Illuminating U2snRNA Mutations: A Fluorescent Splicing Reporter System

Kristina Oh, Meredith Stevers, Melissa Jurica - University of Santa Cruz, Molecular, Cellular and Developmental Biology

Abstract

Splicing is a highly conserved process across eukaryotes. Mitigated by the spliceosome, it is a mega-dalton complex composed of snRNPs that facilitate intron recognition, excision, and exon ligation. Specifically, the U2 snRNP recognizes degenerate sequences along the intronic branchpoint. In yeast, branch-point interactions between pre-mRNA and U2snRNA are conserved. Human splice sites are more obscure, containing non-canonical sites within the intron that can also be spliced. Mutations along these sequences lead to rare genetic diseases and cancers, attributed to the use of non-canonical splice sites.

A major question that we hope to answer is the effects of base pairing on the stringency of U2snNRA and intron interactions. We hypothesize that mutations that increase the strength of base pairing between the U2snRNA and the intronic branch point will decrease splicing efficiency. Conversely, mutated introns that result in weaker base pairing with the U2snRNA will increase splicing efficiency. To investigate this hypothesis, our project aims to develop a fluorescent splicing reporter that will visually depict the effect of branch stem-loop mutations within human U2snRNA. The branch stem-loop is a functional module of U2snRNA that forms during intronic branch site recognition. Based on the alternatively spliced SV40 pre-mRNA framework, I have cloned recombinant DNA with green and red fluorescent proteins that will be mutually expressed depending on the splicing at the specific intron within the SV40 pre-mRNA. Upon transfecting this reporter system into human cells, red and green fluorescent protein expression will depend on mutations introduced to the U2snRNA branch stem-loop. Based on fluorescent output quantified by flow cytometry, we want to measure and compare U2snRNA splicing efficiency across various mutations. Fluorescent assays specific to orthogonal U2snRNA are not something the field has been able to do. If successful, the reporter will be the first of its kind, and hopefully lead to more advances in splicing research

I. Intronic branchpoint sequence is critical to splicing

a. Anatomy of an Intron

Introns include three key sequences. To be successfully excised out, they are defined by the 5' and 3' splice sites at the exon-intron junctions. The branch point is located downstream of the 5' splice site and is the binding site for the U2snRNA.

b. Intronic branchpoint contains adenosine used in the first catalytic step of splicing

Once the U2snRNA base pairs with the intron, the branch point adenosine is "bulged" out. This adenosine is primed to perform a nucleophilic attack to initiate the first catalytic step of splicing that also indirectly determines the 3' splice site for the chemistry that occurs at the second step.

c. Overview of U2snRNA mediated branchpoint identification

This schematic depicts the U1, U2, U4, U5 and U6 snRNA assembly on the intron following U2 binding to the branch-point. Complex B depicts snRNAs and associated proteins priming the intron right before the first catalytic step.

What is our research aim?

The current model of U2snRNA and branch point interactions is based on yeast. Yeast introns contain highly conserved single branch points while human introns contain multiple degenerate branch points.
To further uncover the mechanisms of human U2 base-pairing interactions with the intron, we are in the process of creating a fluorescent splicing reporter where RFP (red fluorescent protein) expression can be altered by the splicing initiated exogenous U2snRNA without the interference of endogenous U2

II. Creating a split mCherry Orthogonal Splicing System

a. Wu and Manely's SV40 pre-mRNA framework

Above is Wu and Manely’s SV40 Large T antigen orthogonal splicing system with two 5’ splice sites and one 3’ splice site (1). Splicing at the first 5’ splice site results in the Large T product, whereas slicing at the second 3’ splice site results in the small T product that includes exon 1 and the Large T intron. Although the large T intron has multiple branch points, the singular branch point within the small T intron allows for the observation of exogenous U2snRNA base pairing with the branch point without interference from endogenous U2snRNA.

b. Split mCherry protein

A full-length 708 nt mCherry protein was split so that 579 bases encompassed the exon upstream of the first 5’ splice site and 129nt downstream within the large T intron Splicing at the first 5’ splice site resulting in only a portion of mCherry translation, resulting in only full-length GFP translation and expression. Splicing at the second 5’ splice site results in full-length mCherry and GFP expression. Depending on mutations made at the small T intron, changes in the levels of mCherry expression hope to serve as a qualitative and quantitative indicator of U2snRNA splicing. This reporter was cloned into a pCI vector containing a CMV promoter which is ideal for transfection assays in mammalian cells.

III. Results: mCherry and GFP co-expression in HEK293T cells

a. Sanger Sequencing Data to confirm mCherry and GFP recombinant plasmid

i) Depiction of mCherry and GFP protein recombinant plasmid created using restriction enzyme cloning on Benchling.

ii) Sanger sequencing alignment peaks (below) confirm the presence and orientation of RFP (shown in red) followed by GFP protein (shown in green).

b. Transfection of mCherry + GFP plasmids in HEK293T cells

To test for the expression of mCherry and GFP in the pCI vector, HEK293T cells were cultured and then transfected with the reporter plasmids along with an empty vector control. mCherry and GFP were observed to be co-expressed under RFP and GFP filters respectively. No expression was observed for the empty vector and therefore not included in the figure above.

IV. Utilizing NEB Hi-Fi to Insert Wild-type and Mutant Branch-point Sequences

GFP and RFP expression was confirmed upon observing their expression in HEK293T cells. NEB Hi-Fi protocol was then utilized to insert either an wild type or mutant intron between the mCherry and GFP.

a. Schematic for HiFi Assembly

After the expression of mCherry and GFP protein was confirmed, NEB HiFi assembly was done to insert the small T intron sequence between the encoding regions of the proteins. This intron was PCR amplified from a plasmid containing the SV40 Large T Antigen gene.

Two separate assemblies were done to create an mCherry and GFP split reporter with either a wild type or mutant branchpoint within the small T intron. These plasmids were then transformed with DH5-alpha E.coli strain.

The small T intron contains a stop codon. In the absence of splicing, the ribosome will not be able to translate into the GFP protein. For all plasmids, mCherry will be expressed.

We expect that the WT intron will be spliced by the endogenous U2snRNA. Splicing of the small T intron will give rise to GFP expression.

The reporter with the mutant intron should not be spliced by endogenous U2, meaning that we should expect to see only mCherry expression and no GFP.

b. HEK293T Transfection of HiFi Clones

After many rounds of HiFi cloning, sequencing data confirmed the presence of introns containing either the WT (wild-type) or mutant branch points. HEK293T cells were transfected with the HiFi clones along with the original mCherry and GFP-only plasmid as a positive control.

Interestingly, strong GFP and mCherry signals are observed for the cells expressing the mutant intron while only mCherry expression is observed for the cells expressing the WT intron. This is the opposite of what we expected.

V. Future Directions

Sanger sequencing to re-verify that the WT and mutant plasmids were correctly transfected to the cells
Re-transfect HEK293T cells and then do an RNA extraction followed by an RT PCR to test for the presence of the branch point within the intron
Site-directed mutagenesis to insert the first 5' splice site within the mCherry
Visualize fluorescence through flow cytometry and quantify fluorescence levels using qPCR

Acknowledgments

Jurica Lab members
PI: Melissa Jurica
My mentor: Meredith Stevers

References Cited

Wu J, Manley JL. Mammalian pre-mRNA branch site selection by U2 snRNP involves base pairing. Genes Dev. 1989 Oct 3(10):(1553-61)
Wu J, Manley JL. Multiple functional domains of human U2 small nuclear RNA: strengthening conserved stem I can block splicing. Mol Cell Biol. 1992 Dec;12(12): (5464-73)
Mathur, M., Kim, C.M., Munro, S.A. et al. Programmable mutually exclusive alternative splicing for generating RNA and protein diversity. Nat Commun 10, 2673 (2019).

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