Post-transcriptional and Translational Regulation - SGA - 2024.pptx

st-transcriptional and anslational Regulation Thomas J. Last, Ph.D. CBFM 2024 Presentation created in collaboration with colleagues Drs. Borghaei and Jenny  Material Students Are Expected To Review Pr...

st-transcriptional and anslational Regulation Thomas J. Last, Ph.D. CBFM 2024 Presentation created in collaboration with colleagues Drs. Borghaei and Jenny  Material Students Are Expected To Review Prerequisite lectures to review: Gene Expression Epigenetics and transcriptional regulation Chapter 7: “From DNA to Protein: How Cells Read the Genome”, and relevant sections of Chapter 8: “Control of Gene Expression”, in Essential Cell Biology 5th edition. Alberts, Hopkin, Johnson, Morgan, Raff, Roberts, Walter; W.W. Norton, 2019, IBSN 0393680363 Relevant sections from Chapter 31: “RNA Structure, Synthesis, and Processing”, Chapter 32: “Protein Synthesis”, Chapter 33: “Regulation of Gene Expression” Lippincott's Illustrated Reviews: Biochemistry, 8th edition. Abali, Cline, Franklin, Viselli; Wolters Kluwer, 2021. ISBN: 9781975155063 Students are recommended to watch the linked videos listed in the “Resources” section of this presentation  1. Review the structure of mRNA. 2. Describe the different types of modifications made in maturing mRNA. 3. Describe mRNA splicing and the spliceosome. Describe the importance of alternative splicing and distinguish constitutive vs regulated forms of alternative splicing. Learning 4. Explain the importance of the 3’UTR and polyA tail in regulation of mRNA stability. 5. Describe the importance and mechanism of nonsense Outcome mediated decay of defective mRNAs. 6. Describe the mechanism of miRNA/siRNA regulation of s gene expression. 7. Describe mechanisms of regulation of gene expression in response to levels of iron through elements in the 5’ or 3’ UTR, affecting translation or mRNA stability, respectively (ferritin / transferrin receptor). 8. Define RNA editing and describe the mechanisms, including cell/tissue specificity. 9. Describe translational regulation via eIF2 phosphorylation.  Gene expression is regulated by multiple levels of post- transcriptional and translational control Post-transcriptional control: RNA processing RNA transport and localization mRNA stability/degradation Translational control: Regulate initiation of translation Role of microRNAs Image from: https://documen.site/download/post-transcriptional-gene-control_pdf  Processing of messenger RNAs in Polyadenylation eukaryotic cells Four modifications to heterogeneous nuclear RNA (hnRNA): 1.5’methyl cap 2.Splicing 3. 3′ polyadenylatio n 4.RNA Editing  The 5’ Methyl Cap Guanine is first added by the enzyme guanylyl transferase “Backward” 5’ to 5’ linkage Then the guanine is methylated to 7-methylguanine The first 1 or 2 ribonucleotides of mRNA also methylated Added by capping enzymes that are carried on the phosphorylated C terminal domain of RNA pol II. (In addition to mRNAs, microRNAs also have methyl caps) Functions: Marks the 5’ end of mRNA mRNA stability Transport of mature mRNA from nucleus to cytoplasm Necessary for efficient translational initiation Transcriptional Termination and Polyadenylation mRNA is cleaved downstream of a polyadenylation signal (AAUAAA) Poly-A-polymerase adds ~200 A’s to the 3’ end, poly A binding protein binds to poly-A tail RNA pol continues to transcribe downstream of the cleavage site A nuclease binds the uncapped 5’ end and degrades the trailing RNA. When the nuclease reaches RNA pol, transcription is terminated. The Poly A tail is added in the nucleus, but in some cases, it can be lengthened in the cytosol Functions of the poly A tail mRNA stability Involved in transport of mature mRNA from nucleus to cytosol Contributes to efficient translational initiation  mRNA splicing Exons are defined by consensus sequences: Splice Donor: GU at the 5’ boundary of the intron Splice Acceptor: AG at the 3’ boundary of the intron Branch Point: A  snRNPs recognize splice sites Small nuclear RNAs (snRNAs) form a complex called a spliceosome with small nuclear ribonucleoproteins (snRNPs) and other proteins. Process requires extensive ATP hydrolysis U1-U6 RNA portions recognize splice sites via complementary base-pairing  Systemic lupus erythmatosis (SLE), is an autoimmune disease that causes chronic inflammation and tissue damage 98% of patients have autoantibodies directed against nuclear antigens (ANA), which include anti-snRNP antibodies  Alternative RNA splicing Alternative splicing produces multiple related proteins, or isoforms, from a single gene. Utilizing different combinations of intron/exon splice junctions in pre-mRNA to produce more than 1 potential mature mRNA Example: Alternative splicing of the mRNA encoding a kinase: alternative exons target the protein to different cellular locations 10 Krebs, Genes XI, Fig 21.22  Alternative splicing Constitutive (intron retention)  Constitutive (including intron retention) results from intron sequence ambiguity and limiting amounts of splicing factors o “better” sites are recognized first often makes defective mRNAs that are quickly degraded Zheng Jian-Tao, Lin Cui-Xiang, Fang Zhao-Yu, Li Hong-Dong, Intron Retention as a Mode for RNA-Seq Data Analysis, Frontiers in Genetics 11;586, 2020, DOI: 10.3389/fgene.2020.00586 Several versions of the mRNA (and possibly the protein) may be made in Regulated the same cell at the same time.  Regulated usually tissue- or developmental stage- specific often involves addition or removal of a functional domain of the protein involves splicing activators or repressors usually only one version of the protein Figure 6-27 Molecular Biology of the Cell (© Garland Science 2008) expressed in a cell 11  Regulated alternative splicing involves cell-specific activators or repressors Mutations that affect the binding sites of splicing activators or repressors can cause disease due to abnormal splicing 12 Figure 7-96 Molecular Biology of the Cell (© Garland Science 2008)  RNA editing changes the sequence of some mRNAs after transcription May affect > 1600 genes Takes place in the nucleus Requires deaminases, which are expressed in cell/tissue or developmental phase specific manner C to U– creates a stop codon o ApoB100 (LDL) vs ApoB48 (chylomicrons) o ApoB48, made in intestines, lacks the domain that binds the LDL receptor A to Inosine - changes the amino acid o Especially important in brain – receptors and ion channels o Altered editing patterns associated with inflammation, epilepsy, depression, gliomas, ALS Christof and Zaravinos J Transl Med (2019) 17:319 https://doi.org/10.1186/s12967-019- 13 2071-4 FYI Editing can modify protein function, generate new protein products and alter gene regulation. https://yuminthu.github.io/training_class/docs/RNA_editing.html  The mature mRNA has 3 key sections 1. 5’ UTR (or leader) with methyl cap 2. Open Reading Frame (ORF) region to be translated Starts with the first codon of the gene: AUG codon (ATG in the DNA) Ends with a stop codon: (UAA, UAG, UGA) 3. 3’ UTR (or trailer) with poly A tail A gene encoded for a 50 amino acid protein. The 5’ UTR is 50 nucleotides in length and the 3’ UTR is 250 nucleotides in length. The gene has an intron of 500 nucleotides. How long would the mature Add the three mRNA together: sections be? 1. 5’ UTR = 50 nt 2. ORF = 3 x 50 = 150 nt (don’t forget 1 aa = 3 nt!!) 3. 3’ UTR = 250 nt 5’UTR + ORF + 3’UTR = 50 + 150 + 250 = 450 nt Only properly processed mRNAs are exported to the cytosol Proteins mark successful modifications of the mRNA Cap-binding protein (CBC) Poly A binding protein (PABP) Exon-junction complex (EJC) Correct capping, splicing and poly-A tail required for nucleus export proteins to guide mRNA out of nucleus through nuclear pores to cytoplasm Eukaryotic initiation factors (eIFs) recognize proteins bound to 5’ and 3’ ends during initiation of translation  Translation-dependent mRNA- surveillance mechanisms Each step in the production of a mature mRNA transcript provides an opportunity for introducing errors. To preserve translational fidelity, the cell has evolved means for detecting and degrading aberrant transcripts, thereby protecting it from potentially toxic protein products. Three translation-dependent surveillance mechanisms: Nonsense-mediated decay (NMD). Detects and degrades transcripts that contain premature termination codons (PTCs). PTCs can arise from mutations, frame-shifts, inefficient processing, leaky translation initiation and extended 3′ UTRs. These transcripts, if translated, could produce truncated proteins with aberrant functions. Non-stop decay (NSD). Targets mRNAs that lack a stop codon. Such transcripts can be generated by breakage, or by the absence of an in-frame stop codon, causing translation to proceed along the poly(A) tail. No-go decay (NGD). NGD prevents the sequestration of translation factors to faulty transcripts by detecting stalled ribosomes on an mRNA and endonucleolytically cleaving the mRNA near the stall site. This releases the stalled ribosome and mRNA fragments, which are decayed by the exosome and Xrn1 Garneau, N., Wilusz, J. & Wilusz, C. The highways and byways of mRNA decay. Nat Rev Mol Cell Biol 8, 113–126 (2007). https://doi.org/10.1038/nrm2104  Nonsense mediated decay (NMD): a form of RNA Surveillance Decays aberrant or defective mRNAs quickly Early nonsense codon Unspliced intron Extended 3’UTR Exon junction complexes bind at exon-exon junctions after spicing in the nucleus A pioneer round of translation (test run) occurs in the cytosol near the nuclear membrane If all EJC are removed before the stop codon is reached, all is well and the mRNA can be translated normally If translation stops and EJC remains, nucleases bind and degrade the mRNA 18  Nonsense -mediated mRNA decay Failure to eliminate mRNAs with PTCs may result in the synthesis of abnormal proteins that can be toxic to cells through dominant-negative https://commons.wikimedia.org/wiki/File:NMD_-_Nonsense-mediated_decay.png or gain-of-function effects. https:// 19 www.nature.com/  Control of normal mRNA stability The longer it takes for an mRNA to be degraded (i.e. the longer its half-life), the more protein can be translated from it Some mRNAs have long half-lives, are very stable Typically “housekeeping” genes that encode proteins needed constantly for the basic functions of the cell Most mRNAs have much shorter half-lives Some have very short half-lives, are very unstable mRNAs encoding growth factors, transcription factors, regulatory proteins whose expression levels need to change rapidly Degradation of some mRNAs can be regulated, resulting in different half-lives under different conditions Regulation usually depends on sequences in the 3’UTR 20  mRNA Stability & Translation Gene expression can be controlled by regulation of mRNA half-life Changes in mRNA-decay rates account for a large proportion of regulated gene expression. Length of Poly A tail correlates with half life Longer tail = longer half life AU-rich sequences in 3’-untranslated region promotes removal of poly-A tail Specific regulators are sometimes involved with modifying mRNA half life Example: regulation of transferrin receptor mRNA When iron is scarce, transferrin receptor RNA is protected from 21  Mechanisms of normal mRNA degradation The predominant pathway initiates with deadenylation. mRNA degradation usually starts with gradual deadenylation Subsequently, the mRNA can either undergo decapping and 5′→3′ decay, or 3′→5′ decay. A critical threshold of poly-A tail length induces 3’-to-5’ degradation, which may be triggered by the loss of the poly-A binding proteins The mRNA can also be rapidly degraded after removing the 5’ methyl cap The enzymes involved in the 5’ to 3’ pathway are concentrated in P bodies Garneau, N., Wilusz, J. & Wilusz, C. The highways and byways of mRNA decay. Nat P bodies are cytoplasmic Rev Mol Cell Biol 8, 113–126 (2007). https://doi.org/10.1038/nrm2104 structures where RNA decay occurs. These two processes can occur together on the same mRNA molecule Competition between mRNA decay and initiation of translation Anything that affects the efficiency of translational initiation will tend to have opposite effects on mRNA degradation and vice versa. 23  Regulation of mRNA stability by 3’UTR ARE sequences AUUUA elements (AREs) are https://mol- recognized by proteins that biol4masters.ma sters.grkraj.org/ html/ increase the rate of Ribose_Nucleic_A cid8- Stability_of_mRN deadenylation As_and_its_Regul ation.htm At certain times other proteins compete for binding and stabilize the mRNA These RNA-binding proteins are regulated by extracellular signals, such as growth factors and hormones. https://www.semanticscholar.org/paper/Macromolecular-Degradation-Systems- and-Aging.-Nakayama-Nishida/ d529894d7562bff125467c753807aaee313b9396/figure/3 24 Regulation of Translation 25 Translational regulation Effects protein levels more quickly than transcriptional regulation More useful for short-term regulation Important form of developmental regulation Some mRNAs are very abundant in oocytes, but their translation is prevented until the appropriate time Especially important in enucleated cells (ie rbc) in which transcriptional control is not possible Initiation is the rate-limiting step, most regulated 26 Overview of translational regulatory mechanisms Regulation includes translational repressor proteins and noncoding regulatory RNAs (microRNAs and siRNAs). Translation rates can be controlled by initiation factors and translational repression Examples of control by initiation factors: Phosphorylation of eukaryotic initiation factor 2 (eIF-2) inhibits translation globally mTOR phosphorylating eIF4E-binding protein 1 (4E-BP) increases translation globally Translational repression Ferritin has an iron response element (IRE) Regulating mRNA stability Global translational activity of cells is modulated in response to cell stress, nutrient availability, and growth factor stimulation. 27  Regulation of translation initiation by phosphorylation of eIF2 and eIF2B Translation can also be regulated by modification of initiation factors. This results in global effects on overall translational activity rather than translation of specific mRNAs. Phosphorylation of eIF2 and eIF2B by regulatory protein kinases blocks exchange of bound GDP for GTP, inhibiting initiation of translation. Prevents recycling of eIF-2 Four different eIF2 kinases cause global inhibition of translation of most mRNAs in response to different types of cell stress  Regulation of Example: Heme levels control eIF2 phosphorylati translation by eIF2 1. 2. phosphorylation status Certain conditions, such as: Absence of nutrients Absence of heme Viral infection Accumulation of misfolded proteins cause eIF2 phosphorylation, which in turns halt translation 1. Heme prevents phosphorylation and inactivation of eIF2 by binding to HCI (Heme Controlled Inhibitor of eIF2). 2. Absence/deficiency of heme causes eIF2 phosphorylation by HCI (Heme controlled inhibitor/ kinase). When eIF2 is phosphorylated by heme kinase, it is inactive, and protein synthesis cannot be initiated, which in turn halts29  The unfolded protein response (UPR, aka the ER stress response) Global decreased rates of translation for most proteins By phosphorylating eIF2-GDP eIF Prevents recharging of eIF-2 to 2 the GTP-bound state However, selective increased synthesis of chaperones and transcription factors that regulate their expression Increased protein degradation to clean up the mess If efforts to resolve ER stress are chaperones unsuccessful, the cell undergoes apoptosis 30  Regulation of translation initiation by eIF4E and mTOR signaling Regulation of eIF4E: growth factors activate protein kinases that phosphorylate regulatory proteins (eIF4E binding proteins, or 4E-BPs). AKT/TSC/Rheb/mTORC1 pathway In the absence of growth factors, the nonphosphorylated 4E-BPs bind to eIF4E and inhibit translation. by mTOR  Iron Metabolism & Transport Transferrin Receptor is a carrier protein for transferrin. Transferrin is an iron-binding protein Translation regulated by controlling mRNA stability Ferritin is an intracellular protein that stores iron and releases it in a controlled fashion. Keeps iron in a “safe” nontoxic form. Translation regulated by controlling initiation of translation http://www.nature.com/nrm/journal/v9/n1/fig_tab/ nrm2295_F4.html 32 Repression of initiation of translational of ferritin Ferritin has an iron response element (IRE) Low iron causes IRE binding protein (cytosolic aconitase) to bind RNA and block translation Remember Ferritin & Five prime both begin with “F” (IRE in 33 5’ UTR)  Regulation of Transferrin Receptor mRNA stability When iron is scarce, transferrin receptor RNA is protected from degradation Remember: Transferrin and Three prime both start with “T” IRE binding protein (cytosolic aconitase) protects the RNA from 34 degradation when iron is scarce  Small noncoding microRNAs regulate gene expression microRNAs (miRNAs): class of single-stranded RNAs, about 19-25 nucleotides long, produced by cellular genes for the purpose of inhibiting the translation of mRNAs produced by other genes Human genome encodes approximately 2,600 mature microRNAs (miRBase v.22) A particular microRNA may target many different mRNAs A particular messenger RNA may bind to a variety of microRNAs, either simultaneously or in context-dependent fashion Involved in regulation of up to 60% of protein-coding genes – “Fine-tuning” reduction of protein synthesis The miRNA precursors are synthesized by RNA polymerase II and are capped and polyadenylated They then undergo a special type of processing, after which the miRNA is assembled with a set of proteins to form an RNA-inducing silencing complex or RISC. Once formed, the RISC seeks out and binds to complementary sequences on its mRNA target The mRNA target is either inhibits translation and/or induces degradation of target mRNAs.  miRNA processing and mechanism of action The precursor miRNA, through complementarity between one part of its sequence and another forms a double- stranded structure. This is cropped while still in the nucleus, and then exported to the cytosol, where it is further cleaved by the Dicer enzyme to form the miRNA proper. Argonaute, in conjunction with other components of RISC, initially associates with both strands of the miRNA and cleaves and discards one of them. The other strand guides RISC to specific mRNAs through base-pairing If the RNA:RNA match is extensive, Argonaute cleaves the target mRNA However, if there is less extensive base pairing between the miRNA and mRNA, translation of the mRNA will be inhibited Figure 7-112 Molecular Biology of the Cell (© Garland Science 2008) 36  miRNAs can https://www.researchgate.net/ figure/Regulatory-mechanisms-of- oncogenic-and-tumor-suppressor- function as tumor microRNAs-in-tumorigenic- events_fig2_336311311 suppressors or oncogenes  An miRNA that normally targets a proto-oncogene can be said to function as a tumor suppressor – if it gets inactivated the oncogene will be over-expressed  An miRNA that normally targets a tumor suppressor can be an oncogene – if it becomes over-expressed, the tumor suppressor will 37 be under-expressed  RNA interference is a cell defense mechanism Many of the proteins that participate in the miRNA regulator mechanisms also serve a second function as a defense mechanism: they orchestrate degradation of foreign mRNA molecules. This mechanism is called RNA interference (RNAi) Helps in the defense against viruses and transposable elements RNA interference utilizes short RNAs (siRNAs) to silence the expression of genes containing complementary base sequences siRNA act as a guide to locate and destroy foreign RNA 38  Resources & Supplementary Material  Draw it to Know it Resources RNA Transcription I: https://drawittoknowit.com/course/cell-biology/protein-synthesis/transcription/1 163/rna-transcription-i/video?autoplay=true&curriculum=cell-biology RNA Transcription II: https://drawittoknowit.com/course/biochemistry/genetic-information/central-do gma/1164/rna-transcription-ii?curriculum=biochemistry Gene Silencing by microRNAs https://youtu.be/t5jroSCBBwk Regulation of Intracellular Iron Homeostasis https://youtu.be/KyPDyziSetg RNA editing of human apolipoprotein B https://pubmed.ncbi.nlm.nih.gov/22347292/  Regulation of translation initiation by mTOR signaling pathway The mTOR pathway stimulates translation initiation by phosphorylating two key targets: 1. eIF4E binding protein 1 (4EBP) 2. S6K kinase Kong, J., Lasko, P. Translational control in cellular and developmental processes. Nat Rev Genet 13, 383–394 (2012). https://doi.org/10.1038/nrg3184  Translation regulation of ferratin Translation of ferritin (a protein that stores iron) mRNA is regulated by repressor proteins. When iron is absent, iron regulatory protein (IRP) binds to a the iron response element (IRE) in the 5′ UTR, blockingFerritin Remember translation. & Five prime both begin with “F” (IRE in 5’ UTR) https://biology-forums.com/index.php?action=gallery;sa=view;id=460  Iron Conditions LOW cellular iron HIGH cellular iron concentrations : concentrations: Cells need to Cells need to increase their Translation decrease their production of occurs production of RNA degraded transferrin transferrin No translation receptor protein receptor to help transport protein. iron into the cell. Cells need to Cells need to decrease their increase their production of Block translation production of Translation ferritin to safely occurs ferritin. bind the surplus https://www.uptodate.com/contents/image?imageKey=HEME cytosolic iron. %2F56268&topicKey=HEME%2F7105&search=iron %20regulation&rank=1~150&source=see_link  Regulation of translation by miRNAs One strand is incorporated into the RNA-induced silencing complex (RISC). Most miRNAs form mismatches that repress translation. miRNAs may interfere with the activity of eIF4A, which unwind secondary structures in the mRNA and allow ribosome scanning to the initiation codon. miRNAs act in networks with signal transduction pathways, transcription factors and epigenetic effectors  Responsible for fine-tuning regulation of protein levels produced from target genes  The genes encoding them are often regulated by epigenetic signals, and genes encoding epigenetic regulators are often targets of miRNAs  miRNAs function as epigenetic effectors when passed to daughter cells, helping to maintain cell memory Megraw, M Plant Cell 28:286 49  mRNAs bound to miRNAs usually are sent to P-bodies mRNA block from translation by miRNA are sent to cytosolic structures called processing bodies (P-bodies). P-bodies are dynamic structures composed of large assemblies of mRNA and RNA-degrading enzymes, and they are believed to be the sites in the cell where the final destruction of most mRNA takes place. mRNA is decapped & degraded Green = decapping enzyme Red = Argonaute Yellow = colocalization of both proteins, the yellow spots are P-bodies Figure 7-114 Molecular Biology of the Cell (© Garland Science 2008) 50  Intercellular Communication by Exosome-Derived microRNAs in Cancer MicroRNAs produced by one cell can regulate translation in a different cell MicroRNAs can move from one cell into a neighboring cell via gap junctions MicroRNAs can be packaged into exosomes and be taken up by distant cells in the body An exosome is a cell-derived vesicle found in body fluids Micro RNAs produced in one tissue can potentially alter rates of translation in a totally different type of recipient cell Image from: Int. J. Mol. Sci. 2013, 14(7), 14240-14269 51 Study Questions The questions on the following slides were taken from ebooks in the PCOM library or otherwise found online. Some are simple recall questions, some are more like questions you might expect to find on board exams. They are reproduced here as a convenience for you, presented in no particular order and without any attempt to match the level of difficulty to CBFM exams and quizzes. Use them to test your comprehension, but make sure you study all of the Learning Outcomes, even those that aren’t well represented by these study questions. A genetic study to identify loci that increase susceptibility to type II diabetes isolated an SNP in the fourth intron of the 17-exon gene TCF7L2. This gene produces a protein involved in regulating T-lymphocyte function. Examination of the molecular effect of the SNP in the fourth intron demonstrated that in addition to the full-length TCF7L2 transcript, a shorter transcript, truncated on the 3′ end in comparison to the full-length transcript, was present in cells with the SNP. The truncated transcript ends not far from the location of the SNP. Which of the following was created by the SNP in the fourth intron that would explain why the SNP results in the production of a truncated transcript of TCF7L2? A. A new exon–intron boundary (splice donor or acceptor site) B. A new polyadenylation signal C. A new translational initiation codon D. A new transcriptional promoter E. A new transcriptional regulatory protein binding site A resident on the pediatric service asks you to consult on a 6-month-old female infant. She was brought to the clinic with a chief complaint of “bumps” on hands and feet and in her mouth. Her mother reports that the child has had a mild fever and been “very sleepy.” At meal time, she appears cranky and disinterested in eating. Examination of the oral mucosa reveals erythematous macules approximately 2 mm in diameter, some of which appear to be ulcerated. Similar small erythematous papules are visible on her hands and feet. You reassure the mother the condition will resolve but to bring the child back if she doesn’t get better in a week. The responsible pathogen, foot and mouth disease virus (FMDV), expresses an enzyme that specifically degrades the proteins in the mRNA cap- binding complex (eIF4F) in infected human cells. Which of the following processes will be diminished in cells infected with FMDV, leading in part to the lesions observed on the child’s skin and mucosa? A. Addition of the 7-methly-guanosine cap to mRNA B. Polyadenylation of mRNA C. Splicing of mRNA D. Transcription of DNA into mRNA E. Translation of mRNA into protein A 66-year-old woman presents with mild jaundice and upper abdominal pain with radiation to her back, unexplained weight loss, and thrombophlebitis. Following imaging studies, which lead to a diagnosis of pancreatic cancer, she is treated by surgical resection followed with combined irradiation and chemotherapy. You identify her as a candidate for a clinical trial with an investigational new drug that is a small interfering RNA (siRNA) directed against the RAS oncogene that is likely to be expressed in any remaining cancer cells she has. If the drug works as designed, which of the following processes will be most directly affected by the siRNA targeting RAS? A. DNA synthesis B. mRNA splicing C. mRNA synthesis D. Protein stability E. Protein synthesis The pathology report on a patient you suspect has iron deficiency anemia reveals mean corpuscular volume of 72 fL (reference range: 83–97 fL) and mean corpuscular hemoglobin concentration of 23 g/dL (reference range: 32– 36 g/dL). Platelet count is slightly elevated and white blood cell count is within reference range. The concentration of heme in reticulocytes directly regulates the expression of globin proteins, though mRNA levels for globin proteins do not vary regardless of heme concentration. Which of the following mechanisms might account for suppression of globin protein expression in this patient’s reticulocytes? A. Reduced expression of a specific miRNA B. Reduced histone acetylation C. Increased DNA methylation D. Reduced activity of eukaryotic initiation factor 2 alpha (eIF2alpha) E. Reduced activity of eukaryotic initiation factor 4G (eIF4G; 7-methyl-guanosine cap- binding complex) If you replace the 3′UTR sequence of an mRNA with a half-life of 20 min with the 3′UTR of an mRNA with a half-life of 10 h, then the resultant mRNA will have a half-life of A. 10 min. B. 20 min. C. 5 h and 10 min. D. 10 h. E. 10 h and 20 min. In iron deficiency anemia, ferritin levels are low because ferritin mRNA is A. Degraded rapidly in the cytoplasm. B. Not transcribed from the ferritin gene. C. Prevented from being translated. D. Retained in the nucleus. E. Transcribed only in small amounts. Which of the following is the basis for the intestine-specific expression of apolipoprotein B-48? A. DNA rearrangement and loss B. DNA transposition C. RNA alternative splicing D. RNA editing E. RNA interference Deletion of a gene coding for a miRNA precursor is likely to result in A. Increased translation of mRNAs B. Misreading of the genetic code C. Reduced stability of mRNAs D. Acetylation of histones and enhanced transcription E. Repression of retroposition by mobile elements