RESUMEN
Ribosome biogenesis is a highly energy-demanding process in eukaryotes which requires the concerted action of all three RNA polymerases. In RNA polymerase II transcription, the general transcription factor TFIIH is recruited by TFIIE to the initiation site of protein-coding genes. Distinct mutations in TFIIH and TFIIE give rise to the degenerative disorder trichothiodystrophy (TTD). Here, we uncovered an unexpected role of TFIIE in ribosomal RNA synthesis by RNA polymerase I. With high resolution microscopy we detected TFIIE in the nucleolus where TFIIE binds to actively transcribed rDNA. Mutations in TFIIE affects gene-occupancy of RNA polymerase I, rRNA maturation, ribosomal assembly and performance. In consequence, the elevated translational error rate with imbalanced protein synthesis and turnover results in an increase in heat-sensitive proteins. Collectively, mutations in TFIIE-due to impaired ribosomal biogenesis and translational accuracy-lead to a loss of protein homeostasis (proteostasis) which can partly explain the clinical phenotype in TTD.
Asunto(s)
Nucléolo Celular/genética , Regulación de la Expresión Génica , Biogénesis de Organelos , Factor de Transcripción TFIIH/genética , Factores de Transcripción TFII/genética , Síndromes de Tricotiodistrofia/genética , Línea Celular Transformada , Nucléolo Celular/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Genes Reporteros , Calor , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Mutación , Complejo de la Endopetidasa Proteasomal/metabolismo , Biosíntesis de Proteínas , Estabilidad Proteica , Proteostasis/genética , ARN Polimerasa I/genética , ARN Polimerasa I/metabolismo , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Factor de Transcripción TFIIH/metabolismo , Factores de Transcripción TFII/deficiencia , Transcripción Genética , Síndromes de Tricotiodistrofia/metabolismo , Síndromes de Tricotiodistrofia/patologíaRESUMEN
TAR DNA binding protein 43 (TDP-43) is closely related to the pathogenesis of amyotrophic lateral sclerosis (ALS) and translocates to stress granules (SGs). The role of SGs as aggregation-promoting "crucibles" for TDP-43, however, is still under debate. We analyzed TDP-43 mobility and localization under different stress and recovery conditions using live cell single-molecule tracking and super-resolution microscopy. Besides reduced mobility within SGs, a stress induced decrease of TDP-43 mobility in the cytoplasm and the nucleus was observed. Stress removal led to a recovery of TDP-43 mobility, which strongly depended on the stress duration. 'Stimulated-emission depletion microscopy' (STED) and 'tracking and localization microscopy' (TALM) revealed not only TDP-43 substructures within stress granules but also numerous patches of slow TDP-43 species throughout the cytoplasm. This work provides insights into the aggregation of TDP-43 in living cells and provide evidence suggesting that TDP-43 oligomerization and aggregation takes place in the cytoplasm separate from SGs.