Page 141 - CSIR-IGIB Annual Report 2020-21

RNA Biology 3’end of MALAT1 aids in localization of NCL in
MALAT1 (Metastases Associated Lung speckles, where it gets acetylated and
Adenocarcinoma Transcript 1) is one of the most orchestrates alternative splicing.
extensively studied long non-coding RNA
(lncRNA). The 3’ end of this lncRNA is known to Nucleic Acid chemistry and detection
promote metastases and is important for its We have developed CRISPR-Cas
localization to nuclear speckles. Bioinformatic Ribonucleoprotein complex for detecting single
analysis revealed that three putative G- nucleotide variants in RNA or DNA or more
quadruplex forming motifs are present at the broadly, any DNA or RNA fragment, without the
3’end of this transcript and are well conserved need for sequencing. The principle of
across species. In our study, we first validated the discrimination is derived from the natural
potency of the three stretches to form G- property of the enzyme being used for the
quadruplex in vitro by using traditional biophysical invention, Francisella novicida Cas9 (FnCas9)
techniques such as CD and UV-Visible which shows very low binding affinity to
spectroscopy. To understand the biological mismatched substrates. DNA is isolated either
relevance, we performed RNA pull-down assay from blood, saliva, or any other biological sources
followed by mass spectrometry using these G- like bacteria and amplified if required. For virus
quadruplex forming sequences, as well as RNA- infected patients, samples are collected as a nasal
immunoprecipitation via which we identified that swab and inactivated. Total RNA isolated from the
Nucleolin protein (NCL) is a bonafide cellular sample is converted to cDNA using the reverse
partner. Using immuno-cytochemistry, we transcriptase enzyme. The DNA (when test
observed that the localization of NCL to speckles is material is DNA) or cDNA (when test material is
directly dependent on the presence of the RNA, like for COVID-19) is subjected to Polymerase
quadruplex structures of MALAT1. We conducted Chain reaction, amplifying using specific primers
immunocytochemistry studies to specifically and tagging the amplified DNA products with a
detect acetylated NCL, by which we demonstrated ligand of choice. The detection mix consists of
that NCL shuttles to speckles and gets acetylated labelled PCR products, sgRNA-fnCAS9 complex.
by the Histone Acetyl Transferases (HATs) already The detection complex can be visualized using a
present in the speckle protein complex. MALAT1 wide array of technologies like lateral flow, gel
in nuclear speckles was previously known to be based cleavage assay, fluorescence based
involved in regulating alternative splicing of pre- detection, in both low, medium or plate based
mRNAs. We followed the splicing pattern for the high-throughput format. The most important
specific pre-mRNA transcripts and found that G- advantage of the present invention as a detection
quadruplex of MALAT1 contributes to this tool over closest prior art is the combination of
regulatory role via its protein partner NCL. Our speed, reliability, robustness and universal
data indicates that quadruplexes present at the applicability for all DNA and RNA variations.

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