Page 61 - Annual report 2021-22

Annual Report 2021-22 |

Niyati Jain

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Niyati Jain studies structure function relationship underlying RNA- protein interactions.

Long non-coding RNA (lncRNA) HOTAIR and RNA binding proteins (RBPs) are critical factors that play
a pivotal role in tumor development and metastasis. However, how specific RBPs influence the
biological function of lncRNA in cancer development still remains unknown. Characterization of
HOTAIR structural organization and identification of specific features involved in binding interactions
with RBPs will be of paramount importance to defining the functional mechanism of lncRNA in cancer
cells. There is a notable dearth of structural studies on lncRNA because of technical challenges in the
structural analysis of these large biomolecules in general and RNA in particular. A combination of the
NMR and SAXS approaches can provide an understanding of the structural basis of HOTAIR and the
influence of RBPs on its function in cancer cells by providing the complete thermodynamic footprint
of RBPs/HOTAIR lncRNA interactions, and also solving co-structures of RBPs (hnRNPA2/B1 and HuR
proteins)/HOTAIR lncRNA elements. The hnRNPB1/A2 protein consists of two RNA recognition
motifs(RRM1- 2) which are held together by a linker that is seven amino acids long and glycine-rich C-
terminal region. The difference between the hnRNPB1 and A2 protein is that B1 has extra 12 amino
acids at its N-terminal region. Full length and mutant plasmid constructs, a kind gift from Akeli
Mayeda, Japan were used in these studies. Derivative constructs including the hnRNPB1 RRM1, RRM2,
RRM1-linker, linker-RRM2, RRM1-2, and full length construct that include the glycine rich C-terminal
end and also the hnRNPA2 RRM1, RRM2, RRM1-linker, linker-RRM2, RRM1-2, and full-length protein
and the mutant constructs wherein the mutations are within these RRM regions were created.

The lncRNA HOTAIR RNA is prepared by in vitro transcription i.e. stem-loop H12 which is a target for
hnRNPB1 recognition domain 1 of HOTAIR. To check for the integrity of H12 RNA first denaturing and
native gel are run. Followed by thermal melting studies to check the stability of H12 RNA which
showed two melting temperatures at 62°C and 82°C. Electrophoretic mobility shift assays (EMSAs)
showed that the hnRNPB1(1-278) binds to H12 to form more than one complex as evident from
supershifting which represents that RNA target can accommodate multiple hnRNPB1 proteins. As all
the protein constructs that include RRM1, RRM2, RRM1-linker-RRM2 protein constructs are being
studied to check which region of the protein is involved in binding to H12 stem-loop region of lncRNA
HOTAIR. The thermodynamic contribution of these domains to this interaction with H12 was also
studied through isothermal calorimetry. The binding of hnRNPB1 (1-278) where RGG domain shows
high binding affinity with a biphasic curve is due to positive charged residues in RGG region binding to
the negatively charged phosphate backbone. Further, the RRMs by themselves only showed 4-5 uM
binding showing that both the RRMs together is needed for tight binding. The circular dichroism data
for the free RNA and complex was also collected. The minimal change in the intensity of peak for free
RNA versus the complex with the B1 constructs (1- 195, 1-278) shows that RNA structure is mostly
intact. The knowledge gained from this research will enhance our fundamental understanding of the
biology of RBPs/HOTAIR interactions. It may also lead to novel strategies to inhibit these interactions
that might be important in cancer development.