Studies on mRNA surveillance and its role in alternative splicing.pptx
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This document outlines a Ph.D. research proposal on studying mRNA surveillance and its role in alternative splicing. The presentation includes background on mRNA surveillance mechanisms like nonsense-mediated decay (NMD), a review of literature on gaps in understanding NMD and its relationship to alternative splicing, objectives to study NMD factors and their role in degrading defective transcripts and interacting with alternative splicing, and proposed methodologies like protein purification and analysis. The expected outcomes are to better understand the mechanistic role of NMD in zebrafish and how it regulates alternative splice variants across eukaryotes.
Studies on mRNA surveillance and its role in alternative splicing.pptx
- 1. Studies on mRNA surveillance and it’s role in alternative splicing by SANTOSH KUMAR SAHOO Application No - cutmphd2022DEC_463 Department of Zoology School of Applied Sciences, CUTM, BBSR Ph.D. Admission Seminar (December 2022)
- 2. Background of the study Review literature Objectives Methodology References 03 06 08 09 11 Presentation Outline Expected outcome 10
- 3. Background of the study... The translation of messenger RNA transcripts into proteins is a vital part of the central dogma of molecular biology. mRNA molecules are, however, prone to a host of fidelity errors which can cause errors in translation of mRNA into quality proteins. (Amrani et al., 2015). RNA surveillance mechanisms are methods that cells use to assure the quality and fidelity of the mRNA molecules (Moore, M. J., 2018). In eukaryotes, mRNA surveillance mechanisms are known to function in both the nucleus and cytoplasm. (Fasken, M. B., & Corbett, A. H., 2015). Nonsense-mediated mRNA decay pathway (NMD) is one of the most studied mRNA surveillance mechanisms.
- 4. Background of the study... Nonsense-mediated decay (NMD) is involved in detection and decay of mRNA transcripts which contain premature termination codons (PTCs). Failure to recognize and decay these mRNA transcripts can result in the production of truncated proteins which may be harmful to the organism. NMD and its mechanisms Fig of Canonical NMD pathway (in human) Image credit – Rehwinkel et al., 2019
- 5. Background of the study... It is a key regulatory step of mRNA processing that creates multiple transcript variants produced from a single gene locus. It is predicted to be one of the main drivers of protein diversity in mammals and has been reported as affecting more than 95% of multi- exonic coding human genes (Johnson et al., 2013; Nilsen and Graveley, 2010; Pan et al., 2018) and many noncoding genes (Derrien et al., 2019). Alternative splicing Fig of Alternative RNA splicing Image credit - Benjamin Pierce
- 6. Review literature # Whether all eukaryotes have NMD ??? NMD factors are highly conserved in Eukaryotes and lower Eukaryotes have homologous structure of NMD factors. In contrast, NMD appears to function in Giardia lamblia, parasitic protist, despite the lack of recognizable homolog's of UPF2 and UPF3 (Chen et al., 2018). Although UPF3 seems to be absent from most protists, a UPF3 homolog has been found in Paramecium tetraurelia and it was shown to play a role in NMD (Contreras et al., 2020). # Whether NMD might function independently of EJCs ? Interestingly, NMD in Tetrahymena thermophila has recently been shown to be EJC- independent (Tian et al., 2017).
- 7. Review literature # Whether same mRNA could be targeted by more than one translation- dependent RNA surveillance pathway ? Preliminary evidence supports When endonucleolytic cleavage occurs upstream of a PTC, the resulting 5′RNA fragment was stabilized on depletion of nonstop decay components Pelota and Hbs1 in Drosophila cells (Hashimoto et al., 2017). Along the same lines, it was shown in C. elegans that the degradation of PTC-containing mRNAs as well as endogenous NMD targets is coupled to nonstop decay (Arribere and Fire, 2018). # Is there any role of NMD in Alternative splicing Yes, NMD also degrades alternatively spliced transcripts with reading frame shifts that introduce PTCs, in a process termed AS coupled to NMD (AS-NMD), and this also contributes to regulate gene expression. AS is believed to be the major source for PTC- containing NMD substrates and bioinformatic predictions indicate that about one-third of alternatively spliced human mRNAs contains a PTC (Lewis et al., 2019). Thus, we are still far away from a comprehensive understanding of the biological functions of NMD and its underpinning molecular mechanism(s).
- 8. Objectives of the study Study the molecular process of nonsense- mediated mRNA decay (NMD) i.e. the steps involved in the recognition of NMD targets and the activation of NMD. 01 The specific objectives and approaches of the proposed research plan includes 02 03 04 Role of UPF1, a crucial NMD factor in the molecular elimination of defective transcripts. Future prospective of the interaction between mRNA surveillance and protein quality control via UPF1. To revel the relationship between NMD and alternative splicing.
- 9. Cloning, Expression and Purification of Protein Protein Immunoprecipitation and western blotting RNA isolation and RT-PCR Confocal Laser Scanning Microscopy 1 2 3 4 Methodology
- 10. EXPECTED OUTCOME Role of NMD in Zebrafish remains blurred. The study will allow us to unrevel the mechanistic role of mRNA surveillance in Zebrafish. The role of RNA helicase factor i.e. UPF1 in maintaining the level of physiological mRNA can be known. The regulation of alternative splice variants mediated by NMD in Eukaryotes can be known since the NMD factors are highly conserved across Eukaryotic species.
- 11. References Amrani, N., Sachs, M. S., & Jacobson, A. (2015). Early nonsense: mRNA decay solves a translational problem. Nature reviews Molecular cell biology, 7(6), 415-425. Moore, M. J. (2018). From birth to death: the complex lives of eukaryotic mRNAs. Science, 309(5740), 1514-1518. Fasken, M. B., & Corbett, A. H. (2015). Process or perish: quality control in mRNA biogenesis. Nature structural & molecular biology, 12(6), 482-488. Delhi, P., Queiroz, R., Inchaustegui, D., Carrington, M., & Clayton, C. (2016). Is there a classical nonsense-mediated decay pathway in trypanosomes?. PloS one, 6(9), e25112. Chen, Y. H., Su, L. H., & Sun, C. H. (2018). Incomplete nonsense-mediated mRNA decay in Giardia lamblia. International journal for parasitology, 38(11), 1305-1317. Contreras, J., Begley, V., Macias, S., & Villalobo, E. (2020). An UPF3-based nonsense-mediated decay in Paramecium. Research in microbiology, 165(10), 841-846. Tian, M., Yang, W., Zhang, J., Dang, H., Lu, X., Fu, C., & Miao, W. (2017). Nonsense-mediated mRNA decay in Tetrahymena is EJC independent and requires a protozoa-specific nuclease. Nucleic acids research, 45(11), 6848-6863. Hashimoto, Y., Takahashi, M., Sakota, E., & Nakamura, Y. (2017). Nonstop-mRNA decay machinery is involved in the clearance of mRNA 5′-fragments produced by RNAi and NMD in Drosophila melanogaster cells. Biochemical and biophysical research communications, 484(1), 1-7.
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