Updating the rna polymerase ctd code
For example, the mobilities of the splicing factors U2AF and U1-70K factors or sn RNPs (Chusainow et al. These aforementioned observations are consistent with a stochastic model in which nuclear proteins are permanently scanning the nuclear environment and searching for interaction partners using a combination of continuous diffusion-like movements and transient interactions. In addition, early mass analysis combined with a novel and precise computational approach provided the first evidence of a putative global role of TCERG1 in m RNA processing (Pearson et al. Given these data, TCERG1 has been suggested as a possible factor that coordinates transcription and splicing (Montes et al.Consistent with this model and in the absence of transcription and splicing activity, nuclear factors move freely throughout the nucleoplasm, with higher dynamic rates due to the absence of pre-m RNA molecules or other factors engaged in pre-m RNA processing. 2012a), a hypothesis that we recently supported by demonstrating that TCERG1 regulates alternative splicing of the gene by modulating the rate of RNAPII transcription (Montes et al. Here, we investigated the dynamic properties of TCERG1 in real time in live cells.() Effect of the α-amanitin treatment on TCERG1 dynamics.Exo1 cells were transfected with GFP-TCERG1, Tat and MS2-mcherry and were then either untreated or treated with 0.1 µg/µL α-amanitin for 2 h and processed for FRAP experiments in the nucleoplasm and the nuclear speckle regions.To test whether TCERG1 modifies its dynamic behavior in response to transcriptional inhibition, the cells were treated with α-amanitin, which strongly inhibits RNAPII-dependent transcription and leads to the destruction of its large subunit.
Next, we quantitatively analyzed the recovery of TCERG1 in the nucleoplasmic and speckle regions of the cells with or without α-amanitin treatment.
In this condition, spliceostatin A has no effect, indicating that TCERG1 rapidly binds and dissociates from stalled spliceosomal complexes and that the mobility properties of TCERG1 do not depend on events occurring after the initial spliceosome formation.
Taken together, these data suggest that TCERG1 binds independently to elongation and splicing complexes, thus performing their coupling by transient interactions rather than by stable association with one or the other complexes.
Next, we sought to analyze the mobility properties of GFP-TCERG1 in the nucleus using the well-established Exo1 cell line, which is a derivative of U2OS cells that express an MS2-tagged reporter RNA (Boireau et al. The FRAP method involves photobleaching a small area of the cell and monitoring the recovery of fluorescence intensity over a selected time interval (Fig. Fluorescence recovery indicates the movement of unbleached molecules into the bleached area.
For these experiments, we selected cells with a low level of GFP fluorescence to avoid potential overexpression artifacts.