Page 79 - CSIR-IGIB Annual Report 2020-21
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Dissecting the binding mechanism of nucleolin colocalization studies for NCL and Telo DNA G-
Nucleolin (NCL) is an evolutionarily conserved quadruplex.
RNA-binding protein found predominantly in
the nucleolus and is involved in ribosome Structure-function dynamics of CONCR and
biogenesis, microRNA processing, mRNA DDX11
stability, and transcriptional regulation. The The lncRNA CONCR (cohesion regulator
RNA binding ability of NCL is attributed to its noncoding RNA) is found to be essential for
four RBD (RNA-binding domains) and the highly sister chromatid cohesion establishment during
disordered RGG domain. NCL RBDs bind to both cell division. CONCR was upregulated in multiple
canonical and non-canonical nucleic acid cancer types and is closely associated with p53.
structures, such as stem-loop structures, bulges, Deletion of p53 causes an upregulation in
and G-quadruplexes. This diverse substrate CONCR expression. CONCR was found to be
promiscuity is likely to result from its modular necessary for proper DNA replication and its
feature which can use a combination of its depletion leads to severe sister chromatid
various domains for binding different cohesion defects. CONCR is transcribed in
substrates. However, particular domains in the antisense from the DDX11 promoter. DDX11 is a
protein structure essential for its preferential superfamily 2 DNA helicase involved in proper
binding and specificity towards different nucleic chromosome cohesion at both the centromeres
acid structures are still not determined and and along the chromosome arms, as well as
comprehensive information comparing its tight binding of cohesion complexes to
recognition and preferential binding ability chromatin. Loss of this protein leads to
towards particular nucleic acid structures or embryonic lethality. An earlier report had
sequences is lacking. NCL modulates the suggested that CONCR binds to DDX11 and the
expression of several oncogenic genes, thereby, defects caused due to sister chromatid cohesion
it is imperative to dissect the recognition ability loss upon CONCR depletion can be rescued by
of relevant domains for therapeutic purposes. over-expression of DDX11, thus proving that
15 NCL constructs corresponding to different both protein and RNA function in a common
domains of NCL were successfully cloned and pathway. Our aim was to recognize these
protein was purified. Binding assays with Telo conserved RNA motifs in CONCR as therapeutic
and Terra DNA were carried out using EMSA and targets to restore normal sister chromatid
MST (Microscale thermophoresis) assays. ΔNCL cohesion and rescue cohesion defects, as well as
(lacking the N-terminal domain) showed binding delineate its role in cellular processes. We
towards both telomeric DNA and RNA G- looked at the secondary structure of CONCR by
quadruplex structures. Individual RNA using the online platform RNAalifold and
recognition motifs (RRMs) lack the ability to determined that CONCR has a highly organised,
bind to the 21 nt Telo DNA G-quadruplex but modular structure having multiple small
RRM2, RRM3 and RRM4 could retain their secondary structures including stem loops,
binding ability towards the 21nt Terra RNA G- bulges and hairpins. We also identified potential
quadruplex. The shortest domain required for G-quadruplex forming sequences (PQS), which
recognition and binding to Telo DNA G- are highly regulating non-canonical secondary
quadruplex is the RRM4 to C-terminal domain of structure moieties inside the cell, having roles in
the NCL. Through CD studies, we found that gene regulation which were identified through
individual RRMs do not induce any QGRS Mapper. We validated these sequences
conformational change upon binding to Telo for their ability to form quadruplexes, however
DNA G-quadruplex and Terra RNA G- these did not show characteristic signatures as
quadruplex. Future studies include performing G-quadruplexes as seen by UV Melting and CD
studies. Further in this project, we are currently
Annual Report 2020-21 76
