G.E. Whiz (Gene Expression) The Life and Death of Eukaryotic mRNA

Learning goals Students will: Explain the role of post-transcriptional regulatory processes in establishing cell diversity. Describe the steps that regulate the production of a functional mRNA. Use information on splicing to explain the paradox that there are fewer genes than proteins. Use a (mathematical) simulation of synthesis and degradation of mRNA to predict the quantitative regulation of mRNA levels.

Teaching Challenges Students often confuse transcription and translation. Discussion of the regulatory events that occur between these two steps may help differentiate transcription and translation in the students’ minds. Control of gene expression at the post-transcriptional level is often overlooked in introductory biology. Biology students do not often apply quantitative approaches (math) to biological questions.

Assessment Content mastery clicker questions problem solving - RNA splicing Peer instruction (group work) – lots of proteins, few genes Data analysis – northern blot Quantitative application/synthesis – mRNA decay Active Learning ExerciseContribution to Diversity Clicker questionsIndividual participation Splicing exercisesTactile/kinesthetic Data analysisPeer instruction & visual learning RNA stability simulationInterdisciplinary approach (quantitative)

Context Introductory (general biology, genetics, cell biology) Large class size (>250). DNA to RNA to protein General features of transcription and translation Biological macromolecules Cellular structure Differential gene expression determines cell type

AB GreenBlue Transcription Genes mRNA Translation protein Genotype Phenotype Which genes are expressed determines the phenotype of the organism

Gene expression is regulated at multiple levels AB GreenBlue Transcription Genes mRNA Translation protein DNA structure Repressors Enhancers 1. Transcriptional RNA splicing RNA trafficking RNA degradation 2. Post-transcriptional Ribosome binding Termination 3. Translational Folding Modification Trafficking 4. Post-Translational PP

Schedule of Activities for the Entire Teachable Unit Step 1: Background: Importance of mRNA splicing in gene expression – hook, group work. Step 2: Mini lecture on pre-mRNA processing: Mini-lecture and animation – clicker question. Step 3: Alternative splicing: Question-driven discussion and a small group activity. Step 4: Export from the nucleus: A mini-lecture with animation. Step 5: Simulation of mRNA levels in the cytoplasm Clicker questions employing graphs and mathematical simulation.

Transcription Pre-mRNA DNA IntronexonIntronexon Intron mRNA RNA splicing, capping, polyadenylation cap AAAAA cap AAAAA storage degradation AA Post-transcriptional processing Export from nucleus cap AAAAA

IAMAPREMRNQRSTVAWHICHHQPBASTRVASBEEQABDFCTVNSPLICED IAMAPREMRNAWHICHHASBEENSPLICED Exon IntronExon Intron I AM A PRE MRNA WHICH HAS BEEN SPLICED Splicing

Alternative Splicing of Tropomyosin IAMTHEQSRTVHEARTQPBASRTVBRAINQABDEVTROPOMYOSIN In the brain IAMTHEBRAINTROPOMYOSIN (I am the brain tropomyosin) Exon Intron In the heart IAMTHEHEARTTROPOMYOSIN (I am the heart tropomyosin)

Schedule of Activities for the Entire Teachable Unit Step 1: Background: Importance of mRNA splicing in gene expression – hook, group work. Step 2: Mini lecture on pre-mRNA processing: Mini-lecture and animation – clicker question. Step 3: Alternative splicing: Question-driven discussion and a small group activity. Step 4: Export from the nucleus: A mini-lecture with animation. Step 5: Simulation of mRNA levels in the cytoplasm Clicker questions employing graphs and mathematical simulation.

Northern Blot of mRNA

What do you notice about the patterns of the different RNAs over time? Describe one mechanism that could account for these patterns Northern Blot of mRNA

Q: Which of the following curves represents the proportion of remaining ND-5 in the blot? B A C D

Half-life of mRNA What does “half- life” mean? What is the half-life of the mRNA shown for curve C? A. About 1 hour B. About 2 hours C. About 15 minutes C

Half-life of mRNA A B C D

If the half-life increases, will the curve become more steep, less steep, or remain the same? Illustrative simulationsimulation B A C D

Synthesis and degradation of mRNA The amount of any specific mRNA in a cell doesn’t only depend on its half- life Often, new mRNA molecules are also being synthesized, processed, and exported from the nucleus

Homework Download the simulation from the course website.simulation BEFORE moving any of the controls, describe the shape of the curve. Does it always increase, always decrease, or does it reach a limit at some point? Increase the half-life of the mRNA and describe how changing this parameter changes the shape of the curve. Save this curve by pressing the “Save Data” button.

Decrease the production rate of the mRNA and describe how changing this parameter changes the shape of the curve. Click on the “Mystery curve” button, and adjust the parameters so that the blue curve matches the green curve. Record the corresponding parameter values. Explore the simulation to your heart’s content and WRITE A QUESTION that you can ask your groupmates tomorrow. Be prepared to share your answers with the rest of the class.

Decrease the production rate of the mRNA and describe how changing this parameter changes the shape of the curve. Click on the “Mystery curve” button, and adjust the parameters so that the blue curve matches the green curve. Record the corresponding parameter values. Explore the simulation to your heart’s content and WRITE A QUESTION that you can ask your groupmates tomorrow. Be prepared to share your answers with the rest of the class.