A fototerapia com laser em baixa intensidade (FTLBI) é capaz de aumentar o metabolismo celular, o que poderia influenciar na diferenciação ósseo/odontogênica das células-tronco da polpa dentária humada (hDPSCs). O PDGF e o BMP-2 são fatores de crescimento envolvidos na dentinogênese e na reparação tecidual. O PDGF tem papel importante durante o desenvolvimento embrionário, na proliferação e migração celular e na angiogênese, enquanto o BMP-2 está fortemente associado à diferenciação celular em tecidos mineralizados, como o osso e a dentina. Sendo assim, o objetivo do estudo foi analisar os efeitos da FTLBI e dos fatores de crescimento (PDGF-BB ou BMP-2), isolados ou em associação, na diferenciação ósseo/odontogênica das hDPSCs. Para o estudo hDPSCs foram cultivadas em meio regular (G1) e irradiadas (G2), meio mineralizante (G3) e irradiadas (G4), meio mineralizante contendo PDGF-BB (G5) e irradiadas (G6), meio mineralizante contendo BMP-2 (G7) e irradiadas (G8). Para os grupos irradiados, a FTLBI foi realizada no modo pontual e em contato, com um laser de diodo semi-condutor, com área de feixe de 0,028cm2 e comprimento de onda 660nm (InGaAlP-vermelho), utilizando-se os seguintes parâmetros: potência de 20mW, densidade de energia de 5J/cm2, tempo de irradiação de 7 segundos por ponto e 0,14J de energia por ponto. A expressão dos genes relacionados à diferenciação ósseo/odontogênica (DSPP, DMP-1 e OCN) através do PCR quantitativo em tempo real (qRT-PCR), a atividade da fosfatase alcalina e os depósitos de cálcio foram analisados em 3, 7 e 14 dias. Os dados obtidos foram comparados pelo teste ANOVA complementado pelo teste de Tukey (p<0,05). As culturas tratadas com meio mineralizante contendo BMP-2 e irradiadas (G8) foram as que mostraram os maiores índices de diferenciação ósseo/odontogênica nos testes realizados. As expressões de DSPP, OCN e DMP-1, ao menos em 14 dias, foram significantemente maiores no G8 que nos demais grupos experimentais, exceto os grupos G3 e G7. Estes grupos apresentaram expressões de DSPP e OCN semelhantes às do G8 em 14 dias. A maior atividade de ALP foi observada no G8 em 3 dias e a menor no mesmo grupo aos 14 dias. A maior quantidade de depósitos de cálcio também foi encontrada no G8 em 14 dias. A associação de FTLBI e BMP-2 se mostrou capaz de induzir a diferenciação ósseo/odontogênica em células-tronco de polpa dentária humana de forma mais marcante que as demais terapias isoladas ou associadas estudadas. Portanto, o uso de uma terapia associando FTLBI e BMP-2 poderia ser de relevância para o restabelecimento da fisiologia pulpar quando aplicada em casos de exposição deste tecido, uma vez que poderia favorecer a diferenciação das células indiferenciadas da polpa dentária. / Laser phototherapy (LPT) is able to increase cellular metabolism, which in turn could influence the odontogenic differentiation of dental pulp stem cells (hDPSCs). PDGF and BMP-2 are growth factors involved in dentinogenesis and tissue repair. PDGF plays a role in embryonic development, cell proliferation, cell migration, and angiogenesis, whereas BMP-2 is strongly associated with cell differentiation in mineralized tissues such as bone and dentin. The aim of this study was to analyze the effects of LPT and the growth factors PDGF-BB and BMP-2 combined or not on the odontogenic differentiation of hDPSCs. These cells were grown in regular medium (G1) and irradiated (G2), mineralizing medium (G3) and irradiated (G4), mineralizing medium containing PDGF-BB (G5) and irradiated (G6), mineralizing medium containing BMP-2 (G7) and irradiated (G8). For irradiated groups, LPT was performed in punctual and contact mode with a semiconductor diode laser, with a beam spot area of 0.028 cm2 and wavelength of 660nm (InGaAlP-visible red), using the following parameters: power of 20mW, energy density of 5J/cm2 and irradiation time of 7 seconds per point (0,14 J per point). Differentiation was assessed by the following analysis: expression of genes related to odontogenic differentiation (DSPP, DMP-1 and OCN) using quantitative real time PCR (qRT-PCR); alkaline phosphatase activity and calcium deposition using alizarin red staining in 3, 7 and 14 days. Data were compared by ANOVA and Tukey´s test (p<0.05). The cultures treated with mineralizing medium containing BMP-2 and irradiated (G8) showed the highest rate of odontogenic differentiation. The expressions of DSPP, DMP-1 and OCN genes, at least in 14 days, were significantly higher in G8 compared to all other groups, except for the groups G3 and G7. These groups showed similar expressions of DSPP and OCN than G8 in 14 days. G8 showed the highest ALP activity in 3 days and the lowest in 14 days compared to all other groups. The largest amount of calcium deposits was observed in G8 in 14 days. The most striking feature on induction of odontogenic differentiation of hDPSCs was observed when LPT was applied in association with BMP-2. Therefore, the use of a combined LPT and BMP-2 therapy could be of relevance for the re-establishment of pulp physiology when applied in cases of dental pulp exposure by promoting the differentiation of hDPSCs.

Le stress oxydant joue un rôle majeur dans le déclenchement et le développement de la sclérodermie systémique (ScS). Nous avons mis au point un modèle murin où la maladie est déclenchée par divers types de stress oxydant, puis nous avons exploré les différentes voies d'activation des fibroblastes sous l'effet des formes réactives de l'oxygène, afin de déterminer d'éventuelles cibles thérapeutiques. Pour apprécier les effets d’un stress oxydant chronique, des solutions contenant différents oxydants ont été injectées dans la peau de souris BALB/c et BALB/c SCID. Les solutions contenant le radical hydroxyl OH° ou HOCl ont induit une maladie caractérisée, comme la ScS diffuse, par une fibrose cutanée et viscérale, et des auto-anticorps anti-ADN topoisomérase-1. Les sérums de ces souris contenaient de grandes quantités de dérivés oxydés des protéines et induisaient la prolifération des fibroblastes et la production de formes réactives de l’oxygène par les cellules endothéliales. Une fibrose pulmonaire de moindre importance était induite chez les souris BALB/c SCID. Grâce à ce nouveau modèle murin de SSc, nous avons démontré que le stress oxydant était directement responsable des anomalies observées dans les fibroblastes, les cellules endothéliales et le système immunitaire. Nous avons ensuite utilisé ce modèle pour analyser les voies d’activation fibroblastique dans la ScS. Dans les fibroblastes des souris exposées à HOCl, on observe une dérégulation des voies des récepteurs Notch, des récepteurs aux cannabinoïdes, et des récepteurs au PDGF. On observe les mêmes dérégulations ex vivo dans les fibroblastes de patients atteints de SSc diffuse. Nous avons ainsi observé une amélioration clinique significative chez les souris sclérodermiques traitées avec un inhibiteur de l’activation de Notch, avec un agoniste des récepteurs aux cannabinoïdes, et avec des inhibiteurs de tyrosine-kinase ciblant le récepteur au PDGF. Puisque les fibroblastes sclérodermiques ont un phénotype activé et produisent de forts taux de formes réactives de l’oxygène, nous avons enfin mis à profit cette particularité pour induire l’apoptose sélective de ces cellules dans le derme des souris. Le trioxyde d’arsenic, molécule cytotoxique utilisée en thérapeutique humaine, augmente la production cellulaire de formes réactives de l’oxygène au-delà d’un seuil létal et induit ainsi l’apoptose des fibroblastes sclérodermiques. L’utilisation in vivo de cette molécule dans notre modèle murin prévient la fibrose cutanée et viscérale, et les anomalies endothéliales. Le trioxyde d’arsenic a un effet comparable dans le modèle murin de ScS associée à la réaction du greffon contre l’hôte en détruisant les lymphocytes T CD4+ alloréactifs activés et les cellules dendritiques plasmacytoïdes responsables de l’activation du système immunitaire. Les formes réactives de l’oxygène sont donc impliquées dans l’induction des lésions observées au cours de la ScS. Dans notre modèle, le rôle du système immunitaire intervient dans l'auto-entretien et l’extension systémique de la maladie. Le stress oxydant contribue à la dérégulation de diverses voies de signalisation dont les voies des récepteurs Notch, des récepteurs aux cannabinoïdes et du PDGF dans les fibroblastes. La modulation de ces voies permet d’obtenir une amélioration clinique chez les souris sclérodermiques, tout comme l’utilisation du trioxyde d’arsenic qui entraîne la délétion spécifique des fibroblastes sclérodermiques surpoduisant des formes réactives de l’oxygène. Le trioxyde d’arsenic montre également une efficacité intéressante dans le modèle de sclérodermie associée à la maladie du greffon contre l’hôte via la délétion des lymphocytes T CD4+ alloréactifs. / We defend the thesis that the oxidative stress plays a major role in the initiation and the development of systemic sclerosis. To demonstrate this thesis, we designed an original mouse model: BALB/c and BALB/SCID mice were injected intra-dermally with prooxidative agents, bleomycin or PBS for 6 weeks. Hypochlorite and hydroxyl radicals induced cutaneous and lung fibrosis in BALB/c mice, in association with anti-DNA topoisomerase-1 auto-antibodies that characterize human diffuse systemic sclerosis. Pulmonary fibrosis was less extensive in BALB/c SCID mice submitted to the same protocol. In this model of HOCl-induced systemic sclerosis, cutaneous fibroblasts display a hyperactivated phenotype that prompted us to investigate several pathways of cellular activation. The NOTCH pathway and the PGDF-receptor pathways were found upregulated in the skin of HOCl-mice. DAPT (a gamma secretase inhibitor that prevents NOTCH cleavage), Sunitinib (an inhibitor of PGDF-receptor phosphorylation), and WIN-55,212, an agonist of the cannabinoid receptors 1 and 2, dramatically improved the clinical, histological and biological signs of systemic sclerosis in the HOCl model.In our model as in patients with SSc, activated fibroblasts produce reactive oxygen species that exert an autocrine effect on their own proliferation and collagen synthesis. By analogy with tumor cells that undergo apoptosis upon cytotoxic treatment that triggers an oxidative stress beyond a lethal threshold, we showed that activated fibroblasts can be selectively killed by the cytotoxic molecule arsenic trioxide (As2O3) that generates intracellular ROS. In the mouse model of sclerodermatous-graft versus host disease (Scl-GVHD), daily intra-peritoneal injections of As2O3 abrogated the clinical symptoms (diarrhea, alopecia, vasculitis, fibrosis of the skin and visceral organs) and specifically induced the apoptosis of activated CD4+ T cells and plasmacytoid dendritic cells. Those data provide a rationale for the evaluation of As2O3 in the management of patients affected by systemic sclerosis or chronic GVHD.

<p>Stimulation of the T cell receptor (TCR) induces tyrosine phosphorylation of numerous intracellular proteins, leading to activation of the interleukin-2 (IL-2) gene in T lymphocytes. Shb is a ubiquitously expressed adapter protein, with the ability to associate with the T cell receptor and several signaling proteins in T cells, including: the TCR ζ-chain, LAT, PLC-γ1, Vav, SLP-76 and Gads. Jurkat T cells expressing Shb with a mutation in the SH2 domain, exhibited reduced phosphorylation of several proteins and abolished activation of the MAP kinases ERK1, ERK2 and JNK, upon CD3 stimulation. The TCR induced Ca²⁺ response in these cells was abolished, together with the activation of the IL-2 promoter via the transcription factor NFAT. Consequently, IL-2 production was also perturbed in these cells, compared to normal Jurkat T cells. Shb was also seen to associate with the β and γ chains of the IL-2 receptor, upon IL-2 stimulation, in T and NK cells. This association occurred between the Shb SH2 domain and Tyr-510 of the IL-2R β chain. The proline-rich domains of Shb were found to associate with the tyrosine kinases JAK1 and JAK3, which are important for STAT-mediated proliferation of T and NK cells upon IL-2 stimulation. Shb was also found to be involved in IL-2 mediated regulation of apoptosis. These findings indicate a dual role for Shb in T cells, where Shb is involved in both T cell receptor and IL-2 receptor signaling. </p><p>A Shb homologue, Shf was identified, and seen to associate with the PDGF-α-receptor. Shf shares high sequence homology with Shb and a Shd (also of the Shb family) in the SH2 domain and in four motifs containing putative tyrosine phosphorylation sites. When Shf was overexpressed in fibroblasts, these cells displayed significantly lower rates of apoptosis than control cells in the presence of PDGF-AA. These findings suggest a role for the novel adapter Shf in PDGF-receptor signaling and regulation of apoptosis.</p>

Stimulation of the T cell receptor (TCR) induces tyrosine phosphorylation of numerous intracellular proteins, leading to activation of the interleukin-2 (IL-2) gene in T lymphocytes. Shb is a ubiquitously expressed adapter protein, with the ability to associate with the T cell receptor and several signaling proteins in T cells, including: the TCR ζ-chain, LAT, PLC-γ1, Vav, SLP-76 and Gads. Jurkat T cells expressing Shb with a mutation in the SH2 domain, exhibited reduced phosphorylation of several proteins and abolished activation of the MAP kinases ERK1, ERK2 and JNK, upon CD3 stimulation. The TCR induced Ca2+ response in these cells was abolished, together with the activation of the IL-2 promoter via the transcription factor NFAT. Consequently, IL-2 production was also perturbed in these cells, compared to normal Jurkat T cells. Shb was also seen to associate with the β and γ chains of the IL-2 receptor, upon IL-2 stimulation, in T and NK cells. This association occurred between the Shb SH2 domain and Tyr-510 of the IL-2R β chain. The proline-rich domains of Shb were found to associate with the tyrosine kinases JAK1 and JAK3, which are important for STAT-mediated proliferation of T and NK cells upon IL-2 stimulation. Shb was also found to be involved in IL-2 mediated regulation of apoptosis. These findings indicate a dual role for Shb in T cells, where Shb is involved in both T cell receptor and IL-2 receptor signaling. A Shb homologue, Shf was identified, and seen to associate with the PDGF-α-receptor. Shf shares high sequence homology with Shb and a Shd (also of the Shb family) in the SH2 domain and in four motifs containing putative tyrosine phosphorylation sites. When Shf was overexpressed in fibroblasts, these cells displayed significantly lower rates of apoptosis than control cells in the presence of PDGF-AA. These findings suggest a role for the novel adapter Shf in PDGF-receptor signaling and regulation of apoptosis.

Gliomas are the most common primary tumors of the central nervous system believed to arise from glial cells. Invasive growth and inherent propensity for malignant progression make gliomas incurable despite extensive treatment. I have developed a life-like orthotopic glioma model and used this and other in vivo models to study basic mechanisms of glioma development and treatment. Previous studies had indicated that experimental gliomas could arise from glial stem cells and astrocytes. The present thesis describes the making and characterization of a novel mouse model, Ctv-a, where gliomas are induced from oligodendrocyte progenitor cells (OPCs). Our study shows that OPCs have the capacity to give rise to gliomas and suggests in light of previous data that the differentiation state of the cell of origin affects tumor malignancy. CDKN2A encodes p16INK4a and p14ARF (p19Arf in mouse) commonly inactivated in malignant glioma. Their roles in experimental glioma have been extensively studied and both proteins have tumor suppressor functions in glial stem cells and astrocytes. Here, we demonstrate that p19Arf only could suppress gliomagenesis in OPCs while p16Ink4a had no tumor suppressive effect. Functional DNA repair is pivotal for maintaining genome integrity, eliminating unsalvageable cells and inhibiting tumorigenesis. We have studied how RAD51, a central protein of homology-directed repair, affected experimental glioma development and have found that expression of RAD51 may protect against genomic instability and tumor development. Angiogenesis, the formation of new blood vessels from pre-existing ones, is a central feature of malignant progression in glioma. Antiangiogenic treatment by inhibition of vascular endothelial growth factor receptor signaling is used in the clinic for treatment of some cancers. We have investigated the effect of an alternative antiangiogenic protein, histidine-rich glycoprotein (HRG), on glioma development and found that HRG could inhibit the formation of malignant gliomas and completely prevent the formation of glioblastoma.

L’athérosclérose est une pathologie cardiovasculaire chronique impliquant de nombreux acteurs. Les cellules du muscle lisse vasculaire (CMLV) jouent un important rôle dans la pathogénicité. Lors de la formation des plaques athérosclérotiques, ces cellules entraînent l’augmentation de la taille de l’athérome, accentuent la formation du chapeau fibreux et à long terme, contribuent à l’instabilité de la plaque. Dans cette étude, nous nous sommes intéressés à l’impact d’ARF6 sur les cellules du muscle lisse vasculaire et ses implications pathologiques dans l’athérosclérose. Les ARF sont des GTPases agissant comme interrupteurs moléculaires dans divers processus physiologiques tels que le trafic vésiculaire intracellulaire et le remodelage des lipides membranaires. ARF6 est importante pour la prolifération et la migration cellulaire des CMLV, deux phénomènes importants dans le développement de l’athérosclérose. Nous émettons donc l’hypothèse que la GTPase ARF6 est impliquée dans la progression de l’athérosclérose. En premier lieu, nous avons étudié l’effet de la GTPase dans le phénomène de l’invasion cellulaire. Dans l’athérosclérose, plusieurs facteurs environnementaux influencent l’invasion des CMLV. Nous avons voulu vérifier l’effet d’ARF6 sur l’invasion des CMLV médiée par le facteur de croissance dérivé des plaquettes (PDGF-BB) et l’angiotensine II (Ang II). Dans un modèle humain, l’invasion était diminuée en l’absence d’ARF6. Nous avons démontré que ce mécanisme résultait d’un effet d’ARF6 sur l’activité de la métalloprotéinase matricielle MMP14. En second lieu, nous avons voulu évaluer l’effet d’ARF6 dans un modèle in vivo d’athérosclérose. En utilisant un modèle accéléré d’athérosclérose inductible, nous avons inhibé ARF6 dans les cellules du muscle lisse. Après dix semaines de diète riche en gras, nous avons observé une diminution de la taille des lésions athérosclérotiques dans les souris ARF6 KO, accompagnée d’une réduction de l’expression des facteurs pro-inflammatoires tels qu’IL-6. Dans un modèle in vitro, l’absence d’ARF6 réduisait l’absorption lipidique en agissant sur l’expression des transporteurs. De plus, ARF6 régulait des voies de signalisation impliquées dans l’inflammation. En somme, nous avons démontré l’importance d’ARF6 dans la modulation pathologique des CMLV dans l’athérosclérose. Ainsi, ARF6 contribue à la pathogénicité des CMLV en modulant leur invasion cellulaire tout en jouant un rôle pro-inflammatoire. / Atherosclerosis is a chronic cardiovascular disease characterized by an accumulation of lipids, followed by the infiltration of macrophages and vascular smooth muscle cells (VSMC). VSMC are responsible for the increase of lesion size, the formation of a fibrous cap, and eventually contributing to the plaque instability. In this study, we were interested in the role of ARF6 in the vascular smooth muscle cells and its pathological implications in atherosclerosis. ARF are a family of GTPases that act as molecular switches and are involved in diverse physiological mechanisms, such as vesicular traffic and membrane lipid transformation. In VSMC, ARF6 is important for cell proliferation and migration, two processes involved in atherosclerosis. We therefore hypothesize that the GTPase ARF6 is involved in the development of atherosclerosis through its impact on VSMC. First, we studied the role of ARF6 in the mechanism of cell invasion. In atherosclerosis, multiple environmental factors affect VSMC invasion. We verified the impact of ARF6 on platelet-derived growth factor (PDGF-BB) and angiotensin II (Ang II)-mediated invasion. Using a human model, we observed a reduction of invasion in the absence of ARF6. We have demonstrated that this mechanism is due to the effect of ARF6 on the activity of the matrix metalloproteinase MMP14. Second, we wanted to verify the role of ARF6 in atherosclerosis in an in vivo model. Using an accelerated inducible atherosclerosis model, we inhibited ARF6 in smooth muscle cells. After ten weeks of high-fat diet, we observed a reduction in the size of atherosclerotic lesions in ARF6 KO mice. This reduction was accompanied by a decrease in the expression of proinflammatory factors. In our in vitro model, ARF6 depletion reduced lipid uptake by downregulating the lipidic transporter expression. Also, ARF6 was responsible to activate inflammation signaling pathways. In summary, we have demonstrated the impact of ARF6 in the pathological modulation of VSMC in atherosclerosis. Indeed, ARF6 contributes to the pathogenicity of VSMC through its ability to modulate cell invasion and induce proinflammatory actions.

Pre-mRNA splicing is an important level in posttranscriptional gene regulation that is essential for accurate protein synthesis and generating protein diversity. The abundance of cryptic splice sites and long intronic DNA sequences makes their splicing a complex one. The identification of correct exons and introns needs additional information in the form of splicing regulatory elements (SREs) along with canonical splice signals. The interplay among these SREs and the trans factors (which bind to SREs) gives the identity to introns and exons which in turn leads to precise pre-mRNA splicing. Previous studies from our laboratory showed, that when expressed in mammalian cells from an expression vector, PDGF-B ORF was re-spliced at 4/5 exon junction with the downstream SV40 splice acceptor site in the vector. However, deletion of the 66-nt PDGF-B 3’ UTR region resulted in about 25% reduction in re-splicing. Sequence analysis of this region revealed presence of binding sites for splicing factors ASF/SF2 and SRp55, and an AU-rich element (ARE), mutation each of which affected re-splicing partially. In mammals, AREs are commonly found in the 3’UTR of mRNAs encoding proteins involved in diverse functions and are involved in selective mRNA degradation. Several ARE binding proteins are crucial for ARE’s function. Since mutation of the single ARE in the 3’UTR region altered the re-splicing efficiency, the role of AU-rich elements and ARE-binding proteins (AU-BPs) in modulation of splicing was investigated using siRNAs against AU-BPs, BRF1, hnRNPD, HuR, GAPDH and TTP. Down regulation of expression of these factors indeed affected the level of re-spliced product. We have studied the interactions between the full-length splicing factors (U1-70K and U2AF35) and the AU-BPs (BRF1, hnRNPD and HuR) as well as among the AU-BPs using three different assay methods: Yeast-two hybrid, co-immunoprecipitation and pull down assays. Our study has revealed that the BRF1 interacts with U1-70K and U2AF35 as well as the other AU-BPs hnRNPD and HuR but with different affinities. We have also analyzed the ability of AU-BPs to interact with SR proteins SRp20 and 9G8. We did find strong interaction of BRF1 with SRp20 and 9G8. Generation of a large number of nested deletion mutants of all the proteins allowed us to identify the interaction regions on the surface of BRF1, U1-70K, hnRNPD, U2AF35 and HuR. The results of Y2H analyses were further confirmed by pull down assay using purified interacting regions. It was found that a single region from aa 181-254 in BRF1 interacts with multiple partners i.e., splicing factors and the AU-BP hnRNPD. However, the RNA-binding zinc-finger domain from residue 120-181 independently interacts with HuR. Further, the multiple protein interacting region (MPIR) (aa 181-254) in BRF1 exhibits different affinities towards its interacting partners with that for U1-70K and hnRNPD being stronger than that for U2AF35 and HuR. This observation suggests that BRF1 activity can be modulated by interaction with different partners at different sites. U1-70K interacted only with BRF1 among the proteins tested in this study and this interaction appears to be RNA independent .This could have implications in splice site selection and RNA stability since BRF1 has been shown to promote RNA degradation. While the Arg/Glu-rich C-terminal region in U1-70K is sufficient for its interaction with BRF1, U2AF35 requires both the zinc-finger 2 and the arg/Gly/Ser-rich C-terminal regions for its association with BRF1. hnRNPD also interacts with multiple partners that include BRF1, HuR and U2AF35 using the N-terminal region that harbors a Ala-rich domain. The interaction of hnRNPD with HuR is RNA dependent while with BRF1 and U2AF35, it is RNA independentt. Further, its interaction with all the partners is equally strong. This suggests that hnRNPD could exert differential influence depending on the context of its interaction and abundance of the interacting partner. HuR, primarily known as an mRNA stabilizing factor, interacts with both BRF1 and hnRNPD with equal affinity involving the hinge region, the interaction with the former being RNA-independent and the later being RNA-dependent. This differential RNA-dependent and independent interactions with the two AU-BPs using a single interacting domain suggests a balancing act of HuR on the activities of BRF1 and hnRNPD. These interactions can further be differentially modulated by posttranslational modifications on one or all of the interacting partners depending on the physiological status of the cell. We have also analyzed the multiple protein complexes formed in absence of cellular RNA. Though we are unable to see direct protein-protein interaction between HuR and U1-70K in Yeast two hybrid analysis, we could detect the presence of U1-70K in HuR immunoprecipitate. It appears that U1-70K associates with HuR via BRF. We also detected the presence of HuR in U1-70K complexes which could be due to its association with BRF1. We are unable to find hnRNPD and U2AF35 in these complexes indicating that they may have been excluded. In anti-U2AF35 immunoprecipitates, we detected the presence of U1-70K as well as hnRNPD but no HuR. This may be due to RNase treatment as hnRNPD and HuR interactions are RNA dependent. Our findings that AU-rich elements in conjunction with AU-BPs function as intronic splicing modulators or enhancers, reveal hitherto unidentified new players in the poorly understood complex mechanisms that mediate alternative splicing. The possibility of dynamic nature of the interactions among splicing factors and AU-BPs mediated by post-translational modifications provide a basis for rapid cellular responses to changing environmental cues through generation of differentially spliced mRNAs and corresponding protein products that differ in their stability and hence their relative abundance. Our results also unfold enormous possibilities for future investigations on interactions among the many splicing factors and AU-BPs, and in understanding these complex interactions in modulation of pre-mRNA splicing, mRNA translation and degradation. The finding of coupling of AU-BPs to splicing machinery could further lead to better understanding of the mechanism of AU-BP-mediated targeting of mRNAs to processing bodies and ultimate degradation of the mRNAs.