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
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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.
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The specific objectives and approaches of the proposed research plan includes
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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
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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.