MCB 201 Gene Expression - Spring Semester 2003
Lecture 22 (chapter 11 RNA Processing, Nuclear Transport, and Post-transcriptional Control cont.)
Section 11.4 Signal-mediated Transport through Nuclear Pore Complexes continued
12. Figure 1B (in Nover et al, "Arabidopsis and the heat stress transcription factor world: how many heat stress transcription factors do we need?". 2001. Cell Stress & Chaperones 6(3):177-189). This figure shows a diagram of a heat shock transcription factor (protein) from tomato. This factor cycles between the cytoplasm and nucleus and has both an NLS and an NES sequence. In the previous lecture we discussed movement of proteins out of the nucleus. The fact that all nuclear proteins are synthesized in the cytoplasm of the cell tells us that there must be a mechanism for moving proteins into the nucleus. All proteins that are imported through nuclear pore complexes (NPCs) contain nuclear localization signals (NLS). Proteins that move back and forth between nucleus and cytoplasm have both an NLS and a nuclear export signal (NES). The NLS is usually a sequence rich in the basic amino acids arginine and lysine. Sometimes it is one segment enriched for basic amino acids and other NLS's have a bipartite arrangement of two basic segments separated by another sequence, as shown here.
13. Figure 11-35: Demonstration that the NLS of a known nuclear protein, the SV40 (simian virus 40) large T-antigen, can direct pyruvate kinase, a cytoplasmic protein, to the nucleus. Panel A: Control cells showing pyruvate kinase in the cytoplasm, visualized using a fluorescent antibody against this protein. Panel B: Cells transformed with a plasmid carrying a gene in which a piece of the viral gene carrying the NLS of the large T-antigen was added to the gene encoding pyruvate kinase. The protein that is produced has the NSL at its N-terminus and this directs the protein into the nucleus.
14. Figure 11-36: Experimental demonstration that nuclear transport in permeabilized cells requires soluble cytosolic components and ATP as an energy source. Panel A: Cells treated with the detergent digitonin which makes holes in the plasma membrane, allowing soluble cellular components to escape, but does not affect the nuclear envelope and NPCs. Panel B: Here a fluorescent protein carrying an NLS is used as the reporter for nuclear localization. In the first pair of panels, buffer containing the fluorescent reporter protein was added to the permeabilized cells without addition of cell lysate. No fluorescence was detected in the presence or absence of ATP, showing that there is no nuclear transport in the absence of cell lysate. When fluorescent peptide with a NLS sequence was added to the lysate and then the mixture added to permeabilized cells, transport into the nucleus was detected in the presence of both lysate and ATP but not when ATP was omitted. There both lysate and ATP are required for nuclear localization. Once an assay like this is developed for a complex lysate, it is then possible to use it as an assay in the purification of the proteins required for nuclear import. In this way, a nuclear import receptor (a heterodimer composed of importin alpha and beta) for basic NLS's was found. The same GTPase involved in nuclear export, Ran, was also found to be an essential component of import.
15. Figure 11-37: Proposed mechanism for the transport of "cargo" proteins containing a basic nuclear-localization signal (NLS) from the cytoplasm to the nucleus. Importin alpha and beta form a complex with the cargo protein using NLS to bind to its receptor site on importin alpha. This complex is transported into the nucleus through the NPC by an ATP dependent (energy requiring) pathway which is thought to involve interactions between the cargo complex and proteins in the NPC. Once inside the nucleus, the cargo is released from the receptor by interaction with Ran(GTP). In the process, Ran(GTP) ends up bound to importin beta. Both Ran(GTP) and the importins must then be exported back into the cytoplasmic to carry out another round of import. What makes this pathway import cargo and not export it, i.e. work in only one direction, is the fact that Ran GAP, a cytoplasmic protein that simulates hydrolysis of GTP to GDP on Ran and RCC1, a nuclear protein that exchanges GDP for GTP, remain in their respective compartments.
Section 11.5: Other mechanisms of post-transcriptional control
16. Figure 11-40: Mechanism of RNA editing in kinetoplast pre-mRNA of trypanosomes. A guide RNA (gRNA), which is complementary to a portion of the pre-mRNA, binds and directs both the addition and deletion of U residues by enzymes. The result of this RNA editing is that the RNA transcript is different in sequence than the gene that encoded this pre-mRNA. This process is encountered only rarely in modern organisms and may be a 'molecular fossil' of a process used extensively in ancient, ancestral organisms.
17. Figure 11-41: Experimental demonstration that the 3' untranslated region of alpha- and beta-actin mRNAs direct localization of a beta-galactosidase reporter mRNA. This figure shows that the 3' untranslated sequences of alpha- and beta-actin mRNA contain localization signals which localize these mRNAs to perinuclear regions and lamellipodia at the edges of cells, respectively. These sequences were added to mRNA encoding beta-galactosidase enzyme. This mRNA became localized in transfected cells and beta-galactosidase enzyme was produced at these locations. This enzyme can be assayed by adding to the cells a substrate that makes a colored localized product when acted on by this beta-galactosidase.
18. Figure 11-43: Gene expression can be controlled by a change in mRNA stability. Control of RNA degradation. Special sequences in the 3' untranslated region (UTR) of unstable mRNAs are responsible for their unusually rapid degradation. As indicated, AU-rich sequences found in the 3' UTR of many short-lived mRNAs cause a rapid removal of the poly-A tail, which in turn makes the RNA unstable. Other mRNAs contain sequences in their 3'UTR that serve as sites for specific endonucleolytic cleavage.
19. Posttranslational control and iron metabolism in animal cells. Two posttranslational controls are mediated by iron. In response to an increase in iron concentration in the cytoplasm, a cell increases its synthesis of ferritin in order to bind extra iron and decreases its synthesis of transferrin receptors in order to import less iron into the cell. Both responses are mediated by the same iron-responsive regulatory protein, aconitase (also called iron response element-binding protein, IRE-BP), which recognizes common features in a stem-and-loop structure in the mRNAs encoding ferritin and transferrin receptor. Aconitase dissociates from the mRNA when it binds iron. Because the transferrin receptor and ferritin are regulated by different types of mechanisms, their levels respond oppositely to iron concentrations even though they are regulated by the same iron-responsive regulatory protein.
A. Figure 11-44: Iron-dependent regulation of the stability of transferrin-receptor (TfR) mRNA. Stem-loop structures have AU-rich regions that promote RNA degradation. These are exposed when aconitase is inactive (when Fe ions are at high concentrations inside cells) and cannot bind to them. The mRNA is rapidly degraded and no receptor is made. This makes sense physiologically since the cell has plenty of iron and does not need a cell surface receptor to bind more to bring into the cell. When iron levels are low, aconitase becomes activated and binds to the stem loops, resulting in stabilized mRNA that is used for translation to produce transferrin receptors, needed to bring iron into cells.
B. Figure 11-45: Iron-dependent regulation of translation of ferritin mRNA. When iron levels are high inside cells, they need more ferritin to bind the iron. Ferritin is an intracellular iron binding and carrier protein. Free iron ions are toxic inside cells. They participate in oxidation reactions which damage proteins and other molecules. This time the stem-loops (IREs) are located in the 5' untranslated region and when aconitase binds to these structures, initiation of translation is inibited. This occurs under conditions of low iron inside cells.
