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Broad specificity of SR (serine/arginine) proteins in the regulation of alternative splicing of pre-messenger RNA.

Author(s) : Bourgeois C, Lejeune F, Stevenin J,
Journal : Prog Nucleic Acid Res Mol Biol
Alternative splicing of pre-messenger RNA (pre-mRNA) is a highly regulated process that allows expansion of the potential of expression of the genome in higher eukaryotes and involves many factors. Among them, the family of the serine- and arginine-rich proteins (SR proteins) plays a pivotal role: it has essential functions during spliceosome assembly and also interacts with RNA regulatory sequences on the pre-mRNA as well as with multiple cofactors. Collectively, SR proteins, because of their capacity to recognize multiple RNA sequences with a broad specificity, are at the heart of the regulation pathways that lead to the choice of alternative splice sites. Moreover, a growing body ofevidence shows that the mechanisms of splicing regulation are not limited to thebasic involvement of cis- and trans-acting factors at the pre-mRNA level, but result from intricate pathways, initiated sometimes by stimuli that are externalto the cell and integrate SR proteins (and other factors) within an extremely sophisticated network of molecular machines associated with one another. This review focuses on the molecular aspects of the functions of SR proteins. In particular, we discuss the different ways in which SR proteins manage to achievea high level of specificity in splicing regulation, even though they are also involved in the constitutive reaction.

Defining an N-terminal activation domain of the orphan nuclear receptor Nurr1.

Author(s) : Nordzell M, Aarnisalo P, Benoit G, Castro D, Perlmann T,
Journal : Biochem Biophys Res Commun
Nurr1 is an orphan nuclear receptor essential for the development of midbrain dopaminergic neurons. Activation of Nurr1 depends on two so-called activation functions (AFs) situated in the N- and C-terminal regions, respectively. The region important for activation within the C-terminal domain has been shown to promote activation in a highly cell-type specific fashion in the absence of added exogenous ligands. In contrast, the region in the N-terminal domain (AF1) has been much less characterized. Here we mutagenized the N-terminal domain of Nurr1to define essential activation regions. The results identified a short core activation region localized close to the N-terminus of Nurr1. In addition, cell-type specific influences by other signaling pathways were analyzed by mutagenesis of specific conserved phosphorylation sites. The results indicate that mitogen-activated protein kinase activity (MAPK) positively influences Nurr1 AF1-dependent transcriptional activation via a conserved phosphorylation site outside the core activation region.

Digging deep into the pockets of orphan nuclear receptors: insights from structural studies.

Author(s) : Benoit G, Malewicz M, Perlmann T,
Journal : Trends Cell Biol
Nuclear receptors comprise a large family of proteins that shares a common structure and mechanism of action. Members of this family, first cloned 20 yearsago, are regulated by small lipophilic signaling molecules such as steroid hormones, retinoids and thyroid hormone. More recently, the characterization of proteins that resemble nuclear receptors (referred to as orphan receptors) has resulted in the determination of novel signaling pathways. However, many orphan-receptor ligands remain unidentified, and recent structural studies of the binding domains for orphan-receptor ligands suggest that not all of these receptors use ligand binding in a classical way. Notably, it is now evident thatsome orphan receptors lack the capacity for ligand binding, which suggests that they are regulated by alternative, ligand-independent mechanisms.

Global transcription analysis of immature avian erythrocytic progenitors: from self-renewal to differentiation.

Author(s) : Damiola F, Keime C, Gonin-Giraud S, Dazy S, Gandrillon O,
Journal : Oncogene
The molecular mechanisms regulating the cell fate decision between self-renewal and differentiation/apoptosis in stem and progenitor cells are poorly understood. Here, we report the first comprehensive identification of genes potentially involved in the switch from self-renewal toward differentiation of primary, non-immortalized erythroid avian progenitor cells (T2EC cells). We used the Serial Analysis of Gene Expression (SAGE) technique in order to identify and quantify the genome fraction functionally active in a self-renewing versus a differentiating cell population. We generated two SAGE libraries and sequenced atotal of 37,589 tags, thereby obtaining the first transcriptional profile characterization of a chicken cell. Tag identification was performed using a newrelational database (Identitag) developed in the laboratory, which allowed a highly satisfactory level of identification. Among 123 differentially expressed genes, 11 were investigated further and for nine of them the differential expression was subsequently confirmed by real-time PCR. The comparison of tag abundance between the two libraries revealed that only a small fraction of transcripts was differentially expressed. The analysis of their functions argue against a prominent role for a master switch in T2EC cells decision-making, but are in favor of a critical role for coordinated small variations in a relativelysmall number of genes that can lead to essential cellular identity changes.

Identification of a novel co-regulator interaction surface on the ligand binding domain of Nurr1 using NMR footprinting.

Author(s) : Codina A, Benoit G, Gooch J, Neuhaus D, Perlmann T, Schwabe J,
Journal : J Biol Chem
The nuclear receptor Nurr1 is a transcription factor essential for the development of midbrain dopaminergic neurons in vertebrates. Recent crystal structures of the Nurr1 ligand binding domain (LBD) and the Drosophila orthologue dHR38 revealed that, although these receptors share the classical LBD architecture, they lack a ligand binding cavity. This volume is instead filled with bulky hydrophobic side chains. Furthermore the "canonical" non-polar co-regulator binding groove is filled with polar side chains; thus, the regulation of transcription by this sub-family of nuclear receptor LBDs may be mediated by some other interaction surface on the LBD. We report here the identification of a novel co-regulator interface on the LBD of Nurr1. We used anNMR footprinting strategy that facilitates the identification of an interaction surface without the need of a full assignment. We found that non-polar peptides derived from the co-repressors SMRT and NCoR bind to a hydrophobic patch on the LBD of Nurr1. This binding surface involves a groove between helices 11 and 12. Mutations in this site abolish activation by the Nurr1 LBD. These findings give insight into the unique mechanism of action of this class of nuclear receptors.

Identitag, a relational database for SAGE tag identification and interspecies comparison of SAGE libraries.

Author(s) : Keime C, Damiola F, Mouchiroud D, Duret L, Gandrillon O,
Journal : BMC Bioinformatics
BACKGROUND: Serial Analysis of Gene Expression (SAGE) is a method of large-scalegene expression analysis that has the potential to generate the full list of mRNAs present within a cell population at a given time and their frequency. An essential step in SAGE library analysis is the unambiguous assignment of each 14bp tag to the transcript from which it was derived. This process, called tag-to-gene mapping, represents a step that has to be improved in the analysis of SAGE libraries. Indeed, the existing web sites providing correspondence between tags and transcripts do not concern all species for which numerous EST and cDNA have already been sequenced. RESULTS: This is the reason why we designed and implemented a freely available tool called Identitag for tag identification thatcan be used in any species for which transcript sequences are available. Identitag is based on a relational database structure in order to allow rapid and easy storage and updating of data and, most importantly, in order to be able to precisely define identification parameters. This structure can be seen like three interconnected modules : the first one stores virtual tags extracted from a given list of transcript sequences, the second stores experimental tags observed in SAGE experiments, and the third allows the annotation of the transcript sequences used for virtual tag extraction. It therefore connects an observed tag to a virtual tag and to the sequence it comes from, and then to its functional annotation when available. Databases made from different species can be connected according to orthology relationship thus allowing the comparison of SAGE libraries between species. We successfully used Identitag to identify tags from our chicken SAGE libraries and for chicken to human SAGE tags interspecies comparison. Identitag sources are freely available on web site. CONCLUSIONS: Identitag is a flexible and powerful tool for tag identification in any single species and for interspecies comparison of SAGE libraries. It opens the way to comparative transcriptomic analysis, an emerging branch of biology.

Inhibition of HIV-1 replication by cell-penetrating peptides binding Rev.

Author(s) : Roisin A, Robin J, Dereuddre-Bosquet N, Vitte A, Dormont D, Clayette P, Jalinot P,
Journal : J Biol Chem
New therapeutic agents able to block HIV-1 replication are eagerly sought after to increase the possibilities of treatment of resistant viral strains. In this report, we describe a rational strategy to identify small peptide sequences owning the dual property of penetrating within lymphocytes and of binding to a protein target. Such sequences were identified for two important HIV-1 regulatory proteins, Tat and Rev. Their association to a stabilizing domain consisting of human small ubiquitin-related modifier-1 (SUMO-1) allowed the generation of small proteins named SUMO-1 heptapeptide protein transduction domain for binding Tat (SHPT) and SUMO-1 heptapeptide protein transduction domain for binding Rev (SHPR), which are stable and efficiently penetrate within primary lymphocytes. Analysis of the antiviral activity of these proteins showed that one SHPR is active in both primary lymphocytes and macrophages, whereas one SHPT is active only in the latter cells. These proteins may represent prototypes of new therapeutic agents targeting the crucial functions exerted by both viral regulatory factors.

Regulation of R7 and R8 differentiation by the spalt genes.

Author(s) : Domingos P, Brown S, Barrio R, Ratnakumar K, Frankfort B, Mardon G, Steller H, Mollereau B,
Journal : Dev Biol
Photoreceptor development begins in the larval eye imaginal disc, where eight distinct photoreceptor cells (R1-R8) are sequentially recruited into each of thedeveloping ommatidial clusters. Final photoreceptor differentiation, including rhabdomere formation and rhodopsin expression, is completed during pupal life. During pupation, spalt was previously proposed to promote R7 and R8 terminal differentiation. Here we show that spalt is required for proper R7 differentiation during the third instar larval stage since the expression of several R7 larval markers (prospero, enhancer of split mdelta0.5, and runt) is lost in spalt mutant clones. In R8, spalt is not required for cell specificationor differentiation in the larval disc but promotes terminal differentiation during pupation. We show that spalt is necessary for senseless expression in R8 and sufficient to induce ectopic senseless in R1-R6 during pupation. Moreover, misexpression of spalt or senseless is sufficient to induce ectopic rhodopsin 6 expression and partial suppression of rhodopsin 1. We demonstrate that spalt andsenseless are part of a genetic network, which regulates rhodopsin 6 and rhodopsin 1. Taken together, our results suggest that while spalt is required for R7 differentiation during larval stages, spalt and senseless promote terminal R8differentiation during pupal stages, including the regulation of rhodopsin expression.

Spalt transcription factors are required for R3/R4 specification and establishment of planar cell polarity in the Drosophila eye.

Author(s) : Domingos P, Mlodzik M, Mendes C, Brown S, Steller H, Mollereau B,
Journal : Development
The establishment of planar cell polarity in the Drosophila eye requires correctspecification of the R3/R4 pair of photoreceptor cells. In response to a polarizing factor, Frizzled signaling specifies R3 and induces Delta, which activates Notch in the neighboring cell, specifying it as R4. Here, we show thatthe spalt zinc-finger transcription factors (spalt major and spalt-related) are part of the molecular mechanisms regulating R3/R4 specification and planar cell polarity establishment. In mosaic analysis, we find that the spalt genes are specifically required in R3 for the establishment of correct ommatidial polarity. In addition, we show that spalt genes are required for proper localization of Flamingo in the equatorial side of R3 and R4, and for the upregulation of Delta in R3. These requirements are very similar to those of frizzled during R3/R4 specification. We show that spalt genes are required cell-autonomously for the expression of seven-up in R3 and R4, and that seven-up is downstream of spalt genes in the genetic hierarchy of R3/R4 specification. Thus, spalt and seven-up are necessary for the correct interpretation of the Frizzled-mediated polarity signal in R3. Finally, we show that, posterior to row seven, seven-up represses spalt in R3/R4 in order to maintain the R3/R4 identity and to inhibit the transformation of these cells to the R7 cell fate.