Ubiquitin recognition by the Cockayne syndrome group B protein: binding will set you free.

Transcription-coupled nucleotide excision repair (TC-NER) requires the coordinated efforts of many proteins. In this issue of Molecular Cell, Anindya et al. (2010) show that the proteins assemble at the site of DNA damage but cannot begin repair until the Cockayne syndrome group B protein (CSB) binds ubiquitin.

Tethering of the conserved piggyBac transposase fusion protein CSB-PGBD3 to chromosomal AP-1 proteins regulates expression of nearby genes in humans.

The CSB-PGBD3 fusion protein arose more than 43 million years ago when a 2.5-kb piggyBac 3 (PGBD3) transposon inserted into intron 5 of the Cockayne syndrome Group B (CSB) gene in the common ancestor of all higher primates. As a result, full-length CSB is now coexpressed with an abundant CSB-PGBD3 fusion protein by alternative splicing of CSB exons 1-5 to the PGBD3 transposase. An internal deletion of the piggyBac transposase ORF also gave rise to 889 dispersed, 140-bp MER85 elements that were mobilized in trans by PGBD3 transposase. The CSB-PGBD3 fusion protein binds MER85s in vitro and induces a strong interferon-like innate antiviral immune response when expressed in CSB-null UVSS1KO cells. To explore the connection between DNA binding and gene expression changes induced by CSB-PGBD3, we investigated the genome-wide DNA binding profile of the fusion protein. CSB-PGBD3 binds to 363 MER85 elements in vivo, but these sites do not correlate with gene expression changes induced by the fusion protein. Instead, CSB-PGBD3 is enriched at AP-1, TEAD1, and CTCF motifs, presumably through protein-protein interactions with the cognate transcription factors; moreover, recruitment of CSB-PGBD3 to AP-1 and TEAD1 motifs correlates with nearby genes regulated by CSB-PGBD3 expression in UVSS1KO cells and downregulated by CSB rescue of mutant CS1AN cells. Consistent with these data, the N-terminal CSB domain of the CSB-PGBD3 fusion protein interacts with the AP-1 transcription factor c-Jun and with RNA polymerase II, and a chimeric CSB-LacI construct containing only the N-terminus of CSB upregulates many of the genes induced by CSB-PGBD3. We conclude that the CSB-PGBD3 fusion protein substantially reshapes the transcriptome in CS patient CS1AN and that continued expression of the CSB-PGBD3 fusion protein in the absence of functional CSB may affect the clinical presentation of CS patients by directly altering the transcriptional program.

SINEs and LINEs: the art of biting the hand that feeds you.

SINEs and LINEs are short and long interspersed retrotransposable elements, respectively, that invade new genomic sites using RNA intermediates. SINEs and LINEs are found in almost all eukaryotes (although not in Saccharomyces cerevisiae) and together account for at least 34% of the human genome. The noncoding SINEs depend on reverse transcriptase and endonuclease functions encoded by partner LINEs. With the completion of many genome sequences, including our own, the database of SINEs and LINEs has taken a great leap forward. The new data pose new questions that can only be answered by detailed studies of the mechanism of retroposition. Current work ranges from the biochemistry of reverse transcription and integration invitro, target site selection in vivo, nucleocytoplasmic transport of the RNA and ribonucleoprotein intermediates, and mechanisms of genomic turnover. Two particularly exciting new ideas are that SINEs may help cells survive physiological stress, and that the evolution of SINEs and LINEs has been shaped by the forces of RNA interference. Taken together, these studies promise to explain the birth and death of SINEs and LINEs, and the contribution of these repetitive sequence families to the evolution of genomes.

An abundant evolutionarily conserved CSB-PiggyBac fusion protein expressed in Cockayne syndrome.

Cockayne syndrome (CS) is a devastating progeria most often caused by mutations in the CSB gene encoding a SWI/SNF family chromatin remodeling protein. Although all CSB mutations that cause CS are recessive, the complete absence of CSB protein does not cause CS. In addition, most CSB mutations are located beyond exon 5 and are thought to generate only C-terminally truncated protein fragments. We now show that a domesticated PiggyBac-like transposon PGBD3, residing within intron 5 of the CSB gene, functions as an alternative 3' terminal exon. The alternatively spliced mRNA encodes a novel chimeric protein in which CSB exons 1-5 are joined in frame to the PiggyBac transposase. The resulting CSB-transposase fusion protein is as abundant as CSB protein itself in a variety of human cell lines, and continues to be expressed by primary CS cells in which functional CSB is lost due to mutations beyond exon 5. The CSB-transposase fusion protein has been highly conserved for at least 43 Myr since the divergence of humans and marmoset, and appears to be subject to selective pressure. The human genome contains over 600 nonautonomous PGBD3-related MER85 elements that were dispersed when the PGBD3 transposase was last active at least 37 Mya. Many of these MER85 elements are associated with genes which are involved in neuronal development, and are known to be regulated by CSB. We speculate that the CSB-transposase fusion protein has been conserved for host antitransposon defense, or to modulate gene regulation by MER85 elements, but may cause CS in the absence of functional CSB protein.

Transference-Focused Psychotherapy for Adolescents With Personality Disorders.

This article presents a conceptualization of personality disorders in adolescence and the adaptation of transference-focused psychotherapy (TFP) for personality disordered adolescents (TFP-A). The model of assessment and treatment presented is based on contemporary psychoanalytic object relations theory developed by Otto F. Kernberg and supported by findings from current evidence-based outcome research. We present a method of assessing personality disorders in adolescents that addresses the variability of personality disorder symptoms and traits among adolescents and their instability over time. We then present the goal of TFP-A and its major phases of implementation. A major focus is therapist interventions.

On the role of a conserved, potentially helix-breaking residue in the tRNA-binding alpha-helix of archaeal CCA-adding enzymes.

Archaeal class I CCA-adding enzymes use a ribonucleoprotein template to build and repair the universally conserved 3'-terminal CCA sequence of the acceptor stem of all tRNAs. A wealth of structural and biochemical data indicate that the Archaeoglobus fulgidus CCA-adding enzyme binds primarily to the tRNA acceptor stem through a long, highly conserved alpha-helix that lies nearly parallel to the acceptor stem and makes many contacts with its sugar-phosphate backbone. Although the geometry of this alpha-helix is nearly ideal in all available cocrystal structures, the helix contains a highly conserved, potentially helix-breaking proline or glycine near the N terminus. We performed a mutational analysis to dissect the role of this residue in CCA-addition activity. We found that the phylogenetically permissible P295G mutant and the phylogenetically absent P295T had little effect on CCA addition, whereas P295A and P295S progressively interfered with CCA addition (C74>C75>A76 addition). We also examined the effects of these mutations on tRNA binding and the kinetics of CCA addition, and performed a computational analysis using Rosetta Design to better understand the role of P295 in nucleotide transfer. Our data indicate that CCA-adding activity does not correlate with the stability of the pre-addition cocrystal structures visualized by X-ray crystallography. Rather, the data are consistent with a transient conformational change involving P295 of the tRNA-binding alpha-helix during or between one or more steps in CCA addition.

Advancements in ocular drug delivery.

This review covers both noninvasive and invasive ophthalmic drug delivery systems that can have application to therapy of veterinary ophthalmic diseases. Noninvasive approaches include gel technologies, permeation enhancement via pro-drug development, solubilization agents and nanoparticle technologies, iontophoresis, microneedles, drug-eluting contact lenses and eye misters, and microdroplets. More invasive systems include both eroding implants and noneroding technologies that encompass diffusion based systems, active pumps, intraocular lenses, suprachoroidal drug delivery, and episcleral reservoirs. In addition to addressing the physiologic challenges of achieving the necessary duration of delivery, tissue targeting and patient compliance, the commercial development factors of biocompatibility, sterilization, manufacturability and long-term stability will be discussed.

Psychotherapy for Borderline Personality Disorder in Adolescents.

Research on borderline personality disorder (BPD) in adolescence has helped to clarify the characteristics of BPD in young people. The considerable emotional and economic cost associated with adolescent BPD supports calls for early intervention and requires that the assessment of personality functioning be an essential component in the psychological evaluation of adolescents. Adult treatment models with demonstrated efficacy have been adapted for adolescents. This article describes the implementation of these treatment approaches, factors that frequently complicate the recognition and diagnosis of BPD in young people, and an overview of research on BPD in adolescents that delineates its clinically relevant features.

tRNA maturation: RNA polymerization without a nucleic acid template.

The CCA-adding enzyme, which builds and repairs the 3' terminal CCA sequence of tRNA, is the only RNA polymerase that can synthesize a defined nucleotide sequence without using a nucleic acid template. New cocrystal structures tell us how this remarkable enzyme works.

Role of the C-terminal domain of RNA polymerase II in U2 snRNA transcription and 3' processing.

U small nuclear RNAs (snRNAs) and mRNAs are both transcribed by RNA polymerase II (Pol II), but the snRNAs have unusual TATA-less promoters and are neither spliced nor polyadenylated; instead, 3' processing is directed by a highly conserved 3' end formation signal that requires initiation from an snRNA promoter. Here we show that the C-terminal domain (CTD) of Pol II is required for efficient U2 snRNA transcription, as it is for mRNA transcription. However, CTD kinase inhibitors, such as 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), that block mRNA elongation do not affect U2 transcription, although 3' processing of the U2 primary transcript is impaired. We show further that U2 transcription is preferentially inhibited by low doses of UV irradiation or actinomycin D, which induce CTD kinase activity, and that UV inhibition can be rescued by treatment with DRB or H7. We propose that Pol II complexes transcribing snRNAs and mRNAs have distinct CTD phosphorylation patterns. mRNA promoters recruit factors including kinases that hyperphosphorylate the CTD, and the CTD in turn recruits proteins needed for mRNA splicing and polyadenylation. We predict that snRNA promoters recruit factors including a CTD kinase(s) whose snRNA-specific phosphorylation pattern recruits factors required for promoter-coupled 3' end formation.

TFOS DEWS II iatrogenic report.

Dry eye can be caused by a variety of iatrogenic interventions. The increasing number of patients looking for eye care or cosmetic procedures involving the eyes, together with a better understanding of the pathophysiological mechanisms of dry eye disease (DED), have led to the need for a specific report about iatrogenic dry eye within the TFOS DEWS II. Topical medications can cause DED due to their allergic, toxic and immuno-inflammatory effects on the ocular surface. Preservatives, such as benzalkonium chloride, may further aggravate DED. A variety of systemic drugs can also induce DED secondary to multiple mechanisms. Moreover, the use of contact lens induces or is associated with DED. However, one of the most emblematic situations is DED caused by surgical procedures such as corneal refractive surgery as in laser-assisted in situ keratomileusis (LASIK) and keratoplasty due to mechanisms intrinsic to the procedure (i.e. corneal nerve cutting) or even by the use of postoperative topical drugs. Cataract surgery, lid surgeries, botulinum toxin application and cosmetic procedures are also considered risk factors to iatrogenic DED, which can cause patient dissatisfaction, visual disturbance and poor surgical outcomes. This report also presents future directions to address iatrogenic DED, including the need for more in-depth epidemiological studies about the risk factors, development of less toxic medications and preservatives, as well as new techniques for less invasive eye surgeries. Novel research into detection of early dry eye prior to surgeries, efforts to establish appropriate therapeutics and a greater attempt to regulate and oversee medications, preservatives and procedures should be considered.

Identification and characterization of the ocular hypotensive efficacy of travoprost, a potent and selective FP prostaglandin receptor agonist, and AL-6598, a DP prostaglandin receptor agonist.

The structure-activity studies that led to the identification of travoprost, a highly selective and potent FP prostaglandin analog, and AL-6598, a DP prostaglandin analog, are detailed. In both series, the 1-alcohol analogs are very effective and are thought to be acting as prodrugs for the biologically active carboxylic acids. The efficacy of amide prodrugs depends on the degree of substitution and the size of the substituents. Selected compounds are profiled in vitro and in vivo preclinically. Clinical studies show that travoprost 0.004% (isopropyl ester) provided intraocular pressure control superior to timolol 0.5% when used as monotherapy in patients with open-angle glaucoma or ocular hypertension. In clinical studies, AL-6598 0.01% provided a sustained intraocular pressure reduction with q.d. application; b.i.d. provided greater intraocular pressure control. The acute and, apparently, conjunctival hyperemia associated with topical ocular AL-6598 can be attenuated while maintaining intraocular pressure-lowering efficacy by formulating with brimonidine.

Comparison of the diurnal ocular hypotensive efficacy of travoprost and latanoprost over a 44-hour period in patients with elevated intraocular pressure.

BACKGROUND: Prostaglandin analogues are effective ocular hypotensive agents and are being used increasingly in the treatment of elevated intraocular pressure (IOP). These agents are typically dosed once daily. OBJECTIVES: A pilot study was conducted to evaluate the duration of travoprost's IOP-lowering efficacy up to 84 hours after the final dose in patients with open-angle glaucoma. A follow-up study was conducted to compare diurnal IOP control with travoprost and latanoprost over a 44-hour period. METHODS: In the open label pilot study, patients received 0.004% travoprost in both eyes at 8 pm daily for 2 weeks. After 2 weeks, IOP was measured before administration of the last daily dose, every 4 hours thereafter for 36 hours, and 60 and 84 hours after the last dose, with no additional ocular hypotensive medication given. In the controlled, double-masked, parallel-group, follow-up study, patients were randomized to self-administer 1 drop of the marketed doses of 0.004% travoprost or 0.005% latanoprost in both eyes at 8 pm daily for 2 weeks. At the end of this period, patients returned to the facility at approximately 8 pm for IOP measurement and administration of the final dose of study medication. IOP was then measured at 4-hour intervals for 44 hours after the last dose, with no additional ocular hypotensive medication given. RESULTS: The pilot study included 21 patients (67% female, 33% male; age range, 35-81 years) with open-angle glaucoma. IOP values were significantly below baseline at all time points up to 84 hours after the final dose of travoprost ( P<0.001). The follow-up study enrolled 35 patients, 1 of whom was excluded for missing data; thus, the intent-to-treat analysis included 34 patients (68% female, 32% male; age range, 36-72 years). At the unmedicated eligibility visit, mean IOP over 24 hours ranged from 21 to 26 mm Hg in each treatment group. After 2 weeks of treatment and 24 hours after the last dose, mean (SD) IOP was 13.1 (2.1) mm Hg (change from eligibility visit, -10.4 [2.7] mm Hg) in the travoprost group and 16.0 (3.1) mm Hg (change from eligibility visit, -7.1 [2.4] mm Hg) in the latanoprost group. The difference in change from baseline was statistically significant between treatment groups (P=0.006). Travoprost lowered IOP significantly at all time points throughout the 44-hour period after the last dose (mean IOP,

Response to travoprost in black and nonblack patients with open-angle glaucoma or ocular hypertension.

Two prospective, controlled, multicenter, double-masked studies--one lasting 6 months (n=594) and the other, 12 months (n=787)--examined the intraocular pressure (IOP)-lowering efficacy of travoprost in 1381 black and nonblack patients with open-angle glaucoma or ocular hypertension. Investigated regimens were travoprost 0.004% once daily, latanoprost 0.005% once daily, and timolol 0:5% twice daily. In both studies, mean IOP was significantly lower in blacks treated with travoprost. The IOP reduction was also significantly greater in blacks after adjustments for age, sex, iris color, diagnosis, and corneal thickness. Timolol lowered mean IOP to a greater extent in nonblack patients. The significantly larger IOP reduction with travoprost compared with timolol in both racial groups was more pronounced in blacks. Travoprost also was superior to latanoprost in blacks. Mean changes from baseline generally were greater for black than for nonblack patients, although the differences did not achieve statistical significance. The response rate to travoprost was higher in blacks. The most common adverse effect was hyperemia.

What role (if any) does the highly conserved CSB-PGBD3 fusion protein play in Cockayne syndrome?

The PGBD3 piggyBac transposon inserted into CSB intron 5 early in the primate lineage. As a result of alternative splicing, the human CSB gene now encodes three proteins: CSB, a CSB-PGBD3 fusion protein that joins the N-terminal CSB domain to the C-terminal PGBD3 transposase domain, and PGBD3 transposase. The fusion protein is as highly conserved as CSB, suggesting that it is advantageous in health; however, expression of the fusion protein in CSB-null cells induces a constitutive interferon (IFN) response. The fusion protein binds in vivo to PGBD3-related MER85 elements, but is also tethered to c-Jun, TEAD1, and CTCF motifs by interactions with the cognate transcription factors. The fusion protein regulates nearby genes from the c-Jun (and to a lesser extent TEAD1 and CTCF) motifs, but not from MER85 elements. We speculate that the fusion protein interferes with CSB-dependent chromatin remodeling, generating double-stranded RNA (dsRNA) that induces an IFN response through endosomal TLR or cytoplasmic RIG-I and/or MDA5 RNA sensors. We suggest that the fusion protein was fixed in primates because an elevated IFN response may help to fight viral infection. We also speculate that an inappropriate IFN response may contribute to the clinical presentation of CS.

PGBD5: a neural-specific intron-containing piggyBac transposase domesticated over 500 million years ago and conserved from cephalochordates to humans.

BACKGROUND: piggyBac domain (PGBD) transposons are found in organisms ranging from fungi to humans. Three domesticated piggyBac elements have been described. In the ciliates Paramecium tetraurelia and Tetrahymena thermophila, homologs known as piggyMacs excise internal eliminated sequences from germline micronuclear DNA during regeneration of the new somatic macronucleus. In primates, a PGBD3 element inserted into the Cockayne syndrome group B (CSB) gene over 43 Mya serves as an alternative 3' terminal exon, enabling the CSB gene to generate both full length CSB and a conserved CSB-PGBD3 fusion protein that joins an N-terminal CSB domain to the C-terminal transposase domain. RESULTS: We describe a fourth domesticated piggyBac element called PGBD5. We show that i) PGBD5 was first domesticated in the common ancestor of the cephalochordate Branchiostoma floridae (aka lancelet or amphioxus) and vertebrates, and is conserved in all vertebrates including lamprey but cannot be found in more basal urochordates, hemichordates, or echinoderms; ii) the lancelet, lamprey, and human PGBD5 genes are syntenic and orthologous; iii) no potentially mobile ancestral PGBD5 elements can be identified in other more deeply rooted organisms; iv) although derived from an IS4-related transposase of the RNase H clan, PGBD5 protein is unlikely to retain enzymatic activity because the catalytic DDD(D) motif is not conserved; v) PGBD5 is preferentially expressed in certain granule cell lineages of the brain and in the central nervous system based on available mouse and human in situ hybridization data, and the tissue-specificity of documented mammalian EST and mRNA clones; vi) the human PGBD5 promoter and gene region is rich in bound regulatory factors including the neuron-restrictive silencer factors NRSF/REST and CoREST, as well as SIN3, KAP1, STAT3, and CTCF; and vii) despite preferential localization within the nucleus, PGBD5 protein is unlikely to bind DNA or chromatin as neither DNase I digestion nor high salt extraction release PGBD5 from fractionated mouse brain nuclei. CONCLUSIONS: We speculate that the neural-specific PGBD5 transposase was domesticated >500 My after cephalochordates and vertebrates split from urochordates, and that PGBD5 may have played a role in the evolution of a primitive deuterostome neural network into a centralized nervous system.

The conserved Cockayne syndrome B-piggyBac fusion protein (CSB-PGBD3) affects DNA repair and induces both interferon-like and innate antiviral responses in CSB-null cells.

Cockayne syndrome is a segmental progeria most often caused by mutations in the CSB gene encoding a SWI/SNF-like ATPase required for transcription-coupled DNA repair (TCR). Over 43Mya before marmosets diverged from humans, a piggyBac3 (PGBD3) transposable element integrated into intron 5 of the CSB gene. As a result, primate CSB genes now generate both CSB protein and a conserved CSB-PGBD3 fusion protein in which the first 5 exons of CSB are alternatively spliced to the PGBD3 transposase. Using a host cell reactivation assay, we show that the fusion protein inhibits TCR of oxidative damage but facilitates TCR of UV damage. We also show by microarray analysis that expression of the fusion protein alone in CSB-null UV-sensitive syndrome (UVSS) cells induces an interferon-like response that resembles both the innate antiviral response and the prolonged interferon response normally maintained by unphosphorylated STAT1 (U-STAT1); moreover, as might be expected based on conservation of the fusion protein, this potentially cytotoxic interferon-like response is largely reversed by coexpression of functional CSB protein. Interestingly, expression of CSB and the CSB-PGBD3 fusion protein together, but neither alone, upregulates the insulin growth factor binding protein IGFBP5 and downregulates IGFBP7, suggesting that the fusion protein may also confer a metabolic advantage, perhaps in the presence of DNA damage. Finally, we show that the fusion protein binds in vitro to members of a dispersed family of 900 internally deleted piggyBac elements known as MER85s, providing a potential mechanism by which the fusion protein could exert widespread effects on gene expression. Our data suggest that the CSB-PGBD3 fusion protein is important in both health and disease, and could play a role in Cockayne syndrome.

Human U2 snRNA genes exhibit a persistently open transcriptional state and promoter disassembly at metaphase.

In mammals, small multigene families generate spliceosomal U snRNAs that are nearly as abundant as rRNA. Using the tandemly repeated human U2 genes as a model, we show by footprinting with DNase I and permanganate that nearly all sequences between the enhancer-like distal sequence element and the initiation site are protected during interphase whereas the upstream half of the U2 snRNA coding region is exposed. We also show by chromatin immunoprecipitation that the SNAPc complex, which binds the TATA-like proximal sequence element, is removed at metaphase but remains bound under conditions that induce locus-specific metaphase fragility of the U2 genes, such as loss of CSB, BRCA1, or BRCA2 function, treatment with actinomycin D, or overexpression of the tetrameric p53 C terminus. We propose that the U2 snRNA promoter establishes a persistently open state to facilitate rapid reinitiation and perhaps also to bypass TFIIH-dependent promoter melting; this open state would then be disassembled to allow metaphase chromatin condensation.