MCB 201 Gene Expression - Spring Semester 2003
Lecture 21 (chapter 11 RNA Processing, Nuclear Transport, and Post-transcriptional Control cont.)
Section 11.3: Regulation of mRNA Processing
1. Conversion of a 5' capped RNA transcript into functional mRNA requires two steps, both of which potentially could be regulated:
a. Cleavage and polyadenylation at the 3' end.
b. Excision of introns and ligation of exons, i.e. RNA splicing.
2. Figure 11-23: Diagram of 3' end of the pre-mRNA encoding U1A protein, a sequence-specific RNA-binding protein that is part of the U1 snRNP. This is one of the best known examples of on-off regulation at the first step, cleavage and polyadenylation. This is a mechanism linked to cell physiology through the levels of U1A protein itself. This protein binds to a sequence in snRNA as a protein component of the snRNP. The same RNA binding sequence appears twice in the pre-mRNA encoding U1A protein. When all of the U1A protein being produced by the cell is being used to assemble snRNPs, then none is left over to bind to these sites. Under these conditions, the poly(A) and cleavage signals are used to produce a polyadenylated, functional mRNA that cells use to produce more U1A protein. When there is more U1A protein in the cell than is needed to make snRNPs, some of the excess binds to the two sites on the pre-mRNA, which blocks the polyadenylation reaction. The result is an unstable mRNA without its poly(A) tail, which is not translated into more U1A protein, but instead, it is rapidly degraded.
3. Figure 9-2. The second step at which mRNA processing can be regulated is during splicing. Recall the complex transcription units which we studied in overview. These primary transcripts can be processed in different ways, i.e. different exons can be removed, to create different mRNAs and ultimately different proteins. About 5% of total transcription units in higher eukaryotes are complex units, so there is significant opportunity for this type of control. It seems to be particularly prevalent in the vertebrate nervous system.
4. Figure 11-27a: Alternative splicing of slo mRNA, which encodes a calcium ion-gated potassium ion channel, in auditory hair cells contributes to the perception of sounds of different frequency. In Panel A, a diagram of the tubular cochlea, or inner ear, is shown.
Figure 22-3 (Alberts). This tissue is composed of 'hair cells', i.e. epithelial cells with clusters of large microvilli called stereocilia, each of which is tuned to respond to a specific frequency of sound. The frequencies range from 50 Hz (apical end of cochlea) to 5,000 Hz (basal end). In mammals these hair cells, produced during embryogenesis, do not divide in adults, so if they are destroyed by disease or loud noise, deafness results. The stereocilia bend in response to pressure waves caused by sound acting on the fluid-filled inner ear. This bending sends transmembrane signals that increase the levels of Ca ions in the cell. The Ca ions bind to potassium channels, and the Ca concentration needed to open the channel determines the frequency with which the membrane potential changes, and therefore, the frequency to which each cell is tuned. Therefore, which cells actually respond to a particular sound is determined by the form of the K ion channel protein they contain, which in turn is determined by the particular splicing reactions in those cells.
Figure 11-27b: The gene encoding this potassium channel (called the slo gene) is expressed as multiple alternatively spliced mRNAs. The various Slo proteins encoded by these alternative mRNAs open at different Ca ion concentrations. Since there is a gradient of cell responses in the cochlea, this suggests that splicing is regulated in response to extracellular signals that give positional information to the cell, i.e. where are you along the cochlea? The molecular mechanism behind the alternative RNA splicing involves binding of sequence-specific RNA-binding proteins near particular splice sites and these can either activate or inhibit splicing at those sites, creating mRNAs with different combinations of introns.
5. CELLebration video, number 3. The Dancing Ear Cell.
Section 11.4 Signal-mediated Transport through Nuclear Pore Complexes
6. Figure 11-28: Nuclear pore complex (NPC). This is another site of regulation of gene expression, since it controls exit and entry of proteins from and into the nucleus. Panel A shows nuclear envelopes from Xenopus oocytes visualized by scanning EM, looking at the cytoplasmic face of the nucleus on left, the nucleoplasmic face in the center, and this face after detergent treatment to remove the nuclear membrane and expose the nuclear lamina, a network of intermediate filaments on the inner surface of the nuclear envelope. Panel B shows a diagram of the NPC. Note attachments of cytoplasmic filaments to cytoplasmic ring and nuclear laminin to the nuclear ring. Point out space around the central plug ( water-filled channel) where uncontrolled diffusion of small molecules and proteins up to about 60 Kilodaltons occurs, and the central plug through which regulated transport occurs.
7. Figure 11-29. Formation of coiled heterogeneous ribonucleoprotein (hnRNP) during synthesis of an insect Balbiani ring (BR) mRNA. Transcription is occurring along this chromatin loop, and as the RNA transcript is made, proteins bind to it to assemble hnRNP particles containing pre-mRNA. After this pre-mRNA is processed by RNA splicing, it becomes an mRNP, messenger RNP. Note how the hnRNP particle becomes larger along the loop of chromatin as the RNA polymerases move along and produce more pre-mRNA for proteins to bind.
8. Figure 11-31. Model for passage of mRNPs through the nuclear pore complex (NPC). The mRNP enters through the terminal ring and it uncoils as it passes through the central plug of the NPC. The 5' end leads the way into the cytoplasmic, deduced from the observation that ribosomes can bind to this end as soon as it appears in the cytoplasm.
9. Figure 11-33. Proposed mechanism for the transport of "cargo" proteins containing a leucine-rich nuclear export signal (NES) from the nucleus to the cytosol. Proteins that can be exported from the nucleus to the cytoplasm have one of several possible sequences as part of their primary amino acid sequence that marks this protein for export. A nuclear export receptor protein, exportin 1, binds to the NES. A small GTPase in its GTP bound form,called Ran-GTP, also binds to this cargo complex. After this complex is transported through the NPC, a protein called Ran GTPase activating protein (RanGAP) stimulates Ran to hydrolyze its bound GTP to GDP, which causes a conformational change in Ran. This results in the disassembly of the cargo complex in the cytosol. Exportin 1 and Ran-GDP then cycle back into the nucleus for use in another cycle of cargo movement. What makes this process work only in the nucleus to cytoplasm direction is that RanGAP stays in the cytosol and another important protein in the cycle, RCC1, a GTP/GDP exchange protein, stays in the nucleus.
10. Figure 11-34. Proposed mechanism for hnRNP protein-mediated export of mRNA from the nucleus. The interesting thing about this process is the remodeling of the protein composition of the RNP along the way. CBC is cap-binding complex, which binds to the 5' end of processed mRNAs. Note that some proteins, that cannot leave the nucleus are stripped off during movement through the NPC. Then other proteins, capable of shuttling back to the nucleus, are removed in the cytosol in a process involving RanGAP. Finally, cytoplasmic proteins are added to the RNP to make a mature cytoplasmic mRNP.
11. It is important to note that pre-mRNAs in spliceosomes are not exported from the nucleus. Since these would not be functional as mRNAs, due to incomplete splicing, it would be wasteful for cells to allow these processing intermediates to be released.