RNA processing and export

Abstract

Messenger RNAs undergo 5' capping, splicing, 3'-end processing, and export before translation in the cytoplasm. It has become clear that these mRNA processing events are tightly coupled and have a profound effect on the fate of the resulting transcript. This processing is represented by modifications of the pre-mRNA and loading of various protein factors. The sum of protein factors that stay with the mRNA as a result of processing is modified over the life of the transcript, conferring significant regulation to its expression.

Figure 1.

mRNA processing is tightly coupled to transcription. The phosphorylation state of the C-terminal domain (CTD) of RNA Polymerase II (Pol II) is given on the right in relation to major steps of transcription. Listed are functional processing sequences (red), components of processing machinery (blue) and factors that are loaded onto the transcript as a result of processing (green).

Figure 2.

In vivo detection of transcription using fluorescence microscopy. Schematic shows a reporter cassette that is integrated as gene arrays into the genome. RFP-LacI labels reveal array locus and immunofluorescence shows that RNA Pol II with three distinct phosphorylation states is recruited to these active transcription sites (B–M). Similarly, the gene array, visualized by CFP-LacI, colocalizes with both MS2-tagged mRNAs, seen by GFP, and mRNA FISH probes targeting exonic and intronic regions (N–Y). (Reprinted, with permission, from Darzacq et al. 2007 [© 2007 Macmillan Publishers, Ltd.].)

Figure 3.

Depiction of several events that coincide with the switch from a promoter-engaged “poised” RNA polymerase to an elongating productive RNA polymerase (shown in purple). Histone modifications and changing CTD phosphorylation states are known to be associated with this switch. Furthermore, transcription is intimately coupled to mRNA processing events such as 5′ capping and splicing. (Reprinted, with permission, from Moore and Proudfoot 2009 [© 2009, Elsevier].)

Figure 4.

Spatial mapping of the interaction of U1 70K with SF2/ASF in vivo. Shown are confocal images of cells transfected with EGFP-U1 70K and cotransfected with either mCherry-C1 or mCherry-SF2/ASF. Mean fluorescence lifetime (in picoseconds) and percentages of FRET efficiency and FRET amplitude are shown (pseudocolor) in these same cells (A). The same experiment was repeated in the presence of 25 µg/ml DBR for 2 hours before imaging (B). FRET efficiencies calculated from FLIM measurements for the interaction of SF2/ASF with U1 70K (C). © Ellis et al., 2008. J. Cell Biol. 181: 921–934.

Figure 5.

In vivo trajectories of single U1 snRNPs within the nucleus of HeLa cells. Fluorescently labeled native U1 snRNPs were microinjected to visualize and track single molecules, recorded at 200 Hz. SF2/ASF-GFP was transiently expressed to distinguish mobile and transiently immobilized U1 snRNP particles within the nucleoplasm and speckles, outlined in gray (A). A 8 µm² area from A is broken down into a short image sequence displaying a single trajectory over time (B). Grunwald et al. 2006, © 2006 by The American Society for Cell Biology.