Gene Expression & Regulation - Part 2 PDF

Gene Expression & Regulation – Part 2 Regulation of mRNA Processing & Decay, and Translation Transcript of Video Presentation SLIDE 1 This presentation is the continuation of our discussion of the regulation of gene expression in human...

Gene Expression & Regulation – Part 2 Regulation of mRNA Processing & Decay, and Translation Transcript of Video Presentation SLIDE 1 This presentation is the continuation of our discussion of the regulation of gene expression in human cells. In the first part, we discussed the regulation of transcription, the first step in gene expression. We looked at how transcription is regulated during cell differentiation and development by transcription factors and changes in chromatin structure. We saw two examples of how transcription can become dysregulated in human disease: In fragile X syndrome, a nucleotide repeat expansion mutation induces heterochromatin formation in the promoter of the FMR1 resulting in the repression of transcription of FMRP mRNA. In beta- thalassemia, a mutation in the promoter of the beta globin gene leads to loss of hemoglobin beta, resulting in severe anemia. SLIDE 2 In this video learning module, we will continue our discussion of the regulation of gene expression by focusing on the control of mRNA processing and protein biosynthesis, known as translation. We will begin with two case presentations, then look at mRNA processing and the effects of mRNA splice site mutations. Then we will consider the regulation of translation initiation and mRNA stability. Lastly, we will return to our cases to use our knowledge of regulation of RNA processing and translation to resolve our cases. SLIDE 3 Here are the learning objectives for our presentation. These objectives form the basis for quizzes and exams. SLIDE 4 Our two cases involve the design of the mRNA vaccine against COVID-19 and the case of a child with impaired mobility. SLIDE 5 In Case 1, one of your fellow students is skeptical about getting vaccinated for COVID-19 by one of the mRNA vaccines because he doesn’t understand how they are made and how they work. You use your knowledge of mRNA structure and function to explain the concepts behind design and mechanism of the vaccine. SLIDE 6 In Case 2, a four-year-old boy is brought to the clinic by his mother because he appears to have gradually lost physical strength in his legs. A maternal uncle developed impaired mobility and cardiac problems at a very young age and died in his teens, although the family does not know the specific diagnosis for the uncle’s condition. SLIDE 7 We will return to these cases later, but now let’s begin our discussion with the regulation of mRNA processing. SLIDE 8 The primary transcript, termed the “pre-messenger RNA or pre-mRNA” contains all the exons and introns encoded in the DNA sequence of a protein-encoding gene. About 95% of human messenger RNAs contain introns, which are non-coding sequences that must be removed prior to translation by a process known as splicing. The pre-mRNA is also devoid of the 7- methylguanosine cap at its 5’ end and the poly(A) tail at its 3’ end. During mRNA processing the 7-Me guanosine cap and the poly(A) tail are added, and all the introns are removed, and the exons are ligated together to form the mature mRNA. SLIDE 9 The 7-Me guanosine cap is added to the 5’ end of the transcript when the transcript is about 20 nucleotides long, just after it emerges from the active site of RNA polymerase II. The factors that are involved in capping sit on a long amino acid sequence comprised of 52 repeats of a 7- amino acid sequence located at the end of the largest subunit of RNAP II, termed the carboxy- terminal domain or CTD. The CTD is rich in the amino acids, serine, threonine, and tyrosine – amino acids that carry hydroxyl groups on their side chains, and which are substrates for phosphorylation. During the movement of RNAP II from the promoter to the transcription termination signal and release of the processed mRNA from the DNA template, these amino acids in the CTD become differentially phosphorylated to sequentially bind different mRNA processing proteins. This sequential binding of the factors onto the CTD and delivery to the pre-mRNA as it is transcribed facilitates the orderly processing of the pre-mRNA to the mature mRNA. SLIDE 10 As shown on the lefthand side of the slide, a unique feature of the 7-MeG cap is the 5’ to 5’ triphosphodiester linkage between the 7-MeG and the first base that was complementary to the DNA template. Normally, all nucleotides in DNA or RNA are linked together by a 5’ to 3’ monophosphodiester linkage. The 7-MeG cap is added post-transcriptionally – that is, it is not encoded in the genome, but is added immediately after transcription begins. The purpose of the 7-MeG is three-fold. First, it protects the 5’ end of the mRNA from degradation from cellular nucleases. Second, the cap acts as a docking signal for the mRNA at the nuclear pore prior to transport of the mRNA from the nucleus to the cytoplasm. Third, the 7-MeG cap is the entry site for the ribosome during translation in the cytoplasm. SLIDE 11 Another post-transcriptional pre-mRNA processing reaction is the addition of a polyadenylate tail or poly(A) tail to the 3’ end of the pre-mRNA. The poly(A) tail protects the mRNA from degradation by cellular exonucleases; the poly(A) tail also acts in consort with the 7-meG cap to confer directionality to mRNA nucleocytoplasmic transport. The capped 5’ and enters the nuclear pore first, and the poly(A) tail is the last part of mRNA to be extruded through the pore. Another function of the poly(A) tail is to facilitate translation of the mRNA, where it loops around to help stabilize the binding of the ribosome to the 7-MeG cap. SLIDE 12 95% of human mRNAs contain introns – ranging in number from just few to dozens or even hundreds in the larger mRNAs. The average number of introns is around 10. A key question is how are the introns sequentially removed so that the exons are spliced together in the correct order? As we saw in the case for the 7-MeG capping reaction, the splicing factors required for removal of the introns are delivered from the CTD on RNA polymerase II to the junctions between the exons and the introns on the pre-mRNA as these sites emerge from the active site on RNA polymerase II. Thus, each intron is removed sequentially as it becomes exposed to the splicing apparatus. If splicing happened after the complete pre-mRNA had been synthesized and released from RNA polymerase, instead of co-transcriptionally, splicing out the introns and putting the exons together in the correct order would be extremely difficult if not impossible to accomplish. SLIDE 13 Splicing is carried out by a large multi-component complex called the spliceosome comprised of over 100 proteins and 6 small nuclear RNAs called the U-rich RNAs. Each of the small nuclear RNAs form individual units with a specific set of proteins to form small ribonuclear protein complexes abbreviated snRNPs and referred to as “snurps.” Each snurp is responsible for an individual step in the splicing reaction. SLIDE 14 In the first step, the 2’ hydroxyl group on an adenosine at a location termed the branch point attacks a phosphodiester bond at the junctions between the 3’ end of the Exon 1 and 5’ end of the Intron 1 forming a covalent bond between the adenosine and the 5’ end of the intron. In the second step, free 3’ hydroxyl group on the end of exon 1 cleaves the 3’ intron-exon splice junction at the 5’ end of Exon 2, releasing the lariat shaped intron. Lastly, the Exons 1 and 2 fuse together. This reaction is repeated sequentially until all introns have been removed. SLIDE 15 One of the most interesting stories in medical research is how the components of the splicing apparatus were identified and the steps in splicing were elucidated – a wonderful example of how sometimes a clinical finding can lead to a basic science discovery. It turns out that patients with the autoimmune disease lupus erythematosus make antibodies to their own tissues and proteins, causing severe chronic inflammation. Some of these antibodies are made to the snRNP spliceosome components. Researchers used these antibodies to purify each of the snRNPs to understand their structure and roles in the splicing reaction. SLIDE 16 This led to the understanding of the stepwise assembly of each of the snRNPs during the splicing reaction and how each step is catalyzed to lead to the final spliced mature mRNA. Each snurp binds sequentially to a different site on the pre-mRNA to guide the cleavage, lariat formation, and ligation reactions. SLIDE 17 When all splice sites in a pre-mRNA are used and all exons are fused sequentially in their original order to make the mature mRNA, we call this constitutive splicing, as shown in the left-hand splice product. However, in almost 90% of mRNAs, alternative splicing patterns can occur, sometimes generating new amino acid sequences in the resulting protein due to the extra codons newly included from the unremoved intron. In this manner, regulated alternative splicing patterns can produce more than one functional protein from a single gene. Thus, one of the surprising and complicating aspects of mRNA splicing is that some introns can become exons, and some exons can become introns, depending on how splicing is regulated. SLIDE 18 Here is an example of how 3 different functional proteins were derived from the same gene by alternative splicing. In protein A, all the introns were removed, and all the exons were fused together, in other words this was constitutive splicing. In protein B, exon 3 was removed as an intron. In protein C, exon 4 was removed as an intron. There are many examples of differential splicing creating new protein sequences, and this is how over 50,000 functionally different proteins with different amino acid sequences can be generated from just 20,000 protein coding genes. SLIDE 19 To underscore the diversity of proteins that alternative splicing can provide, here are several examples of alternative splicing patterns. Notice that splice site utilization is still consecutive in alternative splicing. Exon 4 can never come before exon 1, 2 or 3, but some exons can become introns and be removed, while some introns can be retained and become exons. SLIDE 20 This immediately brings up the question of how alternative splicing is regulated. The regulation of splice site utilization occurs by repressor and activator proteins that bind near splice junctions or at the brand point. The splicing factors either inhibit active splice sites or enhance weaker infrequently used splice sites. The expression of the splicing repressors and activators is tissue specific and often explains why a specific protein is made in one tissue, while an alternative protein from the same gene is generated in another tissue. As we discussed in the session on transcriptional regulation, cell differentiation and organ development are based on differential gene expression, and alternative splicing is almost as important as transcriptional regulation in regulating differential gene expression. SLIDE 21 The clinical significance of mutations that cause dysregulation of mRNA splicing is immense. Dysregulation of mRNA splicing is responsible for a very large number of human genetic diseases. The inclusion of a normally discarded intron into the mature mRNA invariably leads to the generation of a premature translation termination codon in the mature mRNA. The explanation for this is that while evolution normally selects against premature termination codons inn coding regions of genes, no such evolutionary pressure against premature termination codons exists within intron sequences, unless they are used as alternative exons in alternative splicing. We will soon see the significance of abnormal intron inclusion in one of our cases. SLIDE 22 Let’s now look how translation can be regulated. There are many ways to regulate translation, but we will focus exclusively on two prototypical systems that regulate translation at the initiation step. The first example is the regulation of the translation of ferritin mRNA by the iron response element and its binding protein. The second example is the regulation of cell growth- related translation by growth factor signaling via the AKT-mTOR pathway. SLIDE 23 Let’s start with a very brief review of translation in human cells. During translation of protein- coding mRNAs, the 80S ribosome reads mRNAs three nucleotides at a time – referred to as codons. There are 61 codons that specify the various 20 amino acids, with many of the amino acids being specified by multiple codons. Thus, we say that the genetic code is degenerate. SLIDE 24 As the 80S ribosome moves down the mRNA and reads the codons, tRNAs bring amino acids into the active site of the ribosome. There are two sub-sites: The A site is where the next codon of the mRNA will be read and the cognate aminoacyl tRNA will bind. The P site is where the peptidyl tRNA carrying the polymerized peptide chain resides. As the ribosome moves down the mRNA, the peptide chain on the peptidyl tRNA in the P site is transferred to the next amino acid on the tRNA in the A site. The empty tRNA in the P site is ejected, and the newly extended peptidyl tRNA in the A site moves to the empty P site as the ribosome move over one codon. This is known as the peptidyl transferase reaction, and it is one of the most important biochemical reactions in the cell, for it forms the basis for how protein-encoding sequences in the genome direct the synthesis of proteins. SLIDE 25 The step in translation that is most often the target of regulation is initiation. The first step in initiation occurs when the 40S ribosomal subunit gains access to the mRNA via the 7-MeG cap at the 5’ end of the mRNA or in some cases enters internally and finds the translational start site. Internal ribosome entry occurs most often when ribosomes translate viral RNAs without a cap, but it can happen with some cellular mRNAs as well. Once bound to the mRNA, the 40S ribosomal subunits scans in the 5’ to 3’ direction until it finds an AUG start codon. The 40S subunit is then joined by the 60S subunit to form the translationally competent 80S ribosome. SLIDE 26 All the steps in translation initiation are guided by a myriad of translation initiation protein factors. In the original “scanning hypothesis,” which was first formulated to explain translation initiation, it was proposed that the 40S subunit chooses the first AUG that it encounters as the site for translation initiation. But the discovery of many exceptions to this rule led to a reformulation known as the “modified scanning hypothesis” where the AUG must be in the proper context of other flanking bases to be chosen as the start codon. Knowing the details of translation initiation provides a basis for understanding the mechanisms by which translation is regulated. The following are two examples. SLIDE 27 Our first example of the regulation of translation is the control of the synthesis of the iron storage protein, ferritin. Iron is necessary for the function of proteins such as myoglobin and hemoglobin, but free iron cannot be allowed to build up in the bloodstream or in tissues and organs due to its propensity to react and cleave DNA and proteins. To protect the cell, the protein ferritin functions to bind and store iron until it is needed for hemoglobin and myoglobin synthesis. As the level of iron increases due to excessive intake or in certain diseases, the level of ferritin must be increased to allow for proper iron storage. SLIDE 28 The cell controls ferritin synthesis by the action of a repressor protein that binds to a site termed the iron response element or IRE just in front of the start codon for translation of the ferritin mRNA. When iron levels are low and ferritin is not needed in high amounts, the iron response element binding protein binds to the IRE, thereby blocking the scanning 40S ribosome from reaching the ferritin start codon. When iron levels increase and ferritin is needed, iron binds to the IRE binding protein, eliciting a conformational change in the protein, causing the IRE binding protein to dissociate from the IRE, and allowing the 40S ribosome to reach the start site and initiate translation of ferritin. SLIDE 29 Another important example of the regulation of translation is control of cell growth, proliferation, and cell death by the protein AKT, also known as protein kinase B. AKT signaling is often dysregulated in cancer cells. AKT works in conjunction with a protein called “mammalian target of rapamycin” or mTOR. The antibiotic rapamycin is a chemotherapeutic agent that has been used to treat certain cancers and it binds to mTOR. AKT phosphorylates many important proteins in the cell that govern cell growth and proliferation in response to the nutritional status of the cell and damage to the genome. When cells are stressed due to starvation or DNA damage, AKT responds to downregulate cell growth and proliferation, and in extreme cases to upregulate cell death. SLIDE 30 Growth factors stimulate cell growth and proliferation by binding to receptors on the plasma membrane. First, the growth factor binds to a receptor that signals an important cytosolic enzyme phosphotidylinositol 3 kinase or PI3K. PI3K signals the inositol second messengers PIP2 and PIP3 through phosphorylation. This PI3K-mediated signaling at the membrane stimulates AKT activation. AKT works in conjunction with mTOR to activate ribosomal protein synthesis and ribosome biogenesis. This allows for more ribosomes to supply the growing cell’s need for proteins. SLIDE 31 AKT-mTOR removes a protein that normally blocks the entry of the 40S ribosomal subunit at the 7-MeG cap when protein synthesis is turned off by starvation. With ribosome entry at the 7-meG cap now unimpeded, cap-dependent translation of multiple mRNAs involved in cellular function including growth, proliferation, and motility is maximized. It is evident that if the AKT- mTOR pathway becomes constitutive due to mutations in genes encoding pathway regulators, the cell will become independent of growth factors and grow and proliferate out of control leading to cancer. SLIDE 32 The next regulated process we want to explore is the control of mRNA stability. Normal mRNA decay occurs once an mRNA is no longer needed. In some cases, mRNAs can last many hours and be translated multiple times. In other cases, where only a small amount of protein needs to be generated and would be toxic to the cell in higher amounts, there are sites in the 3’ UTR that make such mRNAs unstable. SLIDE 33 Control of mRNA stability is a major regulatory mechanism of gene expression. mRNAs decay through the action of exoribonucleases. Some exoribonucleases can rapidly degrade the mRNA from the 5’ end through removal of the 7-MeG cap, termed decapping, allowing the mRNA to be progressively hydrolyzed. Other exonucleases can slowly degrade the mRNA from the 3’ end by chewing at the poly(A) tail. SLIDE 34 A very important mechanism for control of the stability and translation of selected mRNAs is through the small noncoding RNAs termed microRNAs. The story of the discovery of microRNAs is a wonderful example of how a seemingly unimportant finding in a lower organism can have a huge impact on medicine. In 1993, researchers reported the discovery of a small RNA in the segmented nematode, C. elegans, that stimulated the degradation and translational interference of a specific mRNA. While intrinsically interesting, the discovery did not stimulate much news, primarily because it was found in a worm. Then seven years later a second small RNA was discovered in C. elegans. But this second discovery coincided with the publication of the first draft of the human genome, and in this case the authors of the paper looked to see if a similar RNA was encoded by human DNA – and it was! Since that time, over 2,000 21-22 base microRNAs have been identified in humans. Each miRNA contains an 8-14 base sequence called the “seed sequence” that is complementary to a specific mRNA that it is intended to downregulate. SLIDE 35 Some miRNAs can regulate multiple mRNAs that all carry the same miRNA binding site sequence complimentary to the seed sequence. The miRNA recognitions sequences on most mRNAs are locate in the 3’ UTR of the mRNA, but in some instances, one can find miRNA binding sites in the 5’ UTR or even sometimes in the coding region of the mRNA. A miRNA acts in conjunction with bound proteins. This complex is referred to as the RNA silencing complex, or RISC. Once the miRNA RISC finds its binding site on the mRNA that it is supposed to regulate, the RISC degrades the mRNA, or it directs the mRNA is be moved to a storage compartment in the cytoplasm, hidden from the ribosomes, thereby inhibiting translation. In either case, expression of the mRNA is no longer possible SLIDE 36 There are two pathways for the synthesis of microRNAs. Some microRNAs are initially transcribed from their own genes as much larger precursor transcripts called pri-miRNAs. Other microRNAs are generated from introns during splicing, which underscores that introns are not always junk to be discarded. In either case, the larger pri-miRNA folds back on itself to form a hairpin-shaped structure that is processed in the nucleus to form the pre-miRNA and then exported to the cytoplasm. In the cytoplasm small hairpin miRNA is processed by a nuclease called dicer into a double-stranded 21-22 base pair miRNA. The duplex is then separated, and the so-called passenger strand is discarded, while the final miRNA is incorporated into a complex with the protein called argonaute or Ago. This complex binds to its mRNA target to effect either mRNA degradation or translation inhibition.. SLIDE 37 The importance of miRNAs has become central to our understanding of the regulation of cell differentiation, growth, and development, and is critical to stem cell regulation. mRNA levels are more robustly controlled by transcription initiation and mRNA splicing; however, the fine tuning by miRNAs optimizes gene expression, and is especially important in the differentiation of stem cells. Genes encoding microRNAs have been found to play a central role in cancer, metabolic, cardiovascular, and neurological diseases. In the case of cancer, miRNA expression profiles are different for each tumor and are being explored for use in clinical diagnosis and prognosis. SLIDE 38 Now let’s wrap up our cases. In Case 1 it is important to first consider how the COVID-19 corona virus propagates. SLIDE 39 The virus enters the cell by attaching to the ACE2 receptors of the cells in respiratory tract via the spike protein on the viral capsid and is then endocytosed into the cell. Once inside the cell, the virus disassembles to release its single-stranded RNA genome, which is translated by the cell’s own translation machinery. The viral encoded replicase replicates many copies of the viral genome, which are then packaged by newly translated viral assembly proteins. The new viral particles are released from the infected cells by exocytosis. SLIDE 40 The strategy of the COVID-19 vaccine is to create a synthetic mRNA that encodes the spike protein. The mRNA contains all the essential sequences we have previously described that are required for expression: the 5’ 7-MeG cap, the 5’ UTR, the protein coding region, the 3’ UTR and the poly(A) tail. SLIDE 41 One additional feature of the synthetic viral mRNA is that the uridine residues in the mRNA are substituted with methylpseudouridines, which make the mRNA resistant to cellular nucleases and reduce the antigenicity of the mRNA so that it won’t be recognized as a foreign mRNA to be destroyed. The synthetic spike protein mRNA is delivered to cells in a lipid encased hydrophobic nanoparticle that can penetrate the cell membrane. SLIDE 42 Once inside the cell, the mRNA is translated to make spike protein, which is then displayed on the outside surface of the cell to illicit an immune response from B-cells and T-cells. SLIDE 43 In Case 2, our patient is showing the classic signs and symptoms of Duchenne muscular dystrophy: difficulty walking at a young age; enlarged calf muscles due to substitution of fat cells for dying myocytes; and the Gower sign, which characterizes the difficulty in rising from a lying or sitting position to assume an erect position. He also has a maternal uncle who most likely died of the same or similar disease many years ago, indicating that the disease was passed down by maternal inheritance, and suggesting an X-chromosome linked disorder. SLIDE 44 Duchenne muscular dystrophy, abbreviated DMD, is an inherited myopathy due to mutations in the X-linked DMD gene encoding the muscle cell protein dystrophin. Dystrophin is a large filamentous protein that attaches the sarcomeres to the muscle cell membrane termed the sarcolemma. DMD is relatively common for an inherited myopathy with one case per 3500 births. DMD causes progressive loss of striated muscle function due to death of myocytes and replacement of muscle cells with fat cells. Only males are severely affected because females usually have an unmutated copy of the dystrophin gene. One exception is in females carrying DMD mutation who also have Turner syndrome where there is only one X chromosome. Also, because 50% of cells in a female’s body have one of the X chromosomes inactivated, females carrying a DMD mutation can sometimes show a very mild muscle weakness phenotype. SLIDE 45 The dystrophin gene is expressed primarily in cardiac and skeletal muscle. The DMD gene encodes the largest mRNA in the genome – over 2.5 million bases with 79 exons. The mature mRNA after splicing contains 14,000 bases. Interestingly, many mutations that cause DMD are located within introns or at splice sites. These mutations cause introns to be retained or cause an essential exon to be spliced out. In the case of intron retention, as we have discussed previously, the chances are high that a premature termination codon will be encountered as the ribosome enters the retained intron. The consequences of premature termination codons can be extremely serious. SLIDE 46 There are three termination codons out of the 64 possible codons: UAA, UGA, and UAG. SLIDE 47 If an insertion or deletion mutation in a protein coding sequence leads to translational reading frame-shift mutation, the chances of encountering a premature termination codon are three out of 64 possible codon or about 1 in 20 as the ribosome translate these sequences. Similarly, if an intron is retained, the chances of encountering a stop codon once the ribosome enters the retained intron are 1 in 20 codons. SLIDE 48 Truncated proteins due to premature translation termination are extremely toxic to the cell. Most truncated proteins are not fully functional and can compete with their full-length counterparts, often disrupting protein assemblies and metabolic pathways. Cells have developed an important pathway called the nonsense-mediated decay or NMD pathway to degrade mRNAs with premature termination codons to prevent them to ever being translated. NMD will lead to complete loss of the protein. The mutant truncated protein is almost never detected. SLIDE 49 In addition to DMD, examples of common serious genetic diseases that are due to frameshift mutations in coding regions or retained introns include beta-thalassemia, and BRCA1- associated breast cancer. SLIDE 50 Nonsense mediated decay is carried out by a protein assembly that scans the mRNA looking for premature stop codons, guided by the protein complexes termed the exon junction complexes or EJCs, which are deposited at each exon-intron boundary, SLIDE 51 The efficiency of the EJC in effecting NMD is dependent on its position within the mRNA. If a premature stop codon is located after the last EJC, then NMD is ineffective, and a truncated protein is generated. In some cases, the cell degrades the truncated protein via the proteosome pathway. If not, the truncated protein may disrupt cellular function. SLIDE 52 Because most of the mutations that cause Duchenne muscular dystrophy are frameshift mutations or splicing mutations leading to retained introns, there is a complete loss of dystrophin from the muscle cells, due to nonsense mediated mRNA decay. This is the cause of early death due to cardiac or respiratory failure. In a later session, we will discuss the use of new therapeutic agents designed to correct or bypass translational reading frameshift mutations. SLIDE 53 However, there is a milder form of a disease related to mutations in the DMD gene, called Becker muscular dystrophy or BMD, which is caused by point mutations and short insertions or deletions in the dystrophin gene that do not disrupt the translational reading frame. In BMD, a partially functional dystrophin protein is produced that results in the increased age of the first clinical symptoms and increased lifespan. The age of onset of Duchenne muscular dystrophy is 2-6 years, while the age of onset for Becker muscular dystrophy is usually early to mid-teens and progresses much more slowly. BMD patients can live into their 40s or even 50s. SLIDE 54 These are the take-home messages for this presentation.