Poster abstracts

Poster number 126 submitted by Manu Sanjeev

Role of SR proteins in mRNP packaging and Nonsense Mediated Decay

Manu Sanjeev (Department of Molecular Genetics, The Ohio state University), Guramrit Singh (Department of Molecular Genetics, The Ohio state University)

Abstract:
After their transcription, mRNAs in cells are packaged into large rod-shaped ribonucleoprotein (RNP) complexes with sizes comparable to the small ribosome subunit [1]–[3]. This packaging is crucial for genomic stability, and presumably for mRNA export and translation. The exon junction complex (EJC) is a major mRNP component that plays important roles in multiple levels of gene regulation [4]. The EJC interacts with a variety
of peripheral proteins including several Serine-Arginine-rich (SR) proteins in an RNAindependent manner to form mRNPs that protect RNA from nuclease digestion [5]. Recent work from our lab has shown that EJCs are dynamic and undergo remodeling in the cytoplasm into smaller complexes that contain the EJC core but lack SR proteins [6]. The removal of SR proteins from mRNPs also reduces the Nonsense Mediated Decay (NMD) efficiency of target mRNAs [6], [7]. My overarching goal is to understand how different SR and SR-like proteins assemble on the periphery of the EJC core to form the mRNP in the nucleus, to identify the mechanism by which SR proteins are evicted from mRNPs in the cytoplasm to cause its remodeling, and to determine the effect of mRNP packaging on EJC dependent processes like mRNA export and NMD. Here, I show preliminary results that a major SR protein SRSF1 can be chemically crosslinked with the peripheral EJC factor RNPS1, suggesting a mode of SR protein interaction with the EJC. Further, overexpression of SR protein kinase SRPK1, but not its kinase-dead mutant version, slows down the NMD of a reporter RNA. This suggests that SRPK1 potentially plays a role in the remodeling of mRNPs in the cytoplasm, presumably by phosphorylation and eviction of SR proteins from mRNPs. To understand how SRSF1-RNPS1-EJC nexus
affects NMD efficiency of mRNPs, I have developed a triose phosphate isomerase (TPI)-based reporter mRNAs as described before [8], which contain enhanced SR protein recruitment signals. These reporters undergo NMD with increased efficiency as compared to a normal NMD reporter RNA. I am currently testing if knockdown of SR proteins and RNPS1 slows down the NMD of this reporter. Overall, this study provides insights into three important aspects of gene regulation: (i) how EJC and SR proteins interact to form
mRNPs in the nucleus, (ii) how mRNP remodeling occurs in the cytoplasm, and (iii) how changes in mRNP composition affect NMD efficiency.

References:
[1] G. Singh, G. Pratt, G. W. Yeo, and M. J. Moore, “The Clothes Make the mRNA: Past and Present Trends in mRNP Fashion,” Annu Rev Biochem, vol. 84, pp. 325–354, 2015.
[2] S. Adivarahan et al., “Spatial Organization of Single mRNPs at Different Stages of the Gene Expression Pathway,” Molecular Cell, vol. 72, no. 4, pp. 727-738.e5, Nov. 2018.
[3] M. Metkar et al., “Higher-Order Organization Principles of Pre-translational mRNPs,” Molecular Cell, vol. 72, no. 4, pp. 715-726.e3, Nov. 2018.
[4] L. A. Woodward, J. W. Mabin, P. Gangras, and G. Singh, “The exon junction complex: a lifelong guardian of mRNA fate: EJC: assembly, structure, and function,” WIREs RNA, vol. 8, no. 3, p. e1411, May 2017.
[5] G. Singh et al., “The Cellular EJC Interactome Reveals Higher-Order mRNP Structure and an EJC-SR Protein Nexus,” Cell, vol. 151, no. 4, pp. 750–764, Nov. 2012.
[6] J. W. Mabin et al., “The Exon Junction Complex Undergoes a Compositional Switch that Alters mRNP Structure and Nonsense-Mediated mRNA Decay Activity,” Cell Reports, vol. 25, no. 9, pp. 2431-2446.e7, Nov. 2018.
[7] I. Aznarez et al., “Mechanism of Nonsense-Mediated mRNA Decay Stimulation by Splicing Factor SRSF1,” Cell Reports, vol. 23, no. 7, pp. 2186–2198, May 2018.
[8] J. P. Gudikote, J. S. Imam, R. F. Garcia, and M. F. Wilkinson, “RNA splicing promotes translation and RNA surveillance,” Nat Struct Mol Biol, vol. 12, no. 9, pp. 801–809, Sep. 2005.

Keywords: Nonsense Mediated Decay, SR proteins, mRNP packaging