Updating the rna polymerase ctd code
This finding has conceptual implications for understanding the coupling between transcription and RNA processing. The absence of a defined membrane and the rapid dynamics of nuclear compartments are consistent with a self-organization model in which the structure of a body is determined by the global interactions among its constituents (Misteli 2001; Kaiser et al. One of the most dynamic nuclear compartments is the splicing factor-rich nuclear speckle (Lamond and Spector 2003). Subsequently, FRAP studies of nuclear speckle components revealed that these components move within the nucleus with kinetics ranging from milliseconds to seconds (Phair and Misteli 2000; Rino et al. This high mobility and the morphological changes that occur upon transcription inhibition (O'Keefe et al.Mammalian cell nuclear proteins are concentrated within several stable compartments that are not defined by membranes (Mao et al. Although these compartments are apparently immobile structures, fluorescence recovery after photobleaching (FRAP) analysis has demonstrated that the proteins in these compartments are more mobile than previously assumed and are exchanged continuously between the compartments and the nucleoplasm (Phair and Misteli 2000; Snaar et al. Pioneering time-lapse visualization studies revealed that nuclear speckles are markedly dynamic structures that respond to changes in gene expression (Misteli et al. 1994) indicate that speckles are formed by self-organization, which is achieved by direct interactions between their components, and that speckles then rearrange their structure upon expression of target RNAs. However, FRAP experiments have demonstrated that nuclear proteins exhibit a slower mobility than expected if only diffusion processes act on the proteins probably due to transient interactions with other nuclear components (Kruhlak et al.2012b) and that TCERG1 moves from the nucleoplasm toward enlarged and rounded nuclear speckles upon transcription inhibition (Sánchez-Álvarez et al. We confirmed these results using the GFP-TCERG1 construct and observed the disappearance of TSs upon α-amanitin treatment (data not shown).Next, we quantitatively analyzed the recovery of TCERG1 in the nucleoplasmic and speckle regions of the cells with or without α-amanitin treatment.The half-recovery time under untreated conditions did not significantly differ for the nucleoplasm versus the speckles (Table 1; Fig. Slight and significant increases in the mobility of TCERG1 in the nucleoplasm ( = 0.014) upon inhibition of RNAPII transcription with α-amanitin were observed (Table 1; Fig.2C, D), which are in agreement with the mobility of other splicing factors under these conditions (Phair and Misteli 2000).Coupling between transcription and RNA processing is key for gene regulation.
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.
() 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.
The translocation of particles in the nucleus can be explained by a diffusion-like movement (Seksek et al. 2000; Pederson 2000; Phair and Misteli 2000; Shopland and Lawrence 2000).
This reduced mobility may be dictated by interactions with quasi-immobile structures, such as the chromatin or nuclear matrix, or by integration into larger macromolecular complexes. 2010), reinforced the idea that the dynamic behavior of nuclear factors is largely determined by transient interactions.
The effect of TCERG1 and GFP-TCERG1 overexpression on the alternative splicing of the isoform were increased (Fig. Quantitative analysis of these data is also presented (Fig. Taken together, these results demonstrate that GFP-TCERG1 functions similar to TCERG1 in regulating alternative splicing.