Page 53 - Annual report 2021-22

Annual Report 2021-22 |

Mary Krishna Ekka

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Mary Ekka’s group works in the area of protein-nucleic acid interactions.

Mary Ekka’s group is examining the role of the lncRNA CONCR (cohesion regulator noncoding RNA) in
abolishing birth defects caused due to faulty sister chromatid cohesion. The ability of CONCR-DDX11
to control and rescue defects due to DNA damage while working in a common pathway shows their
relevance in DNA replication. The overall objective of this project is to identify motifs in CONCR that
may modulate its function followed up by mutational approach to analyze minimum regions in CONCR
mediating RNA-protein interaction.

Previously the group has tried multiple methods of protein purification in order to obtain sufficient
quantities of DDX11 protein for biophysical assays. The gene was cloned into vectors expressing
maltose binding protein (MBP) and glutathione S-transferase (GST) to increase the solubility of DDX11.
The GST clones failed to express DDX11 even after several rounds of optimization. The MBP fusion
protein clone gave good expression. However, the protein bound very poorly to the MBP resin and
majority was eluted in the flow through fraction. Since several approaches to improving the protein
purification did not yield desired results, truncated constructs/deletional variants of CONCR and
overlapping constructs of CONCR were made using in vitro transcription (IVT). Recently published
literature that used AlphaFold for protein structure prediction showed that the N-terminus of DDX11
was highly flexible, hence the full-length protein could not be purified from E.coli. Finally, efforts were
shifted to purifying DDX11 from HeLa cells. DDX11 is a DNA helicase and specifically unravels
quadruplex structures. However, its ability to bind to RNA quadruplexes and unravel them is not
known till now. DNA and RNA substrates for G2 quadruplexes and G4 quadruplexes were procured
and DDX11 activity studies in presence of ATP were carried out. DDX11 showed binding with RNA
substrates as seen from EMSA studies.

CHAST is a lncRNA known to be associated with pathological cardiac remodeling or hypertrophy. Mary
Ekka’s lab aims to shed light on the structure-function relation of this lncNRA by identification and
characterization of secondary structures formed by the lncRNA CHAST, identification of novel RNA
binding proteins (RBPs) of CHAST and characterization of interactions between the identified proteins
and CHAST through biophysical and biochemical studies.

Screening of small molecules and ligands that affect CHAST structure, stability and activity is a future
goal of this project. To this end, they have purified two expressed proteins (Rab11 and alpha actinin)
through His-tag based affinity chromatography and after dialysis and subsequent concentration, these
proteins were then purified through size exclusion chromatography for sufficient concentration for
biophysical assays. In vitro transcription (IVT) was performed to get the full-length CHAST, however,
the yield was not sufficient. Efforts to express the full-length CHAST are currently being optimized.
Short oligos (21-mer) harboring putative G-quadruplex structures were procured commercially. EMSA
binding studies were carried out with these oligos (fixed concentration) and the two purified proteins.
Rab11 showed strong binding with CHAST oligos, however alpha-actinin showed moderate binding.
Out of the 5 proteins previously identified from the pull-down experiments, three proteins, Rab11,
alpha actinin-1 and lamin have been cloned and expressed.