Page 59 - Annual report 2021-22

Annual Report 2021-22 |

Souvik Maiti

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Gene editing has scripted a new chapter in post-genomic biology making genetic diseases actionable.
At the core of gene editing technologies lies ribonucleoprotein complexes which bind to DNA to alter
the DNA sequence. A thorough understanding of the biophysical parameters that govern these
interactions allow informed engineering of these systems, to maximize efficacy without compromising
sensitivity in diagnostics and specificity in gene editing, both of which use Cas9 proteins. With these
over-arching goals, Souvik Maiti’s laboratory studies the biophysical principles that govern RNA-
protein, RNA-DNA and DNA- RNA-Protein interactions. Besides CRiSPR/Cas systems, such an approach
is also valuable in understanding the role of secondary structure in lncRNA function.

The triplex towards the 3' region of MALAT1 lncRNA is known to stabilize this lncRNA. Several small
molecules that bind nucleic acid triplexes that could target MALAT1 triplex were identified through
screening. Of these, quercetin showed approximately 50% reduction in MALAT1 levels at a low
concentration. Quercetin was found to lower MALAT1 lncRNA levels in MCF7 cell line and A549 lung
adenocarcinoma cell line. Quercetin showed a similar effect of downregulation of MALAT1 lncRNA, but
A549 needed a ten times higher dose. For biophysical studies on the binding of quercetin MALAT1
triple helix region, a canonical MALAT1 triplex forming 94 nt RNA was compared to a 73nt variant which
formed a duplex structure and was not capable of forming a triplex. Using a battery of techniques,
including UV melting, UV titration, ITC and docking studies a major groove binding mode was found to
be the most stable, lowest energy interaction. Since MALAT1 RNA levels were reduced on quercetin
treatment of cells, RNA FISH was performed. On treatment of MCF7 breast cancer cells with 1 microM
of quercetin and probing with MALAT1 RNA probes, approximately 50% reduction in the number of
MALAT1 puncta of a particular size was evident.

The putative RNA quadruplex (rG4) motif includes three putative rG4 forming motifs named Q1, Q2,
and Q3. To understand whether the rG4s formed in vitro, RNA oligonucleotides for the three putative
rG4 forming sequences and their mutated counterparts were custom synthesized and studied using
CD spectrometry and UV melting study. To validate the formation of these structures in the longer
sequence full-length MALAT1 clone (courtesy: K.V. Prasanth, University of Illinois) was subjected to
site-directed mutagenesis to cause G to A mutations in single rG4 mutant MALAT1 (Q1m, Q2m, Q3m),
double rG4 mutated MALAT1 (Q12m, Q23m, Q13m) and triple rG4 mutated MALAT1 (Q123m). In vitro
synthesized RNA from these plasmids was titrated against an rG4 sensor molecule Thioflavin T (ThT)
to find that the rG4s formed in the longer sequence as well.

As MALAT1 localizes in the nuclear speckles, the initial question was whether the rG4s were acting as
a localization cue for this lncRNA. To test this, in the MALAT1 null cell line from Roderic Guigo and
Rory Johnson's lab (CRG Barcelona) MALAT1 levels were rescued using mutant and wild type
constructs. MALAT1 expression and localization to nuclear speckles was independent of the rG4
structures. RNA pulldown followed by mass spectrometry, led to the RNA binding proteins (RBPs)
Nucleolin (NCL) and Nucleophosmin (NPM). NCL and NPM localize to nuclear speckles, apart from their
major residence in the nucleolus. Immunofluorescence (IF) or Immunocytochemistry (ICC)
experiments were performed to investigate the localization of NPM and NCL in the presence and