Poster number 10 submitted by Kaushik Saha
Pre-mRNA 3D structural scaffold as a contributor to the mammalian splicing code
Kaushik Saha (Department of Chemistry and Biochemistry, University of California San Diego), Mike M. Fernandez (Department of Chemistry and Biochemistry, University of California San Diego), Whitney England (Department of Pharmaceutical Sciences, University of California Irvine), Robert C. Spitale (Department of Pharmaceutical Sciences, University of California Irvine), Gourisankar Ghosh (Department of Chemistry and Biochemistry, University of California San Diego)
The specific recognition of splice signals at or near exon-intron junctions by the early spliceosomal components is not explained by their weak sequence conservation and instead is postulated to require a multitude of features embedded in the pre-mRNA strand. We show that the early spliceosomal components recognize a pre-mRNA 3D structural scaffold beyond the short splice signal sequences. Interestingly, structural remodeling of the pre-mRNA 3D scaffold by the splicing regulatory proteins (SRps) appeared to be critical for its specific recruitment by the early spliceosomal components. We also find that exonic loops immediately upstream of the intron regulate the functional recruitment of SRps and consequent pre-mRNA structural remodeling. These exonic segments are more hybridized upstream of the retained introns compared to the spliced introns across the transcriptome.
Additional investigations into the mechanism of recognition of the pre-mRNA 3D structural scaffold by the early spliceosomal components suggest that the SRps bind the pre-mRNAs cooperatively, generating a substrate that recruits U1 snRNP and U2AF65 in a splice signal-independent manner. Excess U1 snRNP selectively displaces some SR protein molecules from the pre-mRNA generating the substrate for splice signal-specific, sequential recognition by U1 snRNP, U2AF65, and U2AF35.
Overall, the central hypothesis supported by this work is that the early spliceosomal components recognize a regulatable 3D structural scaffold in addition to the short splice signal sequences. This effectively constitutes the newest element of the mammalian splicing code in addition to the splice signal sequences, the splicing regulatory elements, and the pre-mRNA secondary structure.
1. Saha, K., England, W., Fernandez, M.M., Biswas ,T., Spitale, R.C., Ghosh, G. (2020). Structural disruption of exonic stem-loops immediately upstream of the intron regulates mammalian splicing. Nucleic Acids Res 48, 6294-6309. DOI:10.1093/nar/gkaa358
2. Saha, K., Fernandez, M.M., Biswas, T., Joseph, S. and Ghosh, G. (2021) Discovery of a pre-mRNA structural scaffold as a contributor to the mammalian splicing code. Nucleic Acids Res, 49, 7103-7121. DOI:10.1093/nar/gkab533
3. Saha, K. and Ghosh, G. (2022) Cooperative engagement and subsequent selective displacement of SR proteins define the pre-mRNA 3D structural scaffold for early spliceosome assembly. Nucleic Acids Res. Ahead of print. DOI:10.1093/nar/gkac636
Keywords: Pre-mRNA 3D structure, Mammalian splice site recognition, SR proteins