Page 80 - CSIR-IGIB Annual Report 2020-21
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looking at other secondary structures identified phenylephrine did not induce hypertrophy to a
from the modular CONCR structure that may be significant extent. The overexpression of Chast
modulated by small molecules or are essential was validated in isoprenaline treated cells using
for the binding of CONCR to its interacting qPCR using untreated cells as control. We
protein partner in the cell, DDX11. We are also looked at the sequence of Chast using RNA
trying to obtain good quality DDX11 protein alifold and deduced that Chast has a highly
from HeLa cells using FLAG-tag along with organized structure with multiple secondary
making a stable protein expression cell line for structures like stem loops, bulges and hairpins.
detailed RNA-protein interaction studies. The sequence of Chast was also analysed for the
presence of dynamic non-canonical secondary
Binding Interactions Of Chast In Hypertrophy structures like G-quadruplexes using QGRS
Mapper and found that Chast has one G-rich
In this project, we are going to examine the nucleotide stretch that may form a G-
broad role of the long noncoding RNA Chast in quadruplex. This RNA oligo was synthesized
causing cardiac hypertrophy. Expression levels commercially and UV melting and CD studies
of Chast were observed to be higher upon showed that it had characteristic signatures of a
cardiac hypertrophy and silencing of Chast with parallel RNA G-quadruplex. Through literature
GapmeRs led to significant decrease in the mining we identified five proteins whose
hypertrophic response. Chast is a good lncRNA expression was enriched multiple folds upon
target to identify conserved RNA motifs for over-expression of Chast. We cloned and
therapeutic intervention. We obtained HL-1 purified two proteins out of the five, Rab11 and
cells which is an immortalised mouse cardiac alpha actinin from Rosetta DE3 cells.
muscle cell line capable of retaining biochemical Preliminary studies showed tight binding with
and electrophysiological properties with serial in-vitro transcribed (IVT) Chast with binding
passaging. This cell line was cultured in affinity at nM level. Detailed thermodynamic
Claycomb media and hypertrophy was induced studies are on-going. Pull-down studies to
using stress agents isoprenaline and identify specific binding protein partners with
phenylephrine for 48 hours. Size analysis the G-quadruplex sequence are also being
showed that while isoprenaline increased the optimized.
size of the cells to a significant extent,
Publications
Essential biochemical, biophysical and computational inputs on efficient functioning of Mycobacterium
tuberculosis H37Rv FtsY. Shivangi, Ekka MK, Meena LS. Int J Biol Macromol. 2021 Feb 28;171:59-73
Annual Report 2020-21 77
