Fundamentals I: 10:00-11:00Scribe: Ashley Brewington

Friday, September 16, 2009Proof: Myra Dennis

Dr. ChenPost-Transcriptional Regulation Page1 of 5

  1. Alternative mRNA Splicing Creates Protein Isoforms [S40]
  2. There are two modes of splicing.
  3. Constitutive splicing
  4. Alternative splicing
  5. Constitutive splicing means that every intron is removed and every exon is joined together.
  6. There is no exception. You only make one type of mature mRNA because there is no exon that will be skipped.
  7. So, every exon is there and every intron is removed producing only one mature mRNA from the transcript.
  8. However, many eukaryotic genes can give rise to variable forms of mature mRNA. Meaning that one primary transcript can produce several different isoforms of mature mRNA. This is called alternative splicing.
  9. In addition to alternative splicing, you can use different promoters, select different polyadenylation sites, or a combination of these three events and produce different isoforms of mRNA from one gene. Those are the three things that can produce a combination of mature mRNA from a transcript, increasing the coding potential of a gene. One gene can encode several isoforms of a protein.
  1. Constitutive mRNA Splicing [S41]
  2. This is an example of constitutive splicing.
  3. There is a gene here that contains three exons and two introns.
  4. All introns are removed and all three exons are joined together.
  5. There is no exception in A cell or B cell. All exons are joined together.
  1. Alternative mRNA Splicing I [S42]
  2. These exons are optional, they can either be included or excluded/skipped. If all exons are included you will produce a mature mRNA containing three exons. However, if exon II is skipped you only produce an mRNA with exon I and exon III.
  3. Alternative splicing shown on this slide can produce the forms shown.
  4. There is no difference between the A cell or B cell. In A cell and B cell, the same isoforms are produced.
  5. This slide does not show cell-specific alternative splicing.
  1. Alternative mRNA Splicing II [S43]
  2. There is alternative splicing where the pattern is dependent on the cell or tissue type.
  3. This is called tissue specific alternative splicing.
  4. For example, exon II is optional. In the A cell exon II is included, in the C cell this exon is not included, and in the B cell it can be either included or skipped.
  5. The splicing pattern depends on the cell or tissue type.
  6. One gene or primary RNA can produce two isoforms of mature mRNA or protein.
  1. Alternative mRNA Splicing Creates Protein Isoforms [S44]
  2. There is another very striking example, from skeletal muscle troponin T gene.
  3. You can see that there are 18 exons.
  4. 11 of the 18 exons will always be included on the mature mRNA, making them constitutive.
  5. 5 of the exons, exons 4-8, are combinatorial. Either none of the exons will be included, or any other combination can be included skipped. Making 32 possible combinations.
  6. 2 exons, 16 and 17, are mutually exclusive. Meaning, that only one will be present at a time. 16 and 17 will never be present in the same mature mRNA at together.
  7. If everything occurs, 1 out of 32 and 1 out of 2 produces 64 different mature mRNAs from one primary mRNA/gene.
  8. That’s how coding potential can be increased. How alternative splicing can increase coding potential.
  1. Alternative Splicing Expands the Coding Potential of the Genome- Tissue Specific Splicing[S45]
  2. This one shows tissue specific splicing or the usage of the polyadenylation site.
  3. This example includes the -tropomyosin gene.
  4. You see exons and introns
  5. The mature form of -tropomyosin mRNA is different in striated muscle cell and smooth muscle cells.
  6. The difference is because there is alternative splicing and usage of polyadenylation.
  7. In the striated muscle you use one polyadenylation site and smooth muscle uses a different polyadenylation site in addition to alternative splicing.
  8. Even though they are from the same primary RNA/gene, the isoform produced is different between striated and smooth muscle cells.
  1. mRNA Export [S46]
  2. We have finished the three events of mRNA processing that occur in the nucleus. The first is capping methylation, splicing, and polyadenylation is the third.
  3. Now, if all three events occur on the pre-mRNA the mRNA will be mature containing a cap structure, a polyA tail, and introns are all removed and the mRNA is ready for export to the cytosol for translation into protein.
  1. mRNA Transport Through the Nuclear Pore Complex [S47]
  2. Here is the mRNA associated with the RNA binding protein, forming mRNP complex. So one of the proteins called Mex67/Tap will assemble onto the mRNA and the mRNA will be transported from the nucleus to the cytoplasm.
  3. This is a cartoon to show you mRNA with mRNA binding protein being transported across a nuclear pore.
  4. Remember the nucleus contains a nuclear membrane that is not completely sealed, containing pores from protein and mRNA to get in and out.
  1. 5’ End of mRNA Leaves the Nucleus First [S48]
  2. p We know that mRNA export begins from the 5’ end.
  3. The 5’ end is exported first.
  4. Here is mature mRNA with a cap associated with cap binding protein (CBP), polyA tail associated with polyA binding proteins, and in addition there are proteins assembled on the mRNA called RNA export factors which are responsible for export.
  5. In the cytosol, the cap binding protein will be repressed by the translation initiation factor 4 which is responsible for the translation and some of the protein will be exported together with the mRNA.
  1. Two Mechanisms By Which mRNA Export Factors Are Recruited Onto mRNAs [S49]
  2. There are two mechanisms in which mRNA export factors can be assembled onto the mRNA.
  3. One is dependent on the splicing.
  4. The other is dependent on the transcription.
  5. Two cartoons are given to describe how these factors are assembled onto the mRNA.
  1. Splicing-dependent Recruitment of mRNA Export Factors [S50]
  2. One of the proteins called UAP56 is a spliceosome protein which is involved in splicing.
  3. During splicing, this factor is assembled on the splicing machinery and UAP56 recruits ALY that is important for mRNA export and recruits another factor and can work together to export the mRNA together.
  4. If you delete these factors, the mRNA export cannot occur.
  1. Transcription-dependent Recruitment of mRNA Export Factors [S51]
  2. Yra1 is the same as ALY, same protein but with different name.
  3. The protein is also assembled during transcription because factors can associate with RNA polymerase II and are important for transcription elongation. During the transcription, the factors important for the export of mRNA also associate with the mRNA and can export the mRNA out of the nucleus.
  4. Therefore, the export factors can associate with the mRNA during splicing or transcription.
  1. Fates of mRNAs in the Cytoplasm [S52]
  2. There are three possible fates of mRNA in the cytosol.
  3. Translation: to make proteins from mRNA (not going to cover this in his lecture)
  4. Degradation: shut down the expression of mRNA
  5. Localization: transport of mRNA to specific area in cytosol before translation occurs
  1. The Competition Between mRNA Translation and mRNA Decay [S53]
  2. RNA is circularized because the 5’ cap is recognized by the CBP and polyA tail is recognized by polyA binding protein.
  3. This is a protein-protein interaction between the 5’ end and 3’ endand the mRNA is circularized in the cells and the circularization is important for the translation and can protect the RNA from degradation because both ends are protected.
  4. We also know that, mRNA translation and decay is competitive
  5. If you inhibit translation by breaking the complex, exposing the 5’ or 3’ end to the RNAasewhich degrades RNAs
  6. Now the ends are no longer protect because you inhibited translation and the RNAase can degrade the mRNA making a competition between translation and degradation
  1. Two Mechanisms of Eukaryotic mRNA Decay [S54]
  2. There are two possible mechanisms in which RNA can be degraded
  3. One is for maturity of mRNA that is degraded through this mechanism which is called deadenylation dependent, meaning that the polyA tail needs to be removed first
  4. Remember the polyA tail is bound with the polyA binding protein and protects the mRNA for degradtion and by removing the polyA tail, the binding protein can no longer bind to the polyA tail and degradation can be initiated
  5. This mechanism occurs on most mRNAs that are degraded
  6. In rare events a second mechanism degrades mRNA in which the RNA can be degraded by endonucleolytic cleavages, cleaving in the middle of the mRNA by some enzymes
  7. Requires specific enzymes and specific sequences to cleave
  8. Only happens on some mRNA, not many
  9. After removal of the polyA tail or endonucleolytic cleavage, RNA can be degraded from 5’-3’ end or from 3’-5’ end, by different enzymes that degrade RNA in these two directions
  10. The first step is the only difference between these two mechanisms, after the first step the RNA can be degraded in either direction
  1. mRNA Localization [S55]
  2. I already mentioned that mRNA is exported to the cytosol
  3. It will be directed to some part of the cytosol before translation can begin
  4. mRNA needs to be localized to a specific part because you want translation to occur there and increase the protein concentration there
  5. Localization requires at least three features:
  6. cis-acting elements within the mRNA that targets the message to a subcellular region,
  7. a protein-RNA complex that effects localization by recognition of the sequence
  8. and the cytoskeleton that acts as a “road” for RNA movement
  9. Most sequences are located in the 3’ UTR (untranslated region) of mRNA
  1. The Importance of 3’ UTR in mRNA Localization [S56]
  2. There is a RNA encoding the hairy protein that is localized to the apical part of the nuclei
  3. If you manage to label the mRNA containing the 3’UTR with red fluorescent dye (RED) and label the mRNA without a 3’ UTR with green fluorescent dye (cannot direct mRNA to location), co-inject these mRNA into cell together to see where these different mRNA are located
  4. Find that the mRNA with the 3’UTR is localized in the correct location, but the mRNA without 3’UTR stays inside the cytosol
  5. Shows that the sequence in the 3’UTR is important for localization
  1. RNA Editing: Another Way to Increase the Diversity of Genetic Information [S57]
  2. One last event, we call RNA editing
  3. RNA editing is a process that changes the specific nucleotide sequence to another sequence after transcription and splicing
  4. There are two kinds of RNA editing found in mammalian cells, very rare event, but some lower species use a lot of RNA editing
  5. One is where you change A to I
  6. The other changes C to U
  7. Change one single nucleotide, but there are three different effects:
  8. Alter amino acid coding possibilities, change A I or C U changing one codon/amino acid
  9. Introduce stop codon
  10. When normally the full protein would be translated, introducing a stop codon will produce a shorter form of the protein
  11. Introduces premature stop codon
  12. Change splicing site
  13. 5’ splicing site always contains G-U rich site
  14. 3’ splicing site always contain A-G and
  15. Can stop splicing where it normally occurs
  1. RNA Editing – A to I Editing in Mammals [S58]
  2. The A that is to be edited is shown and around this sequence there is a specialize secondary structure and the A is in the exon and will encode a protein
  3. There is an enzyme called adenosine deaminase acting on RNA (ADAR) and RNA binding protein that recognizes the secondary structure and will change A to I
  4. After editing, the amino acid codon will be changed
  1. Editing of Apolipoprotein B (apoB) mRNA – C to U [S59]
  2. Editing of apoB mRNA depends on where it is expressed, if in the liver there is no editing that occurs and can make full protein about 550kDa and only expressed in liver
  3. If expressed in the intestine, editing occurs on mRNA where C will be changed to U, changing the codon from CAA to UAA which is a stop codon and only the mRNA is only translated up to this point making truncated protein in the intestines
  4. mRNA editing does not often occur in mammalian cells, but here are two examples
  1. Unified Theory of Gene Expression [S60]
  2. Finally, to conclude the post-transcriptional regulation
  3. Even though I described the post-transcriptional events in a linear fashion: capping polyA export one by one, it actually occurs while transcription is taking place
  4. Every event is not a separate event, transcription does not have to be completed for capping for example
  5. Post-transcriptional regulation is part of a continuous process that is connected and affect each other
  6. Regulation occurs at multiple levels in this continuous fashion
  1. Figure 29.46 A Unified Theory of Gene Expression [S61]
  2. This last slides describes the previous
  3. Shows RNA polymerase II transcribing mRNA and as soon as 5’ end is transcribed, about 10-20 nucleotides, the capping is already occurring way before completion of transcription.
  4. Reason for capping at this time is that capping factor is associated with C-terminal domain of RNA polymerase II and capping enzyme can act on mRNA as soon as it sees the 5’ end.
  5. So, capping occurs and RNA polymerase II transcribes the introns and the splicing factors can associate with the mRNA even though splicing does not have to be occurring, but same thing here way before the mRNA is cleaved the mRNA is being exported out of the nucleus.
  6. Everything is a continual process; 5’ end capped and then exported and does not wait for transcription or splicing to be complete.
  7. Post-transcriptional regulation is a continuous process, every step affects each other and transcription and export.