Types of RNA's
Types of RNA's
The chemical structure of RNA is very similar to that of DNA, with two differences, (a) RNA contains the
sugar ribose instead of sugar deoxyribose and (b) RNA has the nucleotide uracil in place of DNA’s
thymine .
Most RNA molecules exists as single stranded and can adopt complex three-dimensional structures.
RNA are biosynthesized by a process known as transcription mediated by an enzyme RNA polymerase.
RNA play essential role in many important processes like translation i.e. protein synthesis where mRNA,
r RNA and tRNA play a crucial role by diligently encoding proteins for the message. They also play role in
controlling gene expression, or sensing and communicating responses to cellular signals. In the absence
of proteins, catalyzing enzymes, RNA can also perform biocatalysis.
Messenger RNA (mRNA)
Transfer RNA (tRNA)
In translation
Ribosomal RNA (rRNA)
Transfer-messenger RNA (tmRNA)
Micro RNAs (miRNA)
CRISPR RNAs
Ribozymes
Non-coding RNA
RNAs in Translation
Messenger RNA (mRNA)
Messenger ribonucleic acid (mRNA), like DNA, carries unique codes in different series of patterns that
relay messages to structures within the cell. mRNA is transcribed from a DNA template, and carries
coding information to the sites of protein synthesis: the ribosomes. In mRNA as in DNA, genetic
information is encoded in the sequence of nucleotides arranged into codons consisting of
three bases each. Each codon encodes for a specific amino acid, except the stop codons that
terminate protein synthesis. This process requires two other types of RNA: transfer RNA (tRNA)
mediates recognition of the codon and provides the corresponding amino acid, while ribosomal
RNA (rRNA) is the central component of the ribosome's protein manufacturing machinery.
Structure
Structure of mRNA divided into 5’ cap, untranslated region, coding region and a poly A tail.
The 5' cap is a modified guanine nucleotide added to the "front" (5' end) of the pre-mRNA using a 5'-5'-
triphosphate linkage. This modification is critical for recognition and proper attachment of mRNA to the
ribosome, as well as protection from 5' exonucleases. It is also important for other essential processes,
such as splicing and transport.
Untranslated regions (UTRs) are sections of the mRNA before the start codon and after the stop codon
that are not translated, termed the five prime untranslated region (5' UTR) and three prime untranslated
region (3' UTR), respectively. These regions are transcribed with the coding region and thus are exonic as
they are present in the mature mRNA. Several roles in gene expression have been attributed to the
untranslated regions, including mRNA stability, mRNA localization, and translational efficiency.
Coding regions are composed of codons, which are translated into one protein in eukaryotes and in
prokaryotes usually into several proteins by the ribosome. Coding regions begin with the start codon and
end with a stop codon. The coding regions tend to be stabilised by internal base pairs, this impedes
degradation. In addition to being protein-coding, portions of coding regions may serve as regulatory
sequences in the pre-mRNA asexonic splicing enhancers or exonic splicing silencers.
The 3' poly(A) tail is a long sequence of adenine nucleotides added to the 3' end of the pre-mRNA. This
tail promotes export from the nucleus and translation, and protects the mRNA from degradation.
Figure 1. showing structure of a mature eukaryotic mRNA. A fully processed mRNA includes a 5' cap, 5'
UTR, coding region, 3' UTR, and poly (A) tail.
Function
Structure
The ribosomal RNAs form two subunits, the large subunit and small subunit. mRNA is sandwiched
between the small and large subunits and the ribosome catalyzes the formation of a peptide bond
between the 2 amino acids that are contained in the rRNA.
Function
The structure of tRNA can be decomposed into its primary structure, its secondary structure (usually
visualized as the cloverleaf structure), and its tertiary structure (all tRNAs have a similar L-shaped 3D
structure). It has a 3' terminal site for amino acid attachment. This covalent linkage is catalyzed by
an aminoacyl tRNA synthetase. It also contains a three base region called the anticodon that can base
pair to the corresponding three base codon region on mRNA.
The 5'-terminal phosphate group. The acceptor stem is a 7-base pair stem which contains the CCA 3'-
terminal group used to attach the amino acid. The acceptor stem may contain non-Watson-Crick base
pairs. The D arm is a 4 bp stem ending in a loop that often contains dihydrouridine. The anticodon arm is
a 5-bp stem whose loop contains the anticodon. The T arm is a 5 bp stem containing the sequence TΨC
where Ψ is a pseudouridine.
transfer RNA (tRNA) has role in gene expression. During gene expression, DNA is first transcribed into
messenger RNA. Next, tRNA molecules—each carrying an amino acid as cargo—bind to successive
nucleotides in the messenger RNA. A ribosome links these amino acids together to form a protein, and
the unloaded tRNAs are subsequently released. tRNA also helps control apoptosis, or programmed cell
death. tRNA also binds to cytochrome c, stopping it from binding to Apaf-1 and thereby preventing
apoptosis. tRNA is highly expressed in tumor cells. It inhibits apoptosis in these cells.
Transfer-messenger RNA (tmRNA)
Transfer-messenger RNA abbreviated as tmRNA also known as 10Sa RNA and by its genetic name SsrA. It
is found in bacteria, it exhibits the properties of both tRNA and messenger RNA. The tmRNA forms
a ribonucleoprotein complex with Small Protein B (SmpB), Elongation Factor Tu (EF-Tu), and ribosomal
protein S1.
Structure
Important feature of every tmRNA is the conserved tRNA-like domain (TLD), composed of helices 1, 12,
and 2a which are analogous to the tRNA acceptor stem, T-stem and variable stem. It contains the 5'
monophosphate and alanylatable 3' CCA ends. The mRNA-like region (MLR) is in standard tmRNA a large
loop containing pseudoknots and a coding sequence (CDS) for the tag peptide, marked by the
resume codon and the stop codon. The encoded tag peptide (ANDENYALAA in E. coli) varies among
bacteria, perhaps depending on the set of proteases and adaptors available.
In trans-translation, where ribosome has stalled in the middle of protein synthesis, tmRNA and its
associated proteins bind to bacterial ribosomes and recycles the stalled ribosome, adds a proteolysis-
inducing tag to the unfinished polypeptide, and facilitates the degradation of the aberrant messenger
RNA. In the majority of bacteria these functions are carried out by standard one-piece tmRNAs. In other
bacterial species, a permuted ssrA gene produces a two-piece tmRNA in which two separate RNA chains
are joined by base pairing.
Figure 4. showing trans-Translation stages A through F. A ribosome with its RNA binding sites, designated E, P, and A, is stuck
near the 3' end of a broken mRNA. The tmRNP binds to the A-site, allowing the ribosome to switch templates from the
broken message onto the open reading frame of the tmRNA via the resume codon (blue GCA). Regular translation eventually
resumes. Upon reaching the tmRNA stop codon (red UAA), a hybrid protein with a proteolysis tag (green beads) is released.
Heterogeneous nuclear RNA is also termed as precursor mRNA. It is immature single strand of mRNA. Pre-mRNA
is produced during transcription from DNA template in cell nucleus. After processing of pre-mRNA it is called as
mature mRNA or mRNA. During the processing of eukaryotic pre-mRNA exons are retained while introns are
removed by spliceosomes by a process of splicing. After processing mature mRNA is exported out of the nucleus
where they are translated into proteins by ribosomes.
hnRNA is 4 to 10 times more complex than mRNA. hnRNA has non random and sequence-dependent
ribonucleoproteins.
Regulatory RNAs
MicroRNAs (miRNA)
A miRNA (micro-RNA) is a form of single-stranded RNA which consists of short stretch of RNA which is
20-25 nucleotide long. It is found in all eukaryotic cells. They were initally believed to regulate the
expression of other genes. But it is now clear that they are post transcriptional regulators which are
complementary to the target mRNA sequence. miRNAs are RNA genes which are transcribed from DNA,
but are not translated into protein. They function to translational repression and gene silencing.
Structure
The DNA sequence that codes for an miRNA gene is longer than the miRNA itself. This DNA sequence
includes the miRNA sequence and an approximate reverse complement. When this DNA sequence is
transcribed into a single-stranded RNA molecule, the miRNA sequence and its reverse-complement base
pair to form a double stranded RNA hairpin loop; this forms a primary miRNA structure (pri-miRNA).
In animals, the nuclear enzyme Drosha cleaves the base of the hairpin to form pre-miRNA. The pre-
miRNA molecule is then actively transported out of the nucleus into the cytoplasm by Exportin 5, a
carrier protein. The Dicer enzyme then cuts 20-25 nucleotides from the base of the hairpin to release
the mature miRNA.
In plants, which lack Drosha homologues, pri- and pre-miRNA processing by Dicer probably takes place in
the nucleus, and mature miRNA duplexes are exported to the cytosol by Exportin 5. The pre-miRNA
molecule is then actively transported out of the nucleus into the cytoplasm by Exportin 5, a carrier
protein. The Dicer enzyme then cuts 20-25 nucleotides from the base of the hairpin to release the
mature miRNA. The miRNA in plants has a perfect complimentarity with its target mRNA with a few
mismatches.
Functions
They are primarily involved in regulation of gene expression. That is why miRNA sequence is
complemntary to a part of mRNA or to one or more mRNAs. Animal miRNAs are complemntary to the
mRNA at a site in 3' UTR whereas plant miRNAs are complementary to the coding region of mRNA.
miRNA regulate gene expression by binding to complementary sequence on the mRNA which prevents
mRNA from being translated, sometimes it also causes cleavage of mRNA.
miRNA with partial complimentarity to its target can degrade mRNA by speeding up deadenylation.
Sometimes miRNA also cause histone modification and DNA methylation at promoter site interfering the
expression of targeted genes.
MicroRNAs are significant phylogenetic markers because of their astonishingly low rate of evolution.
MicroRNAs originate predominantly by the random formation of hairpins in "non-coding" regions of
DNA, but also by the duplication and modification of existing microRNAs. Once a microRNA gains a
function it undergoes extreme purifying selection and are rarely lost. This make them ideal candidate to
serve as phylogentic markers.
Small interfering RNAs are also known as short interfering RNA. They are a short stretch of 20-25
nucleotide-long RNA molecules that interfere with the expression of genes. They are synthesized during
RNA interference (RNAi) pathway by the enzyme Dicer. They find extensive use in labs to study
particular gene function, by knocking them down.
Structure
siRNA's have a short of 20-22 nucleotides, double-strand of RNA (dsRNA) with 2-nucleotides overhangs
on either end, including a 5' phosphate group and a 3' hydroxy (-OH) group.
RNA polymerase III promoter, which direct the transcription of small nuclear RNA's. The resulting
molecule has a short hairpin RNA transcript which is further processed by dicer enzyme.
Introduction of too much siRNA can result in non-specific events due to activation of the interferon
pathway. One method of reducing the non-specific effects of siRNA is by turning the shRNA into a micro
RNA. Since miRNA's are naturally occurring and well tolerated by the cell. So by engineering a siRNA
sequence into a miRNA structure, non-specific effects can be eliminated.
Function
The mediators of RNA interference are 21- and 23-nucleotide small interfering RNAs (siRNA). siRNAs
bind to a ribonuclease complex called RNA-induced silencing complex (RISC) that guides the small
dsRNAs to its homologous mRNA target. Consequently, RISC cuts the mRNA approximately in the middle
of the region paired with the antisense siRNA, after which the mRNA is further degraded.
Piwi-interacting RNAs (piRNA)
Argonaute are a class of proteins which are essential components of small RNA silencing pathways. Base
on the similarities in argonaute protein , it can be divided into AGO and PIWI. piRNAs are the largest
class of novel small RNAs which binds specifically to PIWI type of Argonaute proteins, in animals. Both
PIWI type of Argonaute proteins and piRNAs are expressed in gonads of animals where they play a
crucial role in transcriptional gene silencing of retrotransposons and maintains integrity of genome
during gametogenesis.
Structure of piRNA
piRNAs have been found clustered throughout the genome and each cluster contains 10-10,000 of
piRNA. The clustering of piRNA is highly conserved across the species. piRNA have been found out at
cytoplasm and nucleus suggesting their role in both cytoplasmic and nuclear processes.
piRNAs are found both in vertebrates and invertebrates, though their biogenesis is not very clear.
piRNAs have no clear secondary structure motifs, the length of a piRNA is between 26 and
31 nucleotides, and the presence of a 5’ uridine is common to piRNAs in both vertebrates and
invertebrates. piRNAs in C. elegans have a 5’ monophosphate and a 3’ modification that acts to block
either the 2’ or 3’ oxygen.
Function
piRNAs have been known to act as gene silencers specifically of transposons. Their antisense sequences
to transposons further strengthens the statement. In mammals, the activity of piRNAs in transposon
silencing is most important during the development of the embryo, and in both C. elegans and humans,
piRNAs are necessary for spermatogenesis.
CRISPR RNAs
Clustered regularly interspaced short palindromic repeats (CRISPR) RNA. They belong to a class of RNA
regulator, which act through base pairing with RNA. They function to influence the translation and
stability of mRNAs. CRISPR systems have some similarities with eukaryotic siRNA-driven gene silencing,
although they exhibit distinct features as well. They are found in the genomes of a few bacteria and in
most of archaea.
CRISPR contains sequences which are highly variable DNA regions that consist of ~550 bp leader
sequence followed by a series of repeat-spacer units. The repeated DNA can vary from 24 to 47 base
pairs, but the same repeat sequence usually appears in each unit in a given CRISPR array and is repeated
2 to 249 times. Adjacent to the CRISPR DNA array are several CRISPR-associated (CAS) genes. Two to six
core CAS genes seem to be associated with most CRISPR systems, but different CRISPR subtypes also
have specific CAS genes encoded in the flanking region.
Function
CRISPR RNAs interfere with bacteriophage infection and plasmid conjugation and provide a kind of
immunity against infection. They do so by targeting the homologous foreign DNA through an unknown
mechanism. But, few researchers have a belief that they provide immunity by integrating foreign DNA in
between the repeat sequences in the CRISPR locus of the prokaryote. The integrated DNA sequence is
then used by the CRISPR system to identify and silence exogenous genetic elements in a manner
analogous to RNAi in eukaryotic organisms.
The molecular functions of the CAS proteins are still not clearly known but they are contain RNA- or
DNA-binding domains,helicase motifs, and endo- or exonuclease domains.
Recent findings suggests that CAS genes encode for proteins which have function similar to eukaryotic
RNAi enzymes. The CRISPR DNA array is transcribed into a long RNA, which is processed by the Cascade
complex of CAS proteins into a single repeat-spacer unit known as a crRNA. crRNAs would target DNA or
RNA, but CRISPR spacers generated from both strands of phage genes can effectively confer phage
resistance.
Figure 9. showing gene arrangement and regulatory function of CRISPR RNAs
CRISPR arrays are composed of DNA repeats (triangles) separated by unique spacers (speckled boxes). CAS genes (blue), which encode
proteins that function in CRISPR RNA processing and/or DNA silencing, are located nearby. The CRISPR arrays are initially transcribed as a
long RNA, which is subsequently processed by the Cascade complex (blue circles and ovals) to individual repeat-spacer units, called crRNAs.
These crRNAs appear to target foreign DNA through an unknown mechanism likely involving other CAS proteins and the degradation of the
exogenous DNA
The CRISPR system finds a wide use in evolutionary studies. The extreme variability of CRISPR arrays
between organisms and even strains of the same species makes it a useful tool for genotype strains and
to study horizontal gene transfer and microevolution.
The ability of CRISPR system to record the history of recent phage infection and allow differentiation
between strains of the same species. This unique ability of system can be used to identify pathogenic
bacterial strains and track disease progression worldwide, as well as to monitor the population dynamics
of nonpathogenic bacteria.
RNAs in RNA processing
small nuclear RNAs (snRNA)
Small nuclear ribonucleic acid (snRNA) is a class of small RNA molecules that are found only within the
nucleus, they are ~100bp. They are transcribed by RNA polymerase II or RNA polymerase III. They are
generally associated with nuclear proteins to perform some other function (e.g. pre-mRNA splicing or
telomere maintainance) like regulation of transcription factors (7SK RNA) or RNA polymerase II (B2 RNA),
and maintaining the telomeres. They found in association with specific proteins and together they form
a complex known as small nuclear ribonucleoproteins (snRNP). snRNA have modifications other than
5’cap and 3’ poly (A) tail depending on their function.
Function
snRNAs serve as recognition molecules for intron splice sites. They play an
important role in telomere maintenance. Telomerase is special among
polymerases in that it can synthesize DNA without a template. It is able to do
this because telomerase is actually a ribonucleoprotein -- a combination of
protein and RNA. Telomerase contains a ~150 nucleotide RNA subunit which serves as a template for the
telomere sequence. With this internal RNA template, telomerase can accurately extend the telomere
sequence and help maintain the chromosomes' telomeres. The RNA subunit in telomerase is a small
nuclear RNA.
Small nucleolar RNAs are a class of small RNA molecules that has a main function to guide chemical
modifications of other RNAs, mainly ribosomal RNAs, transfer RNAs and small nuclear RNAs. There are
two main classes of snoRNA, the C/D box snoRNAs which are associated with methylation, and the
H/ACA box snoRNAs which are associated with pseudouridylation.
Function of snoRNA
Each snoRNA molecule can guide the modification on only one or in some cases two individual RNA
molecule. To carry out modification of RNA, snoRNA interacts with at least four protein molecules and
forms a RNA/protein complex known as a small nucleolar ribonucleoprotein (snoRNP). snoRNA
molecules are basically a stretch of 10-20 nucleotides (antisense element) which are complementary to
the bases on the target pre-mRNA to be modified. In this way snoRNP recognizes and binds to target
RNA. Once the snoRNP has bound to the target site the associated proteins are in the correct physical
location to catalyse the chemical modification of the target base.
A B
Figure 10. showing structure of snoRNAs. A. C/D Box and b. H/ACA Box.
snoRNAs can also function as miRNAs. It has been shown that human ACA45 is a bona fide snoRNA that
can be processed into a 21 nucleotides long mature miRNA by the RNAse III family
endoribonuclease dicer.
Recently, it has been found that snoRNAs can have functions not related to rRNA. One such function is
the regulation of alternative splicing of the trans gene transcript.
RNAs as enzyme
Ribozymes
A ribozyme can be defined as an RNA molecule which has the capacity to catalyze a chemical reaction in
the absence of catalyzing enzymes (proteins). Ribozymes promote the reaction by increasing their rate
of reaction. Catalytic RNA fold themselves into a 3D structure and they catalyze the transesterification
reaction of phosphate diester and participate in RNA maturation or processing. But majority of RNAs
known perform the function of autocatalysis. The discovery of ribozymes have suggest that ribozymes
would have contributed to self replication and protein synthesis in early evolution and this idea
suggesting the role of RNA in evolution has been termed as RNA World hypothesis.
Catalytic RNAs are found in various biological areas like small catalytic RNAs were found in the
hammerhead, the hairpin, the hepatitis delta virus (HDV), and the Varkud satellite (VS) ribozymes. All
these RNAs perform the same function but they differ in their secondary and tertiary structures. In each
case, the satellite RNA is believed to replicate via a complementary RNA intermediate by a rolling circle
mechanism. The ribozymes participate in the replication by self-cleaving the tandem satellite repeats
into monomer units.
Second and more common type of catalytic RNA consists of self-splicing introns. These types of RNAs
provide the active site necessary to complete their own RNA splicing from an RNA transcipt. Based on
their differences in conserved structure and reaction mechanism they are divided into two groups. In
addition to splicing, both classes of introns can catalyze reverse splicing reactions, which allows them to
act as mobile elements for the horizontal transfer of genetic information.
Third type of ribozymes is ribonuclease P (RNase P). They participate in transfer RNA processing. It acts
in trans to catalyze the removal of nucleotides from the 5′-end of the pre-tRNA. The celular form of this
enzyme consists of both an RNA and a protein component. It is an holoenzyme where catalytic function
is carried out by RNA while protein part increases rate of its catalytic function.
Catalytic RNA consist of a three-dimensional arrangement of nucleotides, metal ions, water molecules
and co-factors. But in reality catalytic RNA acquire more complex structures tan simple single stranded
RNA.RNA can form a pseudo base pair between two consecutive A nucleotides in a single-stranded
region (termed an A-platform) that serves as a platform for tertiary helix-stacking interactions. There are
also examples of pseudoknots, G · U wobble pairing receptors, GAAA tetraloops, and U-turns. The
consensus sequences of these structural motifs are seen repeatedly in the phylogenetic database, which
suggests that they have been used as building blocks to create a variety of RNA structures.
Non coding RNA (ncRNA)
Non-coding RNA genes include highly abundant and functionally important RNAs such as transfer
RNA (tRNA) and ribosomal RNA (rRNA), as well as RNAs such as snoRNAs, miRNA , siRNA and piRNA and
the long ncRNAs that include examples such as Xist and HOTAIR.
Functions
It has been already known that ncRNA are functional in control of chromosome dynamics, RNA editing,
translational inhibition and mRNA destruction. RNA signaling underpins chromatin remodeling and
epigenetic memory, although the mechanisms are unknown. transcription itself may be regulated by
ncRNAs. As noted earlier, RNA polymerase II itself appears to be regulated in part by ncRNA signaling. A
ncRNA has been reported to be required for the repression of RNA polymerase II-dependent
transcription in primordial germ cells in Drosophila. ncRNAs also play a role in stress responses. The
small noncoding transcript B2 is produced by RNA polymerase III from murine short interspersed
elements (SINE) under heat shock. ncRNAs may also act as scaffolding for the assembly of
macromolecular complexes. Examples include rRNA in ribosomes.
Recent discovery also show, the existence of snoRNA, microRNA, piRNA characteristics in a novel non-
coding RNA: x-ncRNA.
References
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