Sánchez-Hernández, N., Boireau, S., Schmidt, U., Muñoz-Cobo, JP, Hernández-Munain, C., Bertrand, E., and Suñé, C.
RNA 2016 22(4). 571-82
The tightly regulated process of precursor messenger RNA (pre-mRNA) alternative splicing is a key mechanism to increase the number and complexity of proteins encoded by the genome. The results of deep sequencing-based expression analyses suggest that more than 90% of multi-exon human genes undergo alternative splicing. Changes in the cis- or trans- regulation of this process can cause multiple pathologies as a result of general or specific aberrant pre-mRNA processing underscoring the fundamental importance of this regulatory process. Evidence gathered in recent years has established that transcription and splicing are physically and functionally coupled and that this coupling may be an essential aspect of the regulation of splicing and alternative splicing.
Studies of the spatial organization and dynamics of transcription and pre-mRNA processing within the highly compartmentalized eukaryotic nucleus are fundamental to fully understand how the regulation of gene expression is exerted in the cell. Therefore, studies aimed to unravel the coupling of transcriptional elongation and alternative splicing directly in living cells are of critical importance. Although some progress has been made in deciphering the functional consequences of this complex network of interacting molecules in the context of nuclear organization, how the proteins and RNA move in the nucleus and how the components of the transcription and RNA processing machineries find their targets are important questions that remain largely unexplored.
In this article, we investigated the factor TCERG1, which coordinates transcriptional elongation with splicing, using the FRAP (Fluorescence Recovery After Photobleaching) technique. FRAP technique is a method to qualitatively and quantitatively study biomolecule dynamics in living cells. FRAP is based on irreversibly bleaching a pool of fluorescent probes with high intensity light and monitoring the recovery in fluorescence due to movement of surrounding intact probes into the bleached spot. Our 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. TCERG1 appears to interact only very transiently with elongation and splicing complexes, thus likely acting by a "hit-and-run" type of mechanism. This finding has conceptual implications for understanding the coupling between transcription and RNA processing. This study was performed in collaboration with the laboratory of Edouard Bertrand, Institut de Génétique Moléculaire of Montpellier (IGM-CNRS).
Dynamic behavior of TCERG1 in different nuclear compartments by FRAP analysis. (Top Panel) Representative images of U2OS cells transiently transfected with the construction EGFP-TCERG1 [1-1098]. Images before (pre-bleach) and after (post-bleach) removing the fluorescence are shown in the splicing factor-rich nuclear speckles (red square) and the nucleoplasm (green square). Amplification of the bleached areas is also shown. (Lower panel) Fluorescence recovery (FRAP) of EGFP-TCERG1 was measured in the nuclear speckles (red) and nucleoplasm (green). The background was removed, the intensities at each time point were corrected for bleaching by dividing them by the total cell fluorescence, and these values were finally normalized by dividing them by the fluorescent intensity before the bleaching. The curves correspond to a pool of at least three independent experiments and the dotted lines represent the DS for each measurement.
The in vivo dynamics of TCERG1, a factor that couples transcriptional elongation with splicing.
RNA 2016 22(4). 571-82.
