Exploring the Structure-Function Relationship in RNA Biochemistry

Megan Filbin, Metropolitan State University of Denver

Location: Colorado

Abstract

Many RNA viruses bypass cellular antiviral responses by hijacking protein synthesis machinery to translate viral proteins for replication and packaging. They accomplish this task using structured regions within the RNA genome that directly bind to a subset of translation factors/ribosomal subunits. The goal of this research is to correlate structure to function using a series of mutations that are predicted to alter the RNA secondary structure. Students will design mutants based on a predicted wild type secondary structure and then test how mutations affect function. Key RNA structures determined by these studies would add to a growing body of knowledge about conserved RNA motifs that bind to and regulate cellular translation machinery. In addition to the research goals, students will also learn tools to effectively communicate their hypotheses, methods and conclusions to a lay audience and expert scientist.

Student Goals

  1. Develop a hypothesis based on the working secondary structure and literature about this virus to target specific sequences/structures of the RNA.
  2. Critically evaluate data gathered from their research and formulate a conclusion as well as a new hypothesis for future research.
  3. Write about their research/conclusions in a way that a lay audience can understand. Then present this work orally (including new directions) to a panel of faculty in the department.

Research Goals

  1. Determine the key sequences/structures necessary to drive protein synthesis from a RNA virus.
  2. Determine how these key sequences/structures are functioning.

Context

Senior Experience in Biochemistry (CHE 4960) is a one semester senior capstone course designed for 15-20 biochemistry majors in their final year of classes. The class meets for 170 minutes per week in a laboratory setting equipped with computers for students to conduct both wet lab work, as well as computational work. Many students in this course will have completed Biochemistry I, which equips them with knowledge about RNA structure and will take Biochemistry II simultaneously, which equips them with knowledge about protein synthesis. This project will be the focus for the entire semester.

Target Audience: Major, Upper Division

CURE Duration:Half a term

CURE Design

How is the function of a biomolecule related to its structure? Much like the structure and function of a sport utility vehicle versus a race car, biomolecules like RNA adopt specific and often intricate structures that facilitate a specific function. In the case of many single-stranded, positive-sense RNA viruses, the genomic RNA adopts a structure that hijacks cellular protein synthesis machinery upon infection. Zoonotic viruses contain internal ribosome entry site (IRES) structures that are found in the 5' UTR or between two cistrons. Plant viruses contain cap-independent translation enhancer (CITE) structures within the 3' UTR, that are hypothesized to deliver translation components to the 5' end of the genome via base-pairing to a 5' stem-loop. Both IRES and CITE mechanism(s) vary as a function of RNA structure. The design of this CURE is to directly correlate how the structure of these viral RNAs dictates their ability to bind to and initiate protein synthesis.

The relationship between RNA structure and its function to hijack cellular protein synthesis will be analyzed via two components: (1) wet- and dry-lab, hypothesis-driven laboratory research and (2) written and oral communication to a lay audience and expert, respectively.

(1) Hypothesis-driven Laboratory Research

For this aspect of the course, groups of 2-4 students will use dry-lab (thermodynamic folding and PCR primer design software) and a variety of wet-lab techniques (mutagenic PCR, transformation/plasmid purification, and in vitro translation coupled with chemiluminescence) to model and empirically correlate RNA structure to function. Students will analyze data (including statistical analysis) and graph results in an Excel workbook. Plans, methods, results and analyses will be recorded in an electronic notebook.

(2) Written and Oral Communication to a Lay Audience and Expert

For this aspect of the course, students will write a summary of their findings using plain English and figures of speech so that a lay (non-scientist) may understand the hypothesis, methods, results and conclusions of the project. Students will also design figures that pictorially represent background information or a predicted model based on the results.

Core Competencies: Analyzing and interpreting data, Asking questions (for science) and defining problems (for engineering), Constructing explanations (for science) and designing solutions (for engineering), Developing and using models, Planning and carrying out investigations, Using mathematics and computational thinking
Nature of Research:Basic Research

Tasks that Align Student and Research Goals

Research Goals →
Student Goals ↓
Research Goal 1: Determine the key sequences/structures necessary to drive protein synthesis from a RNA virus.
Research Goal 2: Determine how these key sequences/structures functioning.


Student Goal 1: Develop a hypothesis and methods for testing the hypothesis, based on the working secondary structure and literature about this virus to target specific sequences/structures of the RNA.

Read one review paper about (general/secondary) RNA structure and three primary research articles about the virus we are studying. Write an annotated bibliography about these papers with the goal of highlighting key RNA structures and what methods were used to determine the RNA structure(s) within the primary research studies. (Learn how to navigate Google Scholar, Web of Science, PubMed, and properly cite literature using Mendeley or Zotero.)

Considering the proposed secondary structure of the wild type RNA, design mutants that either disrupt sequence, structure or make compensatory structures.

With two provided reviews, describe how canonical and noncanonical translation works by generating a figure for each process. (Learn how to make a figure/caption in PowerPoint program.)

Read one review paper about RNA-protein interactions and three primary research articles about how the virus regulates protein synthesis. Write an annotated bibliography about these papers, with the goal of highlighting how the viral RNA functions and what methods were used to determine the function(s) within the primary research studies.

Using the information from the annotated bibliography, predict which sequence/structure of the viral RNA is binding/recruiting a translation factor using software that predicts RNA-protein interactions. (Learn how to use RPISeq.)

Propose a hypothesis for how the chosen mutant will affect the function of the RNA. Create a figure of the designed mutant, as well as a model for this hypothesis.



Student Goal 2: Critically evaluate data gathered from their research and formulate a conclusion as well as a new hypothesis for future research.

Succinctly record goals, methods, results and analyses and conclusions in an electronic format. (Learn how to use an electronic lab notebook, like Benchling or LabArchives.)

Evaluate the predicted structure of each designed mutant using thermodynamic RNA folding software. Correlate differences in free energies to differences in structure. (Learn how to use thermodynamic folding prediction software, ViennaRNA, to predict secondary structure of mutant in comparison to the wild type RNA.)

Design primers for mutagenic PCR and create structural mutants in a plasmid encoding the luciferase gene. Purify the plasmid and verify that the PCR worked properly via sequencing. (Learn how to use primer design software.)

Perform in vitro transcription and translation assays with the wild type and mutant luciferase plasmids for the functional experiments. Importantly, perform control experiments as well!

Process the data after collection, including error calculation (SD versus SEM) and create a graph (e.g. pie chart, bar graph or Venn diagram) figure representing data/control(s). (Learn how to calculate/graph using Excel.)

Evaluate whether the data "says" anything about the hypothesis. Write both the result and the conclusion based on the data.

Propose in writing a new question(s) based on the data.



Student Goal 3: Write about their research/conclusions in a way that a lay audience can understand. Then present this work orally (including new directions) to a panel of faculty in the department.

Describe, in a written paper, the research question, hypothesis, experiments and data in plain English in a way that a nonscientist would understand.

Using the research plan, figures, results, conclusions and new questions they've already written about, present a formal (PowerPoint) 15 minute presentation to faculty members in the department. (Learn how to properly utilize PowerPoint in presentation mode.)


Instructional Materials

Please see the suggested timeline for a 16-week semester: CURE_Timeline.pdf (Acrobat (PDF) 81kB Oct19 19)

I have found the iBiology videos extremely useful for background information that students can watch at their leisure and multiple times throughout the semester. Here is the list of videos I use for this course:

RNA Structure & Function (Anna Marie Pyle, Yale University):

https://www.ibiology.org/biochemistry/rna-structure/

Protein Synthesis (Rachel Green, Johns Hopkins University):

https://www.ibiology.org/biochemistry/protein-synthesis/

I also use a variety of review papers listed below in Resources.

Assessment

Rubric for the Annotated Bibliography: CHE4960_AnnotatedBibRubric.pdf (Acrobat (PDF) 84kB Oct19 19)

Peer review sheet for Lay Summary: CHE4960_LaySummary_PeerReview_CURE.pdf (Acrobat (PDF) 47kB Oct19 19)

Rubric for Lay Summary: CHE4960_LaySummaryRubric_CURE.pdf (Acrobat (PDF) 101kB Oct19 19)

Rubric for Oral Presentation: CHE4960_OralPresRubric_CURE.pdf (Acrobat (PDF) 91kB Oct19 19)

Instructional Staffing

This course can be performed with one instructor, and a TA (depending upon the size of the class).

Author Experience

Megan Filbin, Metropolitan State University of Denver

I became motivated to develop and implement this CURE into an existing literature research senior capstone course in our department called Senior Experience in Biochemistry. I initially altered the course to focus on science communication to both a nonscientist as well as an expert scientist, however I felt that students have struggled to focus on one particular topic of interest. Additionally, students are required to write a research proposal in my course, and I've found they struggle with wanting to actually DO the experiments they propose. So, I decided to develop a CURE about the structure-function analysis of RNA (my own research) as the focus of both the proposal as well as the experiments they will conduct throughout the course.

Advice for Implementation

While I have yet to teach this CURE version of Senior Experience in Biochemistry, I have experience teaching the course with a focus on communicative techniques. It is challenging for students to write a lay summary because by the time they reach senior courses, they have practiced and developed the language used by chemists to communicate with other chemists - not how to translate complex language/topics to a nonscientist. Therefore, I would encourage anyone looking to incorporate a lay summary into a course (particularly a CURE where there is a major focus on research language/content) to really take the time to walk students through using figures of speech and simple, plain English to describe the science (in other words, facilitate a lot of practice and peer review).

The design of this cure is also based on incorporating a lot of new technologies - including predictive softwares for RNA folding, primer design and protein interaction, as well as an electronic laboratory notebook. Encourage students to use these softwares at home as well, so that they really master how to navigate each site, what components can be altered to affect the results (e.g. ionic strength or temperature) and how the notebook could be used in a collaborative way between group members.

Iteration

Throughout this course students should be encouraged to use the software multiple times to verify findings, they will complete the luciferase experiment multiple times for statistical analysis, and fulfill their lab notebook weekly. Throughout each iteration, facilitate a growth mindset by encouraging students to (as a group) propose what to change during each iteration, and then reflect upon the results based on the changes to draw conclusions accordingly.

The course is also designed for multiple drafts of the hypothesis, figures, the lay summary and final presentation. In my experience, giving students few, but significant changes to focus on each round of modification results in substantial growth and well-rounded final assignments. Peer review has been crucial within my courses, as it helps both students learn how to draft higher quality assignments.

Using CURE Data

The data generated from this CURE will be shared at conferences (undergraduate research conference, RNA Society, ASBMB Experimental Biology), used for grant proposals, as well as published in a primary research article. Particularly interesting mutants will be used for more in-depth analysis of RNA structure and binding partners.

Resources

Jackson RJ, Hellen CU, Pestova TV. The mechanism of eukaryotic translatio initiation and principles of its regulation. Nat Rev Mol Cell Biol. 2010, 11(2):113-27. (doi: 10.1038/nrm2838)

Nicholson BL, White KA. 3' Cap-independent translation enhancers of positive-strand RNA plant viruses. Curr Opin Virol. 2011, 1(5):373-80. (doi: 10.1016/j.conviro.2011.10.002)

Filbin ME, Kieft JS. Toward a structural understanding of IRES RNA function. Curr Opin Struct Biol. 2009, 19(3):267-76. (doi: 10.1016/j.sbi.2009.03.005)

ViennaRNA Web Services: http://rna.tbi.univie.ac.at/

Primer Design programs: https://molbiol-tools.ca/PCR.htm

RNA-Protein Interactions: http://pridb.gdcb.iastate.edu/RPISeq/

Benchling Electronic Lab Notebook: https://www.benchling.com/

Mendeley Reference Manager: https://www.mendeley.com/?interaction_required=true

Zotero Reference Manager: https://www.zotero.org/