1.

small nuclear RNA (snRNA)

Small nuclear RNAs are approximately 150 nucleotides long single stranded RNA
molecules with extensive 2’O methylation and pseudouridylation. They are found in
eukaryotic cells. They along with other proteins play critical role in pre-mRNA splicing
during post transcriptional modification of mRNA. In the process, the non-coding sequence
which are called introns are spliced and removed and coding regions called exons are
reattached. (2)

In pre-mRNA splicing process, a complex consisting of five small nuclear RNAs (U1, U2,
U4, U5 and U6) along with other proteins are formed. (2) This complex is know as
spliceosome which can splice RNA in a stepwise process given below-

1. At first, the 5’-end of U1 snRNA recognize and binds to 5’ splice-site of pre-

mRNA by complementary base-pairing.
2. Then U2 snRNA attach to 3’ splice-site sequence of pre-mRNA which causes the
branch point nucleotide (adenosine) to bulge out.
3. Consequently, the other snRNAs- U4/U6.U5 trisnRNP joins the complex.
4. This initiates a series of RNA-RNA rearrangement which consequently destabilize
the U1 and U4 from the complex.
5. This activates the spliceosome where the 2’OH group of the branch point attacks
the 5’SS and the intron is excised and released
6. Following which, the U2, U5 and U6 snRNPs are also released and a mature
mRNA is produced. (2)

The above five steps are illustrated by the figure below :-

Watson, Patricia & Watson, Dennis. (2010). Alternative Splicing in Prostate and Breast Cancer. The
Open Cancer Journal. 3. 62-76. 10.2174/1874079001003010062.

1. microRNA (miRNA)
MicroRNAs are 21-23 ribonucleotides long non-coding RNA which are present in
eukaryotic cells (6). They play major role in post-transcriptional gene regulation. Micro
RNA is also known to cause gene silencing and translational repression as well as
activation. (3)

In mechanism of miRNA mediated gene regulation, the miRNA binds to the 3’ UTR of
the targeted mRNA which in turn may cause translational repression, deadenylation and
decapping of mRNA and mRNA cleavage.

In the mRNA cleavage process, extensive base-pairing occur between miRNA and
mRNA which forms RISC (RNA-induced silencing complex). Following which, Ago2
is recruited in the targated area in the RISC. Ago2 have slicer activity which means it can
cleave RNA. The cleavage of mRNA by Ago2 results in the nucleolytic degradation of
the targeted mRNA. As a result, the gene is not expressed and no protein can be
produced. (14)

14

2. small nucleolar RNA (snoRNA)

 Small nucleolar RNAs are 60-300 nucleotides long non-coding RNAs. They are encoded
by introns and are found mainly in the nucleolus. They play major role in
posttranscriptional modification and cleavage of ribosomal RNAs, snRNA and other
cellular RNAs. This modification involves extensive methylation and pseudouridylation
of rRNAs and snRNAs. The small nucleolar RNAs are divided into 2 classes: C/D box
snoRNAs and H/ACA box. (4) The C/D box snoRNA contains 2 conserved sequences, which
are: C box (RUGAUGA) and D box (CUGA) located at the 5’ and 3’ ends respectively.
These C and D boxes serves as methylation guidelines. The H/ACA box contains 2
conserved sequences, which are: H box (ANANNA) and ACA box (ACA). These H and
ACA boxes serves as pseudouridylation guidelines. (4)(9)

One key role of snoRNA is the modification and maturation of rRNA and snRNA. The
modification involves methylation and pseudouridylation of rRNA and snRNA. In the
methylation process, the C/D box snoRNA and fibrillarin, which is a protein showing
methyl transferase activity is used. (9) At first, the sequence upstream of D box being
complementary to rRNA, attaches to it. Following this, the fibrillarin directs 2’-O-
methylation in rRNA. (9) In the pseudouridylation process, H/ACA snRNA along with
dyskerin, which shows pseudouridine synthase activity. At first, the H/ACA box snoRNA
attaches with the targeted rRNA. Then dyskerin carries out the isomerization of uridine
into pseudouridine. (10)

The binding of snoRNA in 2' -O- methylation and pseudouridylation is illustrated

by the following figure:-
 Yu, Yi-Tao & Terns, Rebecca & Terns, Michael. (2004). Mechanisms and functions of
RNA-guided RNA modification. 10.1007/b105585.

In addition to this, snoRNAs also carries out regulation of mRNA splicing and editing. (4)

1. long non-coding RNA (lncRNA)

Long non-coding RNAs are more than 200 nucleotides long RNAs which play vital role in gene
regulation. They can interact with DNA, RNA as well as protein and regulate chromatin structure
and function. IncRNAs can act as decoys, scaffolds, and enhancer RNAs. In chromatin
regulation, the negatively charged IncRNA binds to the positively charged histone tails, This
causes the histone to loosely pack with the DNA in chromatin and as a result, the gene
expression is switched on in the chromatin. (5) LncRNA is also used as a decoy molecule. They
work by blocking a certain biochemical route by directly binding to some protein molecules,
such as chromosomal folding proteins or transcription regulators which inhibits their function.
LncRNAs can also bind directly to transcription regulators which in turn blocks transcription
factor. This together suppress the downstream gene transcription. IncRNA also acts as scaffold.
For example, an IncRNA called X-inactive specific transcript (Xist) RNA is encoded by the X
chromosome in female. This Xist RNA  recruits two complex PRC1and PRC2, which suppress
the expression of X chromosome. Thus in females, one chromosome remain inactivated. (16)
There are many IncRNA carrying out several different functions. Some of them are listed in the
table below (15) :-

1. Piwi-interacting RNAs (piRNA)

Piwi-interacting RNAs were first found in Drosophila testes in 2001. It is a type of non-coding
RNA which is 24 to 31 nucleotides long and are stabilized by piwi proteins. They are primarily
transcribed in germ line cells as well as somatic cells. (8)(12)

It plays a role in transcriptional gene silencing. At first, the piRNA binds to the targeted
sequence in DNA. Then the piRNA recruits silencing machinery components like Egg and its co-
factor WDE which together add repressive histone to the targeted DNA. Subsequently, a
heterochromatin protein 1 (HP1) is recruited in the site, which induce heterochromatin formation
which is transcriptionally inactive. Another way of gene silencing by piwi complex is
DNA methylation. It recruits DNA methyltransferase to methylate genic CpG
sites, so the gene cannot be transcribed. (12)
https://www.sciencedirect.com/science/article/pii/S0968000415002583

The piRNA along with piwi proteins play pivotal role in spermatogenesis where they silence mobile
genetic elements called transposons. The silencing of transposons are carried out by post-
transcriptional transcript destruction. The piRNA binds to mature transposon transcript with the
help of Aub and Ago3, Then they undergo homology-dependent cleavage where the protein
coding genes between the introns are not silenced.(13)