Arginine methylation analysis of the splicing-associated SR protein SFRS9/SRP30C

The human SFRS9/SRp30c belongs to the SR family of splicing regulators. Despite evidence that members of this protein family may be targeted by arginine methylation, this has yet to be experimentally addressed. In this study, we found that SFRS9 is a target for PRMT1-mediated arginine methylation in vitro, and that it is immunoprecipitated from HEK-293 lysates by antibodies that recognize both mono- and dimethylated arginines. We further observed that upon treatment with the methylation inhibitor Adox, the fluorescent EGFP-SFRS9 re-localizes to dot-like structures in the cell nucleus. In subsequent confocal analyses, we found that EGFP-SFRS9 localizes to nucleoli in Adox-treated cells. Our findings indicate the importance of arginine methylation for the subnuclear localization of SFRS9144657669CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informaçã

IR Surface Brightness Fluctuations of Magellanic Star Clusters

We present surface brightness fluctuations (SBFs) in the near--IR for 191 Magellanic star clusters available in the Second Incremental and All Sky Data releases of the Two Micron All Sky Survey (2MASS), and compare them with SBFs of Fornax Cluster galaxies and with predictions from stellar population models. Our goals are twofold. First, to provide an empirical calibration of near--IR SBFs, given that existing stellar population synthesis models are particularly discrepant in the near--IR. Second, whereas most previous SBF studies have focused on old, metal rich populations, this is the first application to a system with such a wide range of ages (~ 10^6 to more than 10^10 yr, i.e., 4 orders of magnitude), at the same time that the clusters have a very narrow range of metallicities (Z ~ 0.0006 -- 0.01, ie., 1 order of magnitude only). Since stellar population synthesis models predict a more complex sensitivity of SBFs to metallicity and age in the near--IR than in the optical, this analysis offers a unique way of disentangling the effects of age and metallicity. We find a satisfactory agreement between models and data. We also confirm that near--IR fluctuations and fluctuation colors are mostly driven by age in the Magellanic cluster populations, and that in this respect they constitute a sequence in which the Fornax Cluster galaxies fit adequately. Moreover, fluctuation colors display a tendency to redden with age that can be fit by a straight line. Finally, we use for the first time a Poissonian approach to establish the error bars of the fluctuation measurements, instead of the customary Monte Carlo simulations.Comment: 54 pages, 12 figures, accepted by Ap

Root, shoot and leaf traits of the congeneric Styrax species may explain their distribution patterns in the cerrado sensu lato areas in Brazil

Shoot and root lengths, the number of leaves, biomass and leaf area were measured in Styrax ferrugineus Nees and Mart., Styrax camporum Pohl. and Styrax pohlii A. DC cultivated in rhizotrons. Additionally, young individuals of these species were planted in a cerrado sensu stricto (s. str.), at the edge and in the understorey of a cerradao, and in the understorey of a riparian forest. Six months after planting, the specific leaf area (SLA) and the CO(2) assimilation rate were assessed on an area (A(area)) and mass (A(mass)) basis. S. ferrugineus exhibited greater root and lower shoot length in comparison to S. pohlii. The high shoot growth and concomitantly substantial root length of S. camporum may illustrate why this species is widely distributed in the cerrado sensu lato areas, whereas the deep roots of S. ferrugineus could account for its occurrence in the cerrado s. str. In the field, an irradiance-diminishing gradient enlarged the SLA of S. pohlii, which positively influenced its A(mass), and which could partially explain its occurrence in shady habitats. However, a non-plastic trait, such as the high shoot length of S. pohlii, is more likely to be responsible for the success of this species in forest habitats.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Ki-1/57 and CGI-55 ectopic expression impact cellular pathways involved in proliferation and stress response regulation

Ki-1/57 (HABP4) and CGI-55 (SERBP1) are regulatory proteins and paralogs with 40.7% amino acid sequence identity and 67.4% similarity. Functionally, they have been implicated in the regulation of gene expression on both the transcriptional and mRNA metabolism levels. A link with tumorigenesis is suggested, since both paralogs show altered expression levels in tumor cells and the Ki-1/57 gene is found in a region of chromosome 9q that represents a haplotype for familiar colon cancer. However, the target genes regulated by Ki-1/57 and CGI-55 are unknown. Here, we analyzed the alterations of the global transcriptome profile after Ki-1/57 or CGI-55 overexpression in HEK293T cells by DNA microchip technology. We were able to identify 363 or 190 down-regulated and 50 or 27 up-regulated genes for Ki-1/57 and CGI-55, respectively, of which 20 were shared between both proteins. Expression levels of selected genes were confirmed by qRT-PCR both after protein overexpression and siRNA knockdown. The majority of the genes with altered expression were associated to proliferation, apoptosis and cell cycle control processes, prompting us to further explore these contexts experimentally. We observed that overexpression of Ki-1/57 or CGI-55 results in reduced cell proliferation, mainly due to a G1 phase arrest, whereas siRNA knockdown of CGI-55 caused an increase in proliferation. In the case of Ki-1/57 overexpression, we found protection from apoptosis after treatment with the ER-stress inducer thapsigargin. Together, our data give important new insights that may help to explain these proteins putative involvement in tumorigenic event

Ki-1/57 And Cgi-55 Ectopic Expression Impact Cellular Pathways Involved In Proliferation And Stress Response Regulation.

Ki-1/57 (HABP4) and CGI-55 (SERBP1) are regulatory proteins and paralogs with 40.7% amino acid sequence identity and 67.4% similarity. Functionally, they have been implicated in the regulation of gene expression on both the transcriptional and mRNA metabolism levels. A link with tumorigenesis is suggested, since both paralogs show altered expression levels in tumor cells and the Ki-1/57 gene is found in a region of chromosome 9q that represents a haplotype for familiar colon cancer. However, the target genes regulated by Ki-1/57 and CGI-55 are unknown. Here, we analyzed the alterations of the global transcriptome profile after Ki-1/57 or CGI-55 overexpression in HEK293T cells by DNA microchip technology. We were able to identify 363 or 190 down-regulated and 50 or 27 up-regulated genes for Ki-1/57 and CGI-55, respectively, of which 20 were shared between both proteins. Expression levels of selected genes were confirmed by qRT-PCR both after protein overexpression and siRNA knockdown. The majority of the genes with altered expression were associated to proliferation, apoptosis and cell cycle control processes, prompting us to further explore these contexts experimentally. We observed that overexpression of Ki-1/57 or CGI-55 results in reduced cell proliferation, mainly due to a G1 phase arrest, whereas siRNA knockdown of CGI-55 caused an increase in proliferation. In the case of Ki-1/57 overexpression, we found protection from apoptosis after treatment with the ER-stress inducer thapsigargin. Together, our data give important new insights that may help to explain these proteins putative involvement in tumorigenic events.18432944-5

Solution structure of the human signaling protein RACK1

<p>Abstract</p> <p>Background</p> <p>The adaptor protein RACK1 (receptor of activated kinase 1) was originally identified as an anchoring protein for protein kinase C. RACK1 is a 36 kDa protein, and is composed of seven WD repeats which mediate its protein-protein interactions. RACK1 is ubiquitously expressed and has been implicated in diverse cellular processes involving: protein translation regulation, neuropathological processes, cellular stress, and tissue development.</p> <p>Results</p> <p>In this study we performed a biophysical analysis of human RACK1 with the aim of obtaining low resolution structural information. Small angle X-ray scattering (SAXS) experiments demonstrated that human RACK1 is globular and monomeric in solution and its low resolution structure is strikingly similar to that of an homology model previously calculated by us and to the crystallographic structure of RACK1 isoform A from <it>Arabidopsis thaliana</it>. Both sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation techniques showed that RACK1 is predominantly a monomer of around 37 kDa in solution, but also presents small amounts of oligomeric species. Moreover, hydrodynamic data suggested that RACK1 has a slightly asymmetric shape. The interaction of RACK1 and Ki-1/57 was tested by sedimentation equilibrium. The results suggested that the association between RACK1 and Ki-1/57(122-413) follows a stoichiometry of 1:1. The binding constant (KB) observed for RACK1-Ki-1/57(122-413) interaction was of around (1.5 ± 0.2) × 10⁶M^-1and resulted in a dissociation constant (KD) of (0.7 ± 0.1) × 10^-6M. Moreover, the fluorescence data also suggests that the interaction may occur in a cooperative fashion.</p> <p>Conclusion</p> <p>Our SAXS and analytical ultracentrifugation experiments indicated that RACK1 is predominantly a monomer in solution. RACK1 and Ki-1/57(122-413) interact strongly under the tested conditions.</p

Functional association of human Ki-1/57 with pre-mRNA splicing events

The cytoplasmic and nuclear protein Ki- 1 / 57 was first identified in malignant cells from Hodgkin`s lymphoma. Despite studies showing its phosphorylation, arginine methylation, and interaction with several regulatory proteins, the functional role of Ki- 1 / 57 in human cells remains to be determined. Here, we investigated the relationship of Ki- 1 / 57 with RNA functions. Through immunoprecipitation assays, we verified the association of Ki- 1 / 57 with the endogenous splicing proteins hnRNPQ and SFRS9 in HeLa cell extracts. We also found that recombinant Ki- 1 / 57 was able to bind to a poly- U RNA probe in electrophoretic mobility shift assays. In a classic splicing test, we showed that Ki- 1 / 57 can modify the splicing site selection of the adenoviral E1A minigene in a dose- dependent manner. Further confocal and. uorescence microscopy analysis revealed the localization of enhanced green. uorescent protein - Ki- 1 / 57 to nuclear bodies involved in RNA processing and or small nuclear ribonucleoprotein assembly, depending on the cellular methylation status and its N- terminal region. In summary, our findings suggest that Ki- 1 / 57 is probably involved in cellular events related to RNA functions, such as pre- mRNA splicing.Fundacao de Amparo a Pesquisa do Estado Sao Paulo (FAPESP)Conselho Nacional de Pesquisa e Desenvolvimento (CNPq)LNL