Team:MIT/RBPs

• ABOUT US
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• BACKGROUND
  Alternative Splicing CRISPR RNA Binding Proteins REST
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  Guides and ASOs mKate-FF4 2 Exon mKate HBG 3 Exon mKate HBG 3 Exon Dual Fluorescence
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RNA Binding Proteins

In addition to Cas13a, our team considered other non-programmable RNA Binding Proteins to attempt to control alternative splicing. The goal of using an RBP was to stabilize the ASOs so that they could survive in the cell long enough to reach their target. In addition, having another implementation of alternative splicing control would allow us to compare to the efficiency of L. shahii Cas13a. With this in mind, we dove into the realm of RBPs.

What is an RNA Binding Protein?

RNA-binding proteins (RBPs) are proteins that bind to RNA molecules, and are involved in many aspects of post-transcription RNA processing, including alternative splicing as well as stabilization and localization of mRNAs. RBPs control splicing by binding to specific regions on the pre-mRNA and recruiting splicing factors. They can also be used as silencers and enhancers of introns and exons, and are a structural component of the spliceosome along with splicing factors.

There are over five hundred different RBPs, however very few have been studied extensively. Our team focused on two that the Weiss lab or it’s associates have experience with: L7Ae and Ms2.

L7Ae

L7Ae is an RBP that binds to kink turn (k-turn) regions of RNA. K-turns are a class of three dimensional structures used for RNA structural organization. L7Ae binds to k-turns and directs their shape.[1] L7Ae can be used [2] as either a repressor or activator for translation. It can interact with a k-turn in the open reading frame region of a desired target mRNA to repress translation. L7Ae can also be added to compete with translational repressors already found in the RNA, thereby allowing for the activation of translation.[1] L7Ae has a high binding affinity, but is toxic at elevated levels which must be accounted for in conducting research.

Ms2

Ms2 is an RBP from an RNA bacteriophage that binds to the U2-snRNP (small nuclear ribonucleoproteins). U2 snRNP is a region of the spliceosomes that binds with the 5’ branch point of the introns, and causes the intron region to bulge. The bulging region is recognized by other parts of the spliceosome and excised. Ms2 splicing inhibitors binds to the U2-snRNP so it is unable to bind to the branch point of the introns, inhibiting the bulging of the intron. Ms2 splicing inducer binds to the inhibitor, allowing the U2 snRNP to function as normal. [2]

After lengthy consideration, our team decided to use Ms2 for multiple reasons:

Size

Ms2 is relatively small. Its small size limits the chances that the alterations in splicing is due to its size rather than the spot to which it binds. [4]

Known Binding

Known Binding: The target sequence for Ms2 has been well characterized as a 21-nucleotide stem-loop. This fact made designing guide sequences for the protein much simpler. [4]

Lack of Toxicity

As previously mentioned, L7Ae is toxic to cells at elevated levels, whereas high levels of Ms2 doesn’t pose as high of a threat.

References

[1] Saito et al. "Synthetic translational regulation by an L7Ae–kink-turn RNP switch.” Nature Chemical Biology 6 (2010): 71-78.

[2] Kan, Julie L.C., and Michael R. Green. “Pre-mRNA Splicing of IgM Exons M1 and M2 Is Directed by a Juxtaposed Splicing Enhancer and Inhibitor.” Genes & Development 13.4 (1999): 462–471. Print.

[3] Belmont Brian J., Jacquin C. Niles. “Engineering a direct and inducible protein-RNA interaction to regulate RNA biology.” ACS Chemical Biology 5 (9) (2010): 851-861.

[4] Coller, Jeff, and Marv Wickens. “Chapter Fourteen - Tethered Function Assays: An Adaptable Approach to Study RNA Regulatory Proteins”. Methods in Enzymology 429 (2007): 299-321.