the formation of spontaneous telomeric circles, as demonstrated by the increased levels of telomeric circles observed in telomerase-positive WS fibroblasts. The mechanism of circle formation in WS cells does not involve XRCC3 function. Circle formation in WS cells is reduced by reconstitution with wild-type WRN but not mutant forms lacking either exonuclease or helicase activity, demonstrating that both enzymatic activities of WRN are required to suppress telomeric-circle formation in normal cells expressing telomerase reverse transcriptase. Thus, WRN has a key protective function at telomeres which influences telomere topology and inhibits accelerated attrition of telomeres

Lily pollen alkaline phytase is a histidine phosphatase similar to mammalian multiple inositol polyphosphate phosphatase (MINPP)

Phytic acid is the most abundant inositol phosphate in cells; it constitutes 1-5% of the dry weight of cereal grains and legumes. Phytases are the primary enzymes responsible for the hydrolysis of phytic acid and thus play important roles in inositol phosphate metabolism. A novel alkaline phytase in lily pollen (LlALP) was recently purified in our laboratory. In this paper, we describe the cloning and characterization of LlALP cDNA from lily pollen. Two isoforms of alkaline phytase cDNAs, LlAlp1 and LlAlp2, which are 1467 and 1533 bp long and encode proteins of 487 and 511 amino acids, respectively, were identified. The deduced amino acid sequences contains the signature heptapeptide of histidine phosphatases, -RHGXRXP-, but shares \u3c 25% identity to fungal histidine acid phytases. Phylogenetic analysis reveals that LlALP is most closely related to multiple inositol polyphosphate phosphatase (MINPP) from humans (25%) and rats (23%). mRNA corresponding to LlAlp1 and LlAlp2 were expressed in leaves, stem, petals and pollen grains. The expression profiles of LlAlp isoforms in anthers indicated that mRNA corresponding to both isoforms were present at all stages of flower development. The expression of LlAlp2 cDNA in Escherichia coli revealed the accumulation of the active enzyme in inclusion bodies and confirmed that the cDNA encodes an alkaline phytase. In summary, plant alkaline phytase is a member of the histidine phosphatase family that includes MINPP and exhibits properties distinct from bacterial and fungal phytases. © 2006 Elsevier Ltd. All rights reserved

Alkaline phytase from lily pollen: Investigation of biochemical properties

Phytases catalyze the hydrolysis of phytic acid (InsP6, myo-inositol hexakisphosphate), the most abundant inositol phosphate in cells. In cereal grains and legumes, it constitutes 3-5% of the dry weight of seeds. The inability of humans and monogastric animals such as swine and poultry to absorb complexed InsP6 has led to nutritional and environmental problems. The efficacy of supplemental phytases to address these issues is well established; thus, there is a need for phytases with a range of biochemical and biophysical properties for numerous applications. An alkaline phytase that shows unique catalytic properties was isolated from plant tissues. In this paper, we report on the biochemical properties of an alkaline phytase from pollen grains of Lilium longiflorum. The enzyme exhibits narrow substrate specificity, it hydrolyzed InsP6 and para-nitrophenyl phosphate (pNPP). Alkaline phytase followed Michaelis-Menten kinetics with a Km of 81 μM and Vmax of 217 nmol Pi/min/mg with InsP6 and a Km of 372 μM and Vmax of 1272 nmol Pi/min/mg with pNPP. The pH optimum was 8.0 with InsP6 as the substrate and 7.0 with pNPP. Alkaline phytase was activated by calcium and inactivated by ethylenediaminetetraacetic acid; however, the enzyme retained a low level of activity even in Ca2+-free medium. Fluoride as well as myo-inositol hexasulfate did not have any inhibitory affect, whereas vanadate inhibited the enzyme. The enzyme was activated by sodium chloride and potassium chloride and inactivated by magnesium chloride; the activation by salts followed the Hofmeister series. The temperature optimum for hydrolysis is 55°C; the enzyme was stable at 55°C for about 30 min. The enzyme has unique properties that suggest the potential to be useful as a feed supplement. © 2005 Elsevier Inc. All rights reserved

RNA splicing is responsive to MBNL1 dose.

Myotonic dystrophy (DM1) is a highly variable, multi-system disorder resulting from the expansion of an untranslated CTG tract in DMPK. In DM1 expanded CUG repeat RNAs form hairpin secondary structures that bind and aberrantly sequester the RNA splice regulator, MBNL1. RNA splice defects resulting as a consequence of MBNL1 depletion have been shown to play a key role in the development of DM1 pathology. In patient populations, both the number and severity of DM1 symptoms increase broadly as a function of CTG tract length. However significant variability in the DM1 phenotype is observed in patients encoding similar CTG repeat numbers. Here we demonstrate that a gradual decrease in MBNL1 levels results both in the expansion of the repertoire of splice defects and an increase in the severity of the splice alterations. Thus, MBNL1 loss does not have an all or none outcome but rather shows a graded effect on the number and severity of the ensuing splice defects. Our results suggest that once a critical threshold is reached, relatively small dose variations of free MBNL1 levels, which may reflect modest changes in the size of the CUG tract or the extent of hairpin secondary structure formation, can significantly alter the number and severity of splice abnormalities and thus contribute to the phenotype variability observed in DM1 patients

Alkaline phytase from Lilium longiflorum: Purification and structural characterization

Phytases catalyze the hydrolysis of phytic acid (myo-inositol hexakisphosphate), the most abundant inositol phosphate in cells. Phytases are of great commercial importance because their use as food and animal feed supplement has been approved by many countries to alleviate environmental and nutritional problems. Although acid phytases have been extensively studied, information regarding alkaline phytases is limited. Alkaline phytases with unique catalytic properties have been identified in plants, however, there is no report on the purification or structural properties. In this paper, we describe the purification of alkaline phytase from plant tissue. The purification was challenging because of contamination from non-specific phosphatases and acid phytases and low endogenous concentration. The purification of alkaline phytase from pollen grains of Lilium longiflorum involved selective precipitation by heat and ammonium sulfate followed by anion exchange and chromatofocusing chromatography and, finally, gel electrophoresis. Alkaline phytase was purified ∼3000-fold with an overall recovery of 4.2%. The native molecular mass was estimated to be in the range of 118 ± 7 kDa by Ferguson plot analysis and Mr of denatured protein in the range of 52-55 kDa by SDS-PAGE suggesting that the enzyme is a homodimer. Separation by 2-D gel and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometric analysis of separated proteins indicates the presence of multiple mass and charge isoforms with pI values between 7.3 and 8.3. To our knowledge, this is the first alkaline phytase to be purified from plant sources. The unique properties suggest that the enzyme has the potential to be useful as a feed and food supplement. © 2005 Elsevier Inc. All rights reserved

Number and severity of splice defects increase when MBNL1 is silenced incrementally from ∼79% to ∼98%.

<p>SkMC were transfected with siRNAs directed against MBNL1 and cell samples on each subsequent day post-siRNA transfection for a period of 5 days, were divided into 4 aliquots where one aliquot was used to measure MBNL1 levels and total RNA was extracted from each of the three other aliquots. Scrambled siRNA transfected samples were harvested on Day 5, the last time point of the experiment. <b>(A)</b> Total protein (10 µg) was analyzed by western blot to measure the silencing achieved for MBNL1 at 24 h intervals for 5 days. Blots were probed for GAPDH as an internal control. <b>(B)</b> Synthesized cDNAs were subjected to PCR analysis to study RNA splicing as indicated with <i>GAPDH</i> RNA as an internal control. In each case the levels of exon inclusion obtained in the experiment shown are indicated. <b>(C)</b> The results of RNA splicing as a function of MBNL1 levels in SkMC are tabulated.</p

Splice defects in <i>Mbnl1^+/ΔE3</i> and <i>Mbnl1^ΔE3/ΔE3</i> skeletal muscle.

<p>Lower limb skeletal muscles from adult wild-type, <i>Mbnl1^+/ΔE3</i> and <i>Mbnl1^ΔE3/ΔE3</i> mice were harvested and divided into 2 aliquots. One aliquot was used to measure Mbnl1 levels and the other aliquot was used study RNA splicing. <b>(A)</b> Western blot analysis of steady-state Mbnl1 levels in skeletal muscle of wild-type, <i>Mbnl1^+/ΔE3</i> and <i>Mbnl1^ΔE3/ΔE3</i> mice are shown with Gapdh as an internal loading control. <b>(B)</b> cDNAs synthesized from skeletal muscle of wild-type, <i>Mbnl1^+/ΔE3</i> and <i>Mbnl1^ΔE3/ΔE3</i> mice were subjected to PCR analysis as indicated with <i>Gapdh</i> RNA as an internal control. In each case the levels of exon inclusion obtained in the experiment shown are indicated. <b>(C)</b> The results of RNA splicing examined as a function of Mbnl1 levels are tabulated.</p