The splicing regulators Tra and Tra2 are unusually potent activators of pre-mRNA splicing

Sexual differentiation in Drosophila is regulated through alternative splicing of doublesex. Female-specific splicing is activated through the activity of splicing enhancer complexes assembled on multiple repeat elements. Each of these repeats serves as a binding platform for the cooperative assembly of a heterotrimeric complex consisting of the SR proteins Tra, Tra2 and 9G8. Using quantitative kinetic analyses, we demonstrate that each component of the enhancer complex is capable of recruiting the spliceosome. Surprisingly, Tra, Tra2 and 9G8 are much stronger splicing activators than other SR protein family members and their activation potential is significantly higher than expected from their serine/arginine content. 9G8 activates splicing not only through its RS domains but also through its RNA-binding domain. The RS domains of Tra and Tra2 are required but not sufficient for efficient complex assembly. Thus, the regulated assembly of the dsx enhancer complexes leads to the generation of an extended activation domain to guarantee the ‘all or none’ splicing switch that is required during Drosophila sexual differentiation

Tono-Pen XL tonometry during application of a suction ring in rabbits

<p>Abstract</p> <p>Background</p> <p>The purpose of this study is to evaluate the use of Tono-Pen XL in measuring IOP during the application of a suction ring in rabbit eyes with manometrically controlled IOP.</p> <p>Methods</p> <p>Tono-Pen XL was calibrated against direct manometry in 10 rabbit eyes. A suction ring was then applied in 4 rabbit eyes and the IOP was determined manometrically during suction ring application at 350 mmHg vacuum pressure. Finally, in 6 catheterized rabbit eyes the IOP was measured with Tono-Pen XL during suction ring application at suction vacuum from 350 to 650 mmHg, while keeping actual IOP stable at 30 mmHg and 60 mmHg.</p> <p>Results</p> <p>Linear regression analysis revealed that the Tono-pen XL was reliable for IOPs between 10 and 70 mmHg (R²= 0.9855). Direct manometry during suction ring application showed no statistically significant variation of Tono-Pen XL readings when the incanulation manometry intraocular pressure changed from 30 mmHg to 60 mmHg and no statistically significant correlation between suction vacuum and IOP measurements.</p> <p>Conclusion</p> <p>Tono-Pen XL measurements are unreliable during the application of a suction ring on living rabbit eyes even when the actual IOP is forced to be within the validated range of Tono-Pen XL measurements. This inaccuracy is probably related to altered corneal and scleral geometry and stress.</p

A technique to train new oculomotor behavior in patients with central macular scotomas during reading related tasks using scanning laser ophthalmoscopy: immediate functional benefits and gains retention

BACKGROUND: Reading with a central scotoma involves the use of preferred retinal loci (PRLs) that enable both letter resolution and global viewing of word. Spontaneously developed PRLs however often privilege spatial resolution and, as a result, visual span is commonly limited by the position of the scotoma. In this study we designed and performed the pilot trial of a training procedure aimed at modifying oculomotor behavior in subjects with central field loss. We use an additional fixation point which, when combined with the initial PRL, allows the fulfillment of both letter resolution and global viewing of words. METHODS: The training procedure comprises ten training sessions conducted with the scanning laser ophthalmoscope (SLO). Subjects have to read single letters and isolated words varying in length, by combining the use of their initial PRL with the one of an examiner's selected trained retinal locus (TRL). We enrolled five subjects to test for the feasibility of the training technique. They showed stable maculopathy and persisting major reading difficulties despite previous orthoptic rehabilitation. We evaluated ETDRS visual acuity, threshold character size for single letters and isolated words, accuracy for paragraphed text reading and reading strategies before, immediately after SLO training, and three months later. RESULTS: Training the use of multiple PRLs in patients with central field loss is feasible and contributes to adapt oculomotor strategies during reading related tasks. Immediately after SLO training subjects used in combination with their initial PRL the examiner's selected TRL and other newly self-selected PRLs. Training gains were also reflected in ETDRS acuity, threshold character size for words of different lengths and in paragraphed text reading. Interestingly, subjects benefited variously from the training procedure and gains were retained differently as a function of word length. CONCLUSION: We designed a new procedure for training patients with central field loss using scanning laser ophthalmoscopy. Our initial results on the acquisition of newly self-selected PRLs and the development of new oculomotor behaviors suggest that the procedure aiming primarily at developing an examiner's selected TRL might have initiated a more global functional adaptation process

Early Loss of Xist RNA Expression and Inactive X Chromosome Associated Chromatin Modification in Developing Primordial Germ Cells

The inactive X chromosome characteristic of female somatic lineages is reactivated during development of the female germ cell lineage. In mouse, analysis of protein products of X-linked genes and/or transgenes located on the X chromosome has indicated that reactivation occurs after primordial germ cells reach the genital ridges.We present evidence that the epigenetic reprogramming of the inactive X-chromosome is initiated earlier than was previously thought, around the time that primordial germ cells (PGCs) migrate through the hindgut. Specifically, we find that Xist RNA expression, the primary signal for establishment of chromosome silencing, is extinguished in migrating PGCs. This is accompanied by displacement of Polycomb-group repressor proteins Eed and Suz(12), and loss of the inactive X associated histone modification, methylation of histone H3 lysine 27.We conclude that X reactivation in primordial germ cells occurs progressively, initiated by extinction of Xist RNA around the time that germ cells migrate through the hindgut to the genital ridges. The events that we observe are reminiscent of X reactivation of the paternal X chromosome in inner cell mass cells of mouse pre-implantation embryos and suggest a unified model in which execution of the pluripotency program represses Xist RNA thereby triggering progressive reversal of epigenetic silencing of the X chromosome

Evolution from XIST-Independent to XIST-Controlled X-Chromosome Inactivation: Epigenetic Modifications in Distantly Related Mammals

X chromosome inactivation (XCI) is the transcriptional silencing of one X in female mammals, balancing expression of X genes between females (XX) and males (XY). In placental mammals non-coding XIST RNA triggers silencing of one X (Xi) and recruits a characteristic suite of epigenetic modifications, including the histone mark H3K27me3. In marsupials, where XIST is missing, H3K27me3 association seems to have different degrees of stability, depending on cell-types and species. However, the complete suite of histone marks associated with the Xi and their stability throughout cell cycle remain a mystery, as does the evolution of an ancient mammal XCI system. Our extensive immunofluorescence analysis (using antibodies against specific histone modifications) in nuclei of mammals distantly related to human and mouse, revealed a general absence from the mammalian Xi territory of transcription machinery and histone modifications associated with active chromatin. Specific repressive modifications associated with XCI in human and mouse were also observed in elephant (a distantly related placental mammal), as was accumulation of XIST RNA. However, in two marsupial species the Xi either lacked these modifications (H4K20me1), or they were restricted to specific windows of the cell cycle (H3K27me3, H3K9me2). Surprisingly, the marsupial Xi was stably enriched for modifications associated with constitutive heterochromatin in all eukaryotes (H4K20me3, H3K9me3). We propose that marsupial XCI is comparable to a system that evolved in the common therian (marsupial and placental) ancestor. Silent chromatin of the early inactive X was exapted from neighbouring constitutive heterochromatin and, in early placental evolution, was augmented by the rise of XIST and the stable recruitment of specific histone modifications now classically associated with XCI

X Chromosome Inactivation and Differentiation Occur Readily in ES Cells Doubly-Deficient for MacroH2A1 and MacroH2A2

Macrohistones (mH2As) are unusual histone variants found exclusively in vertebrate chromatin. In mice, the H2afy gene encodes two splice variants, mH2A1.1 and mH2A1.2 and a second gene, H2afy2, encodes an additional mH2A2 protein. Both mH2A isoforms have been found enriched on the inactive X chromosome (Xi) in differentiated mammalian female cells, and are incorporated into the chromatin of developmentally-regulated genes. To investigate the functional significance of mH2A isoforms for X chromosome inactivation (XCI), we produced male and female embryonic stem cell (ESC) lines with stably-integrated shRNA constructs that simultaneously target both mH2A1 and mH2A2. Surprisingly, we find that female ESCs deficient for both mH2A1 and mH2A2 readily execute and maintain XCI upon differentiation. Furthermore, male and female mH2A-deficient ESCs proliferate normally under pluripotency culture conditions, and respond to several standard differentiation procedures efficiently. Our results show that XCI can readily proceed with substantially reduced total mH2A content

Xist regulation and function eXplored

X chromosome inactivation (XCI) is a process in mammals that ensures equal transcript levels between males and females by genetic inactivation of one of the two X chromosomes in females. Central to XCI is the long non-coding RNA Xist, which is highly and specifically expressed from the inactive X chromosome. Xist covers the X chromosome in cis and triggers genetic silencing, but its working mechanism remains elusive. Here, we review current knowledge about Xist regulation, structure, function and conservation and speculate on possible mechanisms by which its action is restricted in cis. We also discuss dosage compensation mechanisms other than XCI and how knowledge from invertebrate species may help to provide a better understanding of the mechanisms of mammalian XCI

Analyses of In Vivo Interaction and Mobility of Two Spliceosomal Proteins Using FRAP and BiFC

U1-70K, a U1 snRNP-specific protein, and serine/arginine-rich (SR) proteins are components of the spliceosome and play critical roles in both constitutive and alternative pre-mRNA splicing. However, the mobility properties of U1-70K, its in vivo interaction with SR proteins, and the mobility of the U1-70K-SR protein complex have not been studied in any system. Here, we studied the in vivo interaction of U1-70K with an SR protein (SR45) and the mobility of the U1-70K/SR protein complex using bimolecular fluorescence complementation (BiFC) and fluorescence recovery after photobleaching (FRAP). Our results show that U1-70K exchanges between speckles and the nucleoplasmic pool very rapidly and that this exchange is sensitive to ongoing transcription and phosphorylation. BiFC analyses showed that U1-70K and SR45 interacted primarily in speckles and that this interaction is mediated by the RS1 or RS2 domain of SR45. FRAP analyses showed considerably slower recovery of the SR45/U1-70K complex than either protein alone indicating that SR45/U1-70K complexes remain in the speckles for a longer duration. Furthermore, FRAP analyses with SR45/U1-70K complex in the presence of inhibitors of phosphorylation did not reveal any significant change compared to control cells, suggesting that the mobility of the complex is not affected by the status of protein phosphorylation. These results indicate that U1-70K, like SR splicing factors, moves rapidly in the nucleus ensuring its availability at various sites of splicing. Furthermore, although it appears that U1-70K moves by diffusion its mobility is regulated by phosphorylation and transcription