Altered Nuclear Functions in Progeroid Syndromes: a Paradigm for Aging Research

Syndromes of accelerated aging could provide an entry point for identifying and dissecting the cellular pathways that are involved in the development of age-related pathologies in the general population. However, their usefulness for aging research has been controversial, as it has been argued that these diseases do not faithfully reflect the process of natural aging. Here we review recent findings on the molecular basis of two progeroid diseases, Werner syndrome (WS) and Hutchinson-Gilford progeria syndrome (HGPS), and highlight functional connections to cellular processes that may contribute to normal aging

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

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

Incremental depletion of MBNL1 results in an increase of both the number and severity of RNA splice defects.

<p>RNA splice defects that manifest with the depletion of MBNL1 in SkMC and in <i>Mbnl1^+/ΔE3</i> and <i>Mbnl1^ΔE3/ΔE3</i> skeletal muscle are shown. Line thickness represents the severity of the splice defect.</p

RNA half-life measurements in SkMC.

<p>Normal myoblasts were treated with a combination of actinomycin-D and α-aminitin to inhibit transcription. Myoblasts were harvested at different time-points after treatment (0, 0.5, 4, 8, 16 and 24 h) and RNA was extracted. Synthesized cDNAs were subjected to RT-PCR analysis to measure RNA half-lives as previously described (17,18). <i>MYC</i>, a short-lived RNA and the long-lived 18S RNA were used as controls. Graphical representation of the average percent of RNA plotted against time from two independent experiments is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048825#pone.0048825.s002" target="_blank">Figure S2</a>.</p