RESUMO
Continual spermatogenesis relies on the actions of an undifferentiated spermatogonial population that is composed of stem cells and progenitors. Here, using mouse models, we explored the role of RNA-binding proteins (RBPs) in regulation of the biological activities of this population. Proteins bound to polyadenylated RNAs in primary cultures of undifferentiated spermatogonia were captured with oligo (dT)-conjugated beads after UV-crosslinking and profiled by proteomics (termed mRBPome capture), yielding a putative repertoire of 473 RBPs. From this database, the RBP TRIM71 was identified and found to be expressed by stem and progenitor spermatogonia in prepubertal and adult mouse testes. Tissue-specific deletion of TRIM71 in the male germline led to reduction of the undifferentiated spermatogonial population and a block in transition to the differentiating state. Collectively, these findings demonstrate a key role of the RBP system in regulation of the spermatogenic lineage and may provide clues about the influence of RBPs on the biology of progenitor cell populations in other lineages.
Assuntos
Proteoma/metabolismo , Proteínas de Ligação a RNA/metabolismo , Espermatogônias/citologia , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Testículo/citologia , Regulação para Cima/genéticaRESUMO
SREBPs are master regulators of lipid homeostasis and undergo sterol-regulated export from ER to Golgi apparatus for processing and activation via COPII-coated vesicles. While COPII recognizes SREBP through its escort protein SCAP, factor(s) specifically promoting SREBP/SCAP loading to the COPII machinery remains unknown. Here, we show that the ER/lipid droplet-associated protein Cideb selectively promotes the loading of SREBP/SCAP into COPII vesicles. Sterol deprivation releases SCAP from Insig and enhances ER export of SREBP/SCAP by inducing SCAP-Cideb interaction, thereby modulating sterol sensitivity. Moreover, Cideb binds to the guanine nucleotide exchange factor Sec12 to enrich SCAP/SREBP at ER exit sites, where assembling of COPII complex initiates. Loss of Cideb inhibits the cargo loading of SREBP/SCAP, reduces SREBP activation, and alleviates diet-induced hepatic steatosis. Our data point to a linchpin role of Cideb in regulated ER export of SREBP and lipid homeostasis.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/fisiologia , Retículo Endoplasmático/fisiologia , Complexo de Golgi/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Esteróis/farmacologia , Animais , Proteínas Reguladoras de Apoptose/genética , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/efeitos dos fármacos , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/fisiologia , Retículo Endoplasmático/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Células HEK293 , Células Hep G2 , Homeostase , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Transporte Proteico , Proteína de Ligação a Elemento Regulador de Esterol 1/genéticaRESUMO
Aiming at the miniaturization requirement of shifted excitation Raman spectroscopy test system, a portable grating-coupled external cavity (EC) tunable semiconductor laser in Littrow configuration is designed and fabricated with a commercial 785 nm high-power laser diode as the gain device. By using a new wavelength tuning method, aiming to change the position of gain device relative to the collimating lens in the horizontal direction, a miniaturized device with the size of 140 mm×65 mm×50 mm is designed. Compared to the traditional wavelength tuning method which is to change the light incident angle by rotating the diffraction grating, this new tuning method reduces the translational distance of semiconductor gain device effectively, thus it is conductive to the fast and broad wavelength tuning of portable EC laser. The experimental results show that the EC laser has a wide wavelength tuning range. Under any injection current from 340 to 900 mA, a wavelength tuning range of more than 10 nm can be realized. Especially at 900 mA, good performance including a 11.67 nm-wavelength tuning range from 779.40 to 791.07 nm, a less than 0.2 nm-spectral linewidth, an up to 280 mW-output power, and a more than 25 dB-amplified spontaneous emission suppression ratio is presented, which fully meets the basic testing requirements of shifted excitation Raman spectroscopy. Moreover, 1.35 nm-electric wavelength tuning range is achieved by applying a mini-piezoelectric actuator. This indicates that the home-made 785 nm portable grating-coupled EC tunable semiconductor laser is suitable as the light source of portable shifted excitation Raman spectroscopy testing system to eliminate the fluorescence background of Raman spectrum.
