Mechanism of STMN2 Cryptic Splice-Polyadenylation Assignment

Assignment Task

The questions in this problem set refer to “Mechanism of STMN2 cryptic splice-polyadenylation and its correction for TDP-43 proteinopathies”. These questions are worth 50 points (5 points each). The answers must be in your own words. If you use a reference outside of this research article, cite the reference at the end your answer for that question. Please use MLA format for your references, and include URLs if applicable.  Internet references are OK, but NOT Wikipedia. This problem set is due at 12 noon on May 1. In order for you to receive full credit, I must receive your answers by email by noon on May 1. There are severe penalties for late problem sets.

This article is available free of charge on the LSU Library web site. If you do not know how to get the article from LSU, you can call the library for help.

1.

• Draw or describe the structure of full-length STMN2 mRNA (by the exons).
• Draw or describe the structure of truncated STMN2 mRNA.
• Give the DNA sequence of one TDP-43 binding site on the STMN2 gene.
• What is the function of TDP-43 binding to STMN2 mRNA in healthy cells? Be specific.

2. Show a diagram of the MS2-loop which includes the loop structure with the base sequence.

3. Answer the following for genome editing on the STMN2 gene in the SH-SY5Y cell line.

• Show a diagram of the plasmid that was used to express Cas9 and sgRNA.
• Give the sequence of the sgRNA used to edit the TDP-43 binding site.
• What is Cas9-T2A-GFP? That is, what is T2A and what is GFP?

4. 

• Describe in detail how the authors measured relative RNA expression.
• Give the DNA sequence of the mouse forward Stmn2 PCR primer the authors used.
• Explain the results of Fig. 1B. Explain each part of the figure and describe what the authors concluded from the data.

5.

• What is Dox and what is its function in these experiments?
• What is MCP and what is its function in these experiments?
• What is TDP-43^ΔRRM and what is its function in these experiments?
• What did the authors conclude from the data in these figures and why?

6.

• How were the siRNAs introduced into the cells? What is reverse transfection? What is RNAiMax?
• Explain the results in Figs. 1G, 1H, and 1I.

7.

• What is RfxCas13d?
• How is dCasRx different from RfxCas13d?
• How is dCasRx different from Cas9 in its targeting requirements?
• What is TDP-43^N352S/N352S?
• According to Figs. S2A and S2C, which gRNAs most significantly reduced the cryptically spliced, polyadenylated STMN2 RNA relative to a control TDP43^N352S/N352S line expressing dCasRx without a gRNA? For these, describe the component or area of STMN2 pre-mRNA that the gRNAs targeted (see Fig. S2A).

 8. 

• Describe in detail the Materials and Methods for the experiment in Fig. S2F. What is GAPDH (include the full name)?
• Why do you think the authors used rescue antisense oligonucleotides (rASOs) to continue their research instead of proceeding with dCasRx?

9. 

• What is iPSC?
• What is Smoothened Agonist? (Hint: see Materials and Methods)
• What modifications were made to the ASOs to protect them from ribonuclease H?
• Give the sequences for the Control and TARDBP suppressing ASOs.

10.

• What is the significance of “+TDP-43 ASO” in the experiments? That is, explain what it does to the human motor neurons.
• What are the 2 controls in these experiments?
• What is the significance of the 2 little arrows on top of the diagrams for full-length STMN2 mRNA and truncated STMN2 RNA?
• Which rASO was LEAST effective in restoring full length STMN2 mRNA?

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