Page 80 - Annual report 2021-22

Annual Report 2021-22 |

Beena Pillai

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The vast majority of mammalian genes do not code for a protein. Thousands of non-coding RNAs,
many of them poorly conserved and expressed at low levels participate in gene regulatory functions
as scaffolds for ribo-nucleoprotein complexes, recruiters for chromatin modifiers, sponges of
microRNAs and precursors of circular RNAs and small ORFs. Beena Pillai's lab has previously shown
the anti-HIV potential of microRNAs and the role of inherited non-coding RNAs in the development of
the zebrafish brain. Her lab also works on the mechanistic basis of polyglutamine toxicity using
different neuronal models.

Inherited ncRNAs are RNA transcripts which are longer than 200 nt, do not contain an apparent ORF,
and are found in the gametes. They are a subset of the transcriptome comprising thousands of non-
protein-coding RNA transcripts with a potential role in zygotic gene regulation. Currently, the lab is
exploring the mechanistic details of action of a novel lncRNA, named Durga. Durga originates from
the close vicinity of a protein coding gene called Kalirn. It is nuclear localized and chromatin
associated. Chromatin modification (H3K27ac) marks at the Durga-Kalirn locus were mapped and it
was hypothesized that a GCN5 family member may be a partner in bringing about the modification. A
GCN5 inhibitor, MB3, blocked this activity and knockdown of Kat2A and Kat2B is being pursued now
to test these possible modifiers. Durga expression is restricted to a few cells in the Habenula, a brain
location involved in fear response, and implicated in a variety of neuropsychiatric diseases. To study
the effects of this lncRNA on learning and memory, the novel tank test and the Light - Dark tank test
were set-up using zebrafish. The adult zebrafish from embryos which had been ectopically injected
with Durga RNA at single cell stage, showed subtle defects in the novel tank test. This test is an
estimate of fear or anxiety response, and this observation matches with the finding that Durga is
expressed in the habenula of zebrafish since habenula regulates fear response.

The typical lncRNA is not highly conserved at the sequence level, but maybe functionally conserved
by virtue of the location from which it arises, since they are often involved in regulation of protein
coding genes of the same locus. This approach was used while extrapolating from zebrafish to
mammals, by identifying non-coding RNAs arising from the Kalrn locus. On deeper investigation it was
found that the mammalian Durga locus (in human and mouse) not only gives rise to a number of
protein coding isoforms, but is also transcribed into one circular RNA and seven long non-coding RNAs
which had distinct spatio-temporal expression patterns in the developing mouse brain and in the adult
brain. For instance, Kalnc4 was readily detectable at all developmental stages in the cortex and
hippocampus. A general trend in the expression pattern was that all the lncRNAs from this locus were
least abundant during the period that coincides with the switch from neurogenesis to astrogenesis.
During the later stages when synapses mature, the expression of the Kalrn locus lncRNAs are again
relatively high. This pattern coincides with the decreasing expression of Kalrn isoforms Kal9 and Kal12
whose transcription start site does not overlap with mmDurga. However, the Kalirn isoform Kal7 which
has a TSS overlapping the same region from which Durga originates, showed the opposite effect.
Durga RNA might be involved in shaping the alternative transcript isoform profile of Kalrn mRNA and
thus playing an indirect regulatory role in neuronal maturation. Based on comparative genomics it was
proposed that mammalian genomes express a syntenically conserved lncRNA from the 5'end of the