The Germ Cell Nuclear Proteins hnRNP G-T and RBMY Activate a Testis-Specific Exon

The human testis has almost as high a frequency of alternative splicing events as brain. While not as extensively studied as brain, a few candidate testis-specific splicing regulator proteins have been identified, including the nuclear RNA binding proteins RBMY and hnRNP G-T, which are germ cell-specific versions of the somatically expressed hnRNP G protein and are highly conserved in mammals. The splicing activator protein Tra2β is also highly expressed in the testis and physically interacts with these hnRNP G family proteins. In this study, we identified a novel testis-specific cassette exon TLE4-T within intron 6 of the human transducing-like enhancer of split 4 (TLE4) gene which makes a more transcriptionally repressive TLE4 protein isoform. TLE4-T splicing is normally repressed in somatic cells because of a weak 5′ splice site and surrounding splicing-repressive intronic regions. TLE4-T RNA pulls down Tra2β and hnRNP G proteins which activate its inclusion. The germ cell-specific RBMY and hnRNP G-T proteins were more efficient in stimulating TLE4-T incorporation than somatically expressed hnRNP G protein. Tra2b bound moderately to TLE4-T RNA, but more strongly to upstream sites to potently activate an alternative 3′ splice site normally weakly selected in the testis. Co-expression of Tra2β with either hnRNP G-T or RBMY re-established the normal testis physiological splicing pattern of this exon. Although they can directly bind pre-mRNA sequences around the TLE4-T exon, RBMY and hnRNP G-T function as efficient germ cell-specific splicing co-activators of TLE4-T. Our study indicates a delicate balance between the activity of positive and negative splicing regulators combinatorially controls physiological splicing inclusion of exon TLE4-T and leads to modulation of signalling pathways in the testis. In addition, we identified a high-affinity binding site for hnRNP G-T protein, showing it is also a sequence-specific RNA binding protein

CARACTERISATION DES MECANISMES DE REGULATION DE L'EXPRESSION DE LA PROTEINE SR 9G8 AU NIVEAU DE L'EPISSAGE ALTERNATIF DE SES TRANSCRITS

STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Transcripts dans l'espace et le temps

Les biologistes moléculaires cherchent à comprendre comment fonctionnent les organismes au niveau moléculaire. Le but ultime de ces recherches est d offrir la possibilité de manipuler sans risque des cellules et/ou des organismes afin de combattre des maladies génétiques, d éradiquer les maladies contagieuses ou par example d améliorer les qualités nutritives de l alimentation. Actuellement, la manière la plus précise et pratique de comprendre le fonctionnement d un organisme est d étudier son transcriptome et ses variations dans l espace et le temps. Suivant cette logique, le but de ma thèse de doctorat a été double: (1) estimer l importance de l épissage alternatif qui engendre une diversité des transcripts (2) étudier les transcriptomes de deux organismes modèles : Mus musculus et Drosophila melanogaster, respectivement dans l espace et le temps. Durant ces années de recherche, j ai rassemblé des découvertes intéressantes concernant l expression des gènes et sa régulation. D abord, l épissage alternatif s est avéré être un méchanisme important non seulement en terme de fréquence (des transcripts alternatifs sont générés pour une vaste majorité des gènes, et ce dans de multiples espèces), mais aussi en terme d évolution (l épissage alternatif semble permettre à un gène d évoluer sans conséquences trop négatives pour l organisme). Par ailleurs nous avons prouvé que le niveau d expression de transcripts n est pas en soi synonyme de fonction: il y a en effet une quantité non négligeable d expression neutre, qui doit être prise en compte lors de l assignation d une fonction à un gène, uniquement basée sur la similarité de son profil d expression par rapport à celui d un gène de fonction connue. Enfin, nous avons étudié des séries de puces à ADN appliquées à l embryogenèse de la mouche dans le temps, en utilisant une technique non conventionnelle pour ce type d approche. Nous avons réparti les gènes en différentes classes selon leurs profils d expression. Nous avons pu prouver que ces classes de gènes ont des critères biologiques en commun, ce qui laisse supposer que les gènes inconnus ou mal caractérisés qui tombent dans ces classes sont d interessants points de départ pour de futures recherches. Des découvertes inestimables ont été et seront encore faites en biologie moléculaire grâce à l étude des transcriptomes dans des organismes variés, analysés dans différentes conditions. Cependant, il est devenu clair qu à cause de la présence de nombreuses étapes de régulation après la transcription, dont l épissage alternatif, seule l analyse des protéomes permettra d obtenir une vision complète de la biologie de la cellule.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Metrizamide dissociates nuclear particles containing heterogeneous nuclear RNA

Metrizamide gradients were tested for the possible fractionation of the constitutive units of nuclear particles. Material from 35-55 S monoparticles was indeed distributed along the gradient but rerun experiments, CsCl density determinations, formaldehyde fixation prior to centrifugation suggested that the separation was due to dissociation or (and) action of endogeneous ribonucleases rather than to monoparticle fractionation. That dissociation had indeed occured was confirmed by the study of 60-110 S polyparticles. They were dissociated into ribonucleoproteins rich in phosphoproteins and into free proteins. These products were essentially similar to those obtained after NaCl treatment of the particles though the modes of action of metrizamide and NaCl are likely to be different. The loss of proteins from particles reaches 60-70% and we conclude that metrizamide gradients are not utilizable for the fractionation of the units of nuclear particles

Differential effects of the SR proteins 9G8, SC35, ASF/SF2, and SRp40 on the utilization of the A1 to A5 splicing sites of HIV-1 RNA

Splicing is a crucial step for human immunodeficiency virus, type 1 (HIV-1) multiplication; eight acceptor sites are used in competition to produce the vif, vpu, vpr, nef, env, tat, and rev mRNAs. The effects of SR proteins have only been investigated on a limited number of HIV-1 splicing sites by using small HIV-1 RNA pieces. To understand how SR proteins influence the use of HIV-1 splicing sites, we tested the effects of overproduction of individual SR proteins in HeLa cells on the splicing pattern of an HIV-1 RNA that contained all the splicing sites. The steady state levels of the HIV-1 mRNAs produced were quantified by reverse transcriptase-PCR. For interpretation of the data, transcripts containing one or several of the HIV-1 acceptor sites were spliced in vitro in the presence or the absence of one of the tested SR proteins. Both in vivo and in vitro, acceptor sites A2 and A3 were found to be strongly and specifically regulated by SR proteins. ASF/SF2 strongly activates site A2 and to a lesser extent site A1. As a result, upon ASF/SF2 overexpression, the vpr mRNA steady state level is specifically increased. SC35 and SRp40, but not 9G8, strongly activate site A3, and their overexpression ex vivo induces a dramatic accumulation of the tat mRNA, to the detriment of most of the other viral mRNAs. Here we showed by Western blot analysis that the Nef protein synthesis is strongly decreased by overexpression of SC35, SRp40, and ASF/SF2. Finally, activation by ASF/SF2 and 9G8 was found to be independent of the RS domain. This is the first investigation of the effects of variations of individual SR protein concentrations that is performed ex vivo on an RNA containing a complex set of splicing sites

The CD44 alternative v9 exon contains a splicing enhancer responsive to the SR proteins 9G8, ASF/SF2, and SRp20

The CD44 gene alternative exons v8, v9, and v10 are frequently spliced as a block by epithelial cells. By transfecting minigenes containing only one of these alternative exons, we show that splicing of each of them is under cell type-specific control. By using minigenes carrying short block mutations within exons v8 and v9, we detected a candidate exon splicing enhancer in each of these exons. These candidates activated splicing in vitro of a heterologous transcript and are thus true exon splicing enhancers. We analyzed further a v9 exon splicing enhancer covering approximately 30 nucleotides. This enhancer can be UV cross-linked to SR proteins of 35 and 20 kDa in HeLa nuclear extract. By using individual recombinant SR proteins for UV cross-linking in S100 extract, these proteins were identified as 9G8, ASF/SF2, and SRp20. S100 complementation studies using recombinant 9G8, ASF/SF2, and SRp20 showed that all three proteins can activate splicing in vitro of a heterologous exon containing the v9 enhancer; the strongest activation was obtained with 9G8. Progressive truncation of the 30-nucleotide enhancer leads to a progressive decrease in splicing activation. We propose that 9G8, ASF/SF2, SRp20, and possibly other non-SR proteins cooperate in vivo to activate v9 exon splicing