Post-transcriptional Modifications

Arun Kumar Under the supervision of:

PhD Scholar Dr. Ashok Kumar Yadav
Centre for Molecular Biology Assistant Professor

CENTRAL UNIVERSITY OF Centre for Molecular Biology

JAMMU CENTRAL UNIVERSITY OF JAMMU

 Post-transcriptional Modifications

Almost all major types of RNA synthesized by cellular DNA dependent RNA polymerases undergo changes
before they can carry out their functions. These changes involve addition to or alterations of existing bases or
Sugars and phosphodiester bond cleavage or loss of certain nucleotides from the transcript.
The nascent RNA, also known as primary
transcript, needs to be modified to
become functional tRNAs, rRNAs, and
mRNAs.

Post-transcriptional step can also be

regulated to control gene expression in
the cell.

• The modification is critical to

eukaryotic systems, otherwise if RNA
is not processed, shuttled or translated,
then no protein will be synthesized.
Post-transcriptional modifications of RNA
accomplish two things:

1) During post-transcriptional processing, portions of the RNA chain

that are not supposed to be translated into proteins are cut out of the
sequence.

2) Modifications help the RNA molecule to be recognized by molecules

that mediate RNA translation into proteins.
 Post transcriptional modifications
 RNA Processing
• Processing of Eukaryotic Pre-mRNA
• Processing of Pre-rRNA
• Processing of Pre-tRNA

 mRNA degradation
• Degradosome based
mRNA Splicing
 Processing of Eukaryotic Pre-mRNA
• In humans and other eukaryotes, a freshly made
RNA transcript is not quite ready to go. Instead,
it's called a (pre-mRNA/heteronuclear RNA
(hnRNA)) Primary transcripts of mRNA.
• hnRNA are larger than matured mRNA by many
folds and has to go through some processing
steps to become mature messenger RNA (mRNA)
that can be translated into a protein.

Processing includes:
 5’capping
 3’Polyadenylation
 Splicing
 RNA editing
 5’ Capping
• The 5’- cap structure is found on hnRNA too. The capping process occurs in nuclei.
• The capping occurs prior to the splicing.

Types of Methylated caps

 Two types of methylated caps are found
a) Mono-methylated caps
• commonly found in most eukaryotic mRNAs
b) Tri-methylated caps
• less frequently observed (e.g., only on some non-coding RNAs) but highly conserved throughout the
eukaryotes {e.g., small nuclear (sn) RNAs, small nucleolar (sno) RNAs, and telomerase RNA TLC1}
5’ Capping and Mono-methylation
 The presence of an unusual methylated nucleotide at the 5’
terminus most viral & cellular mRNAs was discovered by A.
Shatkin & co-workers in 1975.
 The entire methylated terminal oligonucleotide is called as
‘cap structure’.
 Site of biogenesis is Nucleus, where by a series of enzymatic
reaction process takes place.
 Capping process is completed before the completion of
nascent transcript.
 TFIIH kinase phosphorylate S-5 at CTD tail (-Tyr1-Ser2-Pro3-
Thr4-Ser5-Pro6-Ser7-)n and recruits enzymes required for 5’
capping of mRNA.
 Primary transcripts (pre-mRNAs or heterogeneous nuclear
RNA) are usually first "capped" by a guanylyl group.
 The reaction is catalyzed by guanylyl transferase.
 Capping G residue is methylated at 7-position.
 Additional methylations occur at 2'-O positions of next two
residues and at 6-amino of the first adenine.
 Function of 5’CAP
• Cap may function in the transport of mRNA from nucleus to cytoplasm (CBP 20/80 interact with
TREX component & facilitate transport of mature mRNA).
• It protect mRNA from phosphatases & nucleases attack and degradation (enzymatic decapping is one
of the major mechanisms in eukaryotic cells to regulate mRNA turnover)
• Required for productive mode of elongation of transcription by RNA Pol II (It represents a checkpoint
for transcription re-initiation from the initial pausing)
• Helps in ribosomal attachment
• The leader sequence & Cap binding proteins (CBP) may play a role in enhancing & stabilizing the
interaction of the mRNA with the ribosome & translational initiation process
 TRIMETHYL GUANOSINE CAP

The tri-methyl-guanosine (TMG) cap modification is highly conserved throughout the eukaryotes and mainly
present in Small nuclear (sn) RNAs, Small nucleolar (sno) RNAs and telomerase RNA TLC1
Example:
• S. Cerevisiae etc.

Site of Biogenesis:
• m7G in nucleus
• Hypermethylation occurs (addition of other two methyl groups on 2’C) in cytosol

Main functions:
• Efficient pre-mRNA splicing & pre-rRNA processing
• Small ribosomal subunit synthesis
• Maintenance of the structural organization of nucleolus
 3’ Polyadenylation
The 3’ ends of Pol II transcribed mRNAs are generated by cleavage followed by polyadenylation

The hexanucleotide sequence

AAUAAA is necessary for
cleavage to generate a 3’ end
for polyadenylation

The unprotected 5’ end gives

signal for transcriptional
termination
Poly-A tailing at 3 - end
There is no poly(dT) sequence on the DNA
template i.e. tailing process dose not depend on the
template.
 The tailing process occurs prior to the splicing.
 Site of Polyadenylation is nucleus.
 pTEFb kinase phosphorylate S-2 at CTD tail (-Tyr1-
Ser2-Pro3-Thr4-Ser5- Pro6-Ser7-)n and recruits the
machinery required for 3’ polyadenylation &
splicing of hnRNA.
 Termination of transcription occurs only after RNA
polymerase has transcribed past a consensus
AAUAAA sequence - the poly(A) addition site.
 10-30 nucleotides past this site, a string of 100 to
200 adenine residues are added to the mRNA
transcript - the poly(A) tail.
 poly(A) polymerase adds these A residues.
Efficiency & Auxillary Elements: Enhance the efficiency of cleavage & polyadenylation
Molecular Components of 3’ end Formation Reaction
• The 3’ polyadenylation complex has been analyzed at the molecular level.
• Some of the identified proteins of the complex with their known functions are listed below:
Schematic representation of the mammalian polyadenylation machinery
 Alternate Polyadenylation
In mRNA of some protein coding genes, the
addition of poly-A tail occurs at one of the
several possible sites. These protein coding
genes have more than one polyadenylation
site, so a gene can code for several mRNAs
that differ in the position of poly a tail. This
process is termed as alternative
polyadenylation. This cause the formation of
more than one transcript from a single gene.
For example: during the development of B-
lymphocytes, the synthesis of antibody
molecules switches from membrane bound
form to secreted form due to switch from one
polyadenylation site to another in pre-mRNA
which results in a short antibody molecule,
lacking hydrophobic membrane spanning part
at c-terminus ends of the heavy chains.
 Cytoplasmic Polyadenylation
Length of the poly-A tail can be regulated in
cytoplasm. In some species, for example the
egg cell (oocytes) stores mRNA in the
cytoplasm for later use after fertilization. The
stored mRNA has a short poly-A tail.
Activation of the mRNA for translation
includes lengthening of poly-A tail. It has also
been suggested that the poly-A tail is involved
in determining how long an mRNA is present
in the cell before it degraded. Cytoplasmic
polyadenylation targets mRNA that already
contain a short poly-A tail, usually 20-30
nucleotides long and catalyzed by a
cytoplasmic poly-A polymerase.
 Functions of Polyadenylation

Maturation of mRNA from nuclear RNA

Stability
a) Protect mRNA from 3’ to 5’ end
b) Provide stability of cap structure
Facilitate export of mRNA from nucleus to cytoplasm
Influences splicing events
Influences translation reaction
 5’Cap: eIF4E: Poly A Tail complex
eIF4G functions as multipurpose adapter
( holds 5’cap+eIF4E complex & PABPI
poly A tract)

eIF4F
(is a heterotrimer of eIF4E+eIF4G+eIF4A)
Many eukaryotic genes are mosaics, consisting of blocks of coding sequences separated from each other by blocks of non-coding sequences.

The coding sequences are called exons and the intervening sequences are called introns.

The sizes of the exons and introns vary as well. Indeed, introns are very often much longer than the exons they separate. Thus, for example,
the mammalian gene for the enzyme dihydrofolate reductase is more than 31 kb long, and within it are dispersed six exons that correspond
to 2 kb of mRNA. Thus, in this case, the coding portion of the gene is <10% of its total length.

EXONS INTRONS
 Term exon applies to any region retained in a mature  Sequence that is present in gene or primary transcript derive from that
RNA, whether or not it is coding. Non-coding exons gene but not present in final Transcript.
include the 5’ and 3’ untranslated regions of an mRNA.  The number of introns found within a gene varies enormously—from 50 in
the case of the chicken collagen gene to as many as 363 in the case of the
 Exons are typically on the order of 150 nucleotides Titin gene of humans.
 Introns—although they too can be short but can be as long as 800,000
nucleotides (800 kb).
5’ UTR 3’ UTR
The length of a gene is defined by the length of primary transcript instead of the length of mature RNA and
mainly because because of the length and number of introns, the primary transcript (pre-mRNA) can be very
long indeed. For example in the extreme case of the human dystrophin gene, RNA polymerase must traverse
2600 kb of DNA to copy the entire gene into RNA (transcription proceeds at a rate of 40 nucleotides per
second, it can readily be seen that it takes a staggering 17 hrs to make a single transcript of this gene)
 R-loop Technique
An R-loop is a three-stranded nucleic acid
structure, composed of a DNA:RNA hybrid and
the associated non-template double-
stranded DNA. The term "R-loop" was given to
reflect the similarity of these structures to D-
loops; the "R" in this case represents the
involvement of an RNA moiety.
 Actively transcribed regions of DNA often form R-loops
that are vulnerable to DNA damage. Introns reduce R-
loop formation and DNA damage in highly expressed
yeast genes. Genome-wide analysis showed that intron-
containing genes display decreased R-loop levels and
decreased DNA damage compared to intron-less genes
of similar expression in both yeast and
humans. Inserting an intron within an R-loop prone
gene can also suppress R-loop formation
and recombination.
 R-loop mapping is a laboratory technique used to
distinguish introns from exons in double-stranded
DNA. These R-loops are visualized by electron
microscopy and reveal intron regions of DNA by creating
unbound loops at these regions.
 Types of Introns in Gene
Prokaryotes (Rare) Eukaryotes (Common)
 Archae bacteria  Nuclear gene
• Archeal Intron  mRNA forming gene
• (GU-AG intron) (mostly)
(Gp.-2 introns mostly)
• (AU-AC intron)
 rRNA forming gene
 Eubacteria • Gp.-1 Intron

• Gp.-1 Intron  tRNA forming gene

• Pre-tRNA intron
• Gp.-2 Intron
 Extranuclear (organelle) gene
• Gp.-1 Intron
• Gp.-2 Intron
• Gp.-3 Intron
• Twintron
Evolutionary relationship of Bacterial, Mitochondrial, Plastid
and nuclear introns from a common ancestor
 Group1 intron
Group I introns are large self-splicing ribozymes. They catalyze their
own excision from mRNA, tRNA and rRNA precursors in a wide range
of organisms. The core secondary structure consists of nine paired
regions (P1-P10). These fold to essentially two domains - the P4-P6
domain (formed from the stacking of P5, P4, P6 and P6a helices) and
the P3-P9 domain (formed from the P8, P3, P7 and P9 helices). The
secondary structure mark-up for this family represents only this
conserved core. Splicing mechanism of group I introns is processed
by two sequential ester-transfer reactions. The exogenous guanosine
or guanosine nucleotide (exoG) first docks onto the active G- binding
site located in P7, and its 3'-OH is aligned to attack the
phosphodiester bond at the 5' splice site located in P1, resulting in a
free 3'-OH group at the upstream exon and the exoG being attached
to the 5' end of the intron. Then the terminal G (omega G) of the
intron swaps the exoG and occupies the G-binding site to organize
the second ester-transfer reaction: the 3'-OH group of the upstream
exon in P1 is aligned to attack the 3' splice site in P10, leading to the
ligation of the adjacent upstream and downstream exons and
release of the catalytic intron.
Group 2 introns Group II introns are a large class of self-catalytic ribozymes and mobile genetic elements
found within the genes of all three domains of life. Ribozyme activity (e.g., self-splicing) can occur under high-
salt conditions in vitro. However, many proteins and intron-intron and intron-exon interactions important for Group-2 introns
splice site positioning for splicing in vivo. Group II introns sub-classified into groups: IIA and IIB, which differ
in: i) splice site consensus sequence ii) and the distance of the bulged adenosine in domain VI (the prospective
branch point forming the lariat) from the 3' splice site. In contrast to group I introns, intron excision occurs in
the absence of GTP and involves the formation of a lariat, with an A-residue branchpoint strongly resembling
that found in lariats formed during splicing of nuclear pre-mRNA. It is hypothesized that pre-mRNA splicing
may have evolved from group II introns, due to the similar catalytic mechanism as well as the structural
similarity of the Domain V substructure to the U6/U2 extended snRNA. The secondary structure of group II
introns is characterized by six typical stem-loop structures, also called domains I to VI or D1 to D6. The
domains radiate from a central core that brings the 5' and 3' splice junctions into close proximity. The
proximal helix structures of the six domains are connected by a few nucleotides in the central region (linker or
joiner sequences). Due to its enormous size, the domain 1 was divided further into subdomains a, b, c, and d.
Sequence differences of group II introns that led to a further division into subgroups IIA and IIB were
identified. Group II introns also form very complicated RNA Tertiary Structure. Group II introns have a very few
conserved nucleotides, and the nucleotides important for the catalytic function are spread over the complete
intron structure. The few strictly conserved primary sequences are the consensus at the 5' and 3' splicing site
(...↓GUGYG&... and ...AY↓...), some of the nucleotides of the central core (joiner sequences), a relatively high
number of nucleotides of D5 and some short-sequence stretches of D1. The unpaired adenosine in D6 marked
by an asterisk (7 or 8 nt away from the 3' splicing site, respectively) is also conserved and plays a central role
in the splicing process. During splicing of Group II introns, all reactants are preorganized before the initiation
of splicing. Group II catalytic introns are found in rRNA, tRNA, and mRNA of organelles (chloroplasts and
mitochondria) in fungi, plants, and protists, and also in mRNA in bacteria. The length of Type II introns can be
up to 3 kb. A subset of group II introns also encode essential splicing proteins in intronic ORFs. Splicing occurs
in almost identical fashion to nuclear pre-mRNA splicing with two transesterification steps. The 2' hydroxyl of
a bulged adenosine in domain VI attacks the 5' splice site, followed by nucleophilic attack on the 3' splice site
by the 3' OH of the upstream exon.
 Group-3 intron
Define as conventional group II-type dVI with a
bulged adenosine, a streamlined dI, no dII-dV,
and a relaxed splice site consensus. Splicing is
done with two transesterification reactions with
a dVI bulged adenosine as initiating nucleophile;
the intron is excised as a lariat. Not much is
known about how they work. Splicing of group
III introns occurs through lariat and circular
RNA formation. Similarities between group III
and nuclear introns include conserved 5'
boundary sequences, lariat formation, lack of
internal structure, and ability to use alternate
splice boundaries.
Group III intron is a class of introns found in
mRNA genes of chloroplasts
in euglenoid protists.
• Group III introns are much shorter than other self-splicing intron classes,
ranging from 95 to 110 nucleotides On the other hand, Christopher and Hallick
stated: By contrast, the smallest Euglena chloroplast group II intron is 277
nucleotides.
• Their conserved sequences proximal to the splicing sites have similarities to
those of group II introns, but have fewer conserved positions.
• They do not map into the conserved secondary structure of group II introns.
(Indeed, Christopher and Hallick were unable to identify any conserved
secondary structure elements among group III introns.)
• They are usually associated with genes involved in translation and transcription.
• They are very A+T rich.
 Twintron
Intron within-intron excised by
sequential splicing reactions. A twintron is
presumably formed by the insertion of a mobile
intron into an existing intron.
Twintron was discovered by Donald W.Copertinol
and Richard B.Hallick as a group II intron within
another group II intron in
Euglena chloroplast genome. They found that
splicing of both the internal and external introns
occurs via lariat intermediates. Additionally,
twintron splicing was found to proceed by a
sequential pathway, the internal intron being
removed prior to the excision of the external intron.
 Spliceosome
Central component of the splicing apparatus for GU-
AG introns ate the snRNAs called U1,U2,U4,U5 and
U6. these short RNA molecules (snRNAs, <250
nucleotides) associate with proteins to form small
nuclear ribonucleoproteins (snRNPs).
Within nucleus, centres of transcriptional
modifications of snRNAs and snRNPs assembly are
present, known as Cajal Bodies.
The snRNPs, together with other non-snRNPs, attach
to the transcript and form a series of complexes.
Collectively all these apparatus, working together to
make a complex machinery known as Spliceosome
within which the actual splicing reaction occurs.
Spliceosomes are multi-megadalton RNA (snRNA)–
protein (snRNPs/snurps) complexes responsible for
the faithful removal of noncoding segments (introns)
from pre-messenger RNAs (pre-mRNAs), a process
critical for the maturation of eukaryotic mRNAs for
subsequent translation by the ribosome.
 Types of spliceosome
Every human cell contains ~100,000 spliceosomes, which are responsible for
removing over 200,000 different intron sequences. Human cells contain two types of
spliceosome: the major spliceosome responsible for removing 99.5% of introns and
the minor spliceosome, which removes the remaining 0.5%.
Major spliceosomes are assembled from U1, U2, U4, U6, and U5 snRNPs (which are
named according to the U snRNA(s) they contain); minor spliceosomes are assembled
from U11, U12, U4atac, U5, and U6atac snRNPs.
How did the various spliceosomal parts get their names?
The U snRNAs were originally discovered as abundant small uridine-rich RNA
molecules present in mammalian nuclei, and they were initially numbered in order of
their apparent abundance. U1, U2, U4, U5, U6, U11, and U12 were later found to be
spliceosome components.
 Major Splicing Apparatus
This complex comprises about 150 proteins and five RNAs and is similar in size
to a ribosome. In performing even a single splicing reaction, the spliceosome
hydrolyzes several molecules of ATP. Strikingly. RNAs locate the sequence
elements at the intron–exon borders and likely participate in catalysis of the
splicing reaction itself.
The U1, U2, U4/U6, and U5 snRNPs are the main building blocks of the major
spliceosome, which is responsible for removing the vast majority of pre-mRNA
introns. Some metazoan species and plants contain a second, minor
spliceosome that is composed of the compositionally distinct but functionally
analogous U11/U12 and U4atac/U6atac snRNPs, with the U5 snRNP shared
between the machineries.
In addition, the U1, U2, U4, and U5 snRNPs all contain seven Sm proteins.

Assembly begins with the ATP-independent binding of the U1 snRNP through

base-pairing interactions of the 5′ end of the U1 snRNA to the 5′ Splice site of
the intron by RNA-RNA base pairing. This interaction in higher eukaryotes is
stabilized by members of the serine-arginine-rich (SR) protein family and
proteins of the U1 snRNP. Indeed, most of the functionally important RNA-RNA
interactions formed within the spliceosome are weak and generally require the
assistance of proteins to enhance their stability. In addition to the U1-5′SS
interaction, the earliest assembly phase of the spliceosome also involves the
binding of the BBP protein and the U2 auxiliary factor (U2AF) to the BPS and
the polypyrimidine tract just downstream of the BPS, respectively. These
proteins bind cooperatively, with BBP interacting with the 65 kDa subunit of
U2AF (U2AF65) through its C-terminal RNA recognition motif (RRM). In
addition, the 35 kDa subunit of U2AF, which is tightly bound to U2AF65 in the
U2AF heterodimer, binds the AG dinucleotide of the 3′SS. Together, these
molecular interactions yield the spliceosomal E complex and play crucial roles in
the initial recognition of the 5′SS and 3′SS of an intron.
After the formation of the spliceosomal E complex, the U2
snRNA engages in an ATP-dependent manner in a base-pairing
interaction with the pre-mRNA's BPS, leading to the formation
of the A complex. Association of U2 leads to the displacement
of SF1/BBP from the BPS. Subsequent to A complex
formation, the U4/U6 and U5 snRNPs are recruited as a
preassembled U4/U6. U5 tri-snRNP, forming the B complex.
Although all snRNPs are present in the B complex, it is still
catalytically inactive and requires major conformational and
compositional rearrangements (catalytic activation) in order
to become competent to facilitate the first trans-esterification
step of splicing. During spliceosome activation, U1 and U4 are
destabilized or released, giving rise to the activated
spliceosome (the B∗ complex). The activated spliceosome
then undergoes the first catalytic step of splicing, generating
the C complex. Prior to the second catalytic step, additional
rearrangements occur in the spliceosomal RNP network. After
the second catalytic step, the spliceosome dissociates,
releasing the mRNA in the form of an mRNP. It also releases
the U2, U5, and U6 snRNPs to be recycled for additional
rounds of splicing.
 GU-AG intron
Shortly after the discovery of split genes in 1977, a conserved sequence feature at both ends of cellular and viral introns was recognized i.e. the presence of GT at the 59 splice site and
AG at the 39 splice site, giving rise to the so-called GT-AG rule

5’ end (donor site) of the intron includes the consensus sequence

5’-GU-3’ and the last two nucleotides sequence at the 3’
end(acceptor site) includes the consensus sequence 5’-AG-3’ (GU-
AG rule).
A branch point locate 18-40 nucleotides upstream from 3’ end of
an intron. This branch point has a pyrimidine rich region called
polypyrimidine tract( UACUAAC).
Splicing of GU-AG intron involves two transesterification reactions.
During first transesterification reaction cleavage of 5’splice site by
2’-OH of adenosine nucleotide (A) located within branch point
sequence, which results in formation of 5’-2’ phosphodiester bond
(between (G) of the 5’-GU-3’ Motif and (A )of branch point
sequence) i.e. intron has now been looped back on itself to create
a lariat structure and this promoted the appearance of 3’-OH group
at end of upstream exon (5’-splice site).
During second transesterification reaction, 3’-OH group attack the
phosphodiester bond at 3’-splice site and releases the intron as the
lariat structure which is subsequently converted back to linear
structure and degraded.
Finally, 3’-end of upstream exon and 5’-end of downstream exon
joins and splicing process gets completed.
 Minor Splicing Apparatus
(A Small Group of Introns Is Spliced by an Alternative Spliceosome
Composed of a Different Set of snRNPs)
Higher eukaryotes (including mammals, plants, etc.) use the major splicing
machinery we have discussed thus far to direct splicing of the majority of their
pre-mRNAs. But in these organisms (unlike in yeast), some pre-mRNAs are
spliced by an alternative, low-abundance form of the spliceosome. This rare
form contains some components common to the major spliceosome, but it
contains other unique components as well. Thus, U11 and U12 components of
the alternative spliceosome have the same roles in the splicing reaction as U1
and U2 of the major form, but they recognize distinct sequences. U4 and U6
have equivalent counterparts in both spliceosome forms— although these
snRNPs are distinct, they share the same names. Finally, the identical U5
component is found in both the major and the alternative— so-called minor—
spliceosome. The minor spliceosome recognizes rarely occurring introns having
consensus sequences distinct from the sequences of most pre-mRNA introns.
It should be emphasized that although these introns are rare, they are widely
distributed—approximately 800 human genes contain at least one minor
intron. Furthermore, mutations in minor snRNAs have recently been found to
underlie some rare human genetic diseases. The minor form of the
spliceosome is also known as the AT-AC spliceosome, because the termini of
the originally identified rare introns contain AU at the 50 splice site and AC at
the 30 site (in RNA or AT and AC in DNA).
 AU-AC INTRON
GU-AG rule holds in most cases, but exceptions have been
found as GC-AG introns, AU-AA introns and AU-AG introns
etc. are processed by the same splicing pathway as
conventional GU-AG introns. It had long been assumed that
removal of all introns from eukaryotic pre-mRNAs took place
by the same splicing pathway, until recent developments
demonstrated the existence of a second pre-mRNA splicing
pathway, for example intron 7 of the gene encoding human
CMP, a cartilage matrix protein (matrilin 1), were the first
reported examples of introns with AU and AC at the intron
ends, instead of GU and AG.
In addition to their distinctive dinucleotide ends, these and
other AU-AC introns have highly conserved 59-splice-site and
presumptive branch site 8-nucleotide sequence elements,
AUAUCCUY and UCCUURAY that are not present in the major
class of introns, respectively. On the basis of these sequence
features, it was proposed that the minor U11 and U12,
U4atac and U6atac snRNAs, which have regions of
complementarity to these elements, are required for splicing
of AU-AC introns. Important aspects of this prediction were
soon verified experimentally and two additional minor
snRNAs involved in the novel pre-mRNA splicing pathway
were discovered.
 VARIANTS OF SPLICING
Trans-Splicing:

In some cases, two exons carried on different RNA molecules can be

spliced together in a process called trans-splicing. Although generally
rare, trans-splicing occurs in almost all of the mRNAs of
trypanosomes. In the nematode worm (Caenorhabditis elegans), all
mRNAs undergo trans splicing and many of them undergo cis-splicing
as well.
Trans-splicing uses the same spliceosomal machinery as normal cis-
splicing, except for U1, which, at least in worms, is not needed for
trans-splicing. We now turn to cases of splicing in which the
machinery is quite distinct.
Alternate Splicing:
• Many genes in higher eukaryotes encode RNAs that can be spliced in alternative ways to generate two or more different
mRNAs and thus different protein products (or isoforms). It is now believed that at least 40% of Drosophila genes and as
many as 90% of human genes undergo alternative splicing.
• Spliced genes generate only two alternative products, but in some cases, the number of potential alternatives that can be
generated from a single gene can be upto hundreds (e.g., in the human Slo gene) or even many thousands (for the
Drosophila Dscam gene). Alternative splicing is sometimes used as away of generating diversity, with alternative forms
being generated stochastically.
 Errors because of
mistakes in splice-site selection
 Exon skipping: This happens if the spliceosome
components bound at the 5’ splice site of one
exon interact with spliceosome components
bound at the 3’ splice site of not the next exon,
but one beyond.

 Pseudo splice-site selection: The effect of

spliceosome components recognizing pseudo-
splice sites—sequences that resemble (but are
not) legitimate splice sites. In the case shown,
the pseudo-site is within an exon and leads to
regions near the 5’ end of that exon being
mistakenly spliced out along with the intron.
 RNA Editing/Modification
Can change the sequence of an RNA after it has been transcribed. Thus, the protein produced upon translation
is different from that predicted from the gene sequence. Stretches of the mRNA being reassorted during
editing, individual bases are either inserted, deleted or changed. That is, the coding information in the RNA is
altered.
There are two mechanisms that mediate editing:
1. Site-specific deamination of adenines or cytosines.
2. Guide RNA–directed uridine insertion or deletion.
 Site Specific Deamination
In one form of site-specific deamination, a specifically
targeted cytosine residue within mRNA is converted
into uridine by deamination. For a given mRNA species
that undergoes editing, that process typically occurs
only in certain tissues or cell types and in a regulated
manner.
This gene has several exons, within one of which is a
particular CAA codon that is targeted for editing; it is
the C within this codon that gets deaminated. That
deamination, performed by the enzyme cytidine
deaminase, converts the C to a U.
Deamination occurs in a tissue-specific manner:
messages are edited in intestinal cells but not in liver
cells. The CAA codon, which is translated as glutamine
in the unedited message in the liver, is thus converted
to UAA—a stop codon—in the intestine.
The result is that the full-length protein (of some 4500
amino acids) is produced in the liver, but a truncated
polypeptide of only about 2100 amino acids is made in
the intestine. Mammalian apolipoprotein B gene
 Guide RNAs Direct the Insertion and Deletion of Uridines
A very different form of RNA editing is found in the RNA transcripts that encode proteins in the mitochondria of trypanosomes. In this case, multiple Us are
inserted into specific regions of mRNAs after transcription (or, in other cases, Us may be deleted). These insertions can be so extensive that in an extreme case,
they amount to as many as half the nucleotides of the mature mRNA. The addition of Us to the message changes codons and reading frames, completely altering
the “meaning” of the message.
As an example, consider the trypanosome coxII gene. In a specific region of the mRNA of this gene, four Us are inserted between adjacent bases at three sites
(two Us at one site and one U at each of two additional sites). These additions alter some codons and cause a “–1” change in the reading frame, a shift that is
required to generate the correct open reading frame. How are these additional bases inserted? Us are inserted into the message by so-called guide RNAs
(gRNAs). These gRNAs range from 40 to 80 nucleotides in length and are encoded by genes distinct from those that encode the mRNAs on which they act. Each
gRNA is divided into three regions. The first, at the 5’ end, is called the anchor and directs the gRNA to the region of the mRNA it will edit; the second determines
exactly where the Us will be inserted within the edited sequence; and the third, at the 3’ end, is a poly-U stretch. The anchor region of the gRNA contains a
sequence that can base-pair with a region of the message immediately beside (3’ to) the region that will be edited. This is followed by the editing “instructions”:
a stretch of gRNA complementary to the region in the message to be edited but containing additional As.
This process is
catalyzed by the
enzyme 3’
terminal uridylyl
transferase
(TUTase)
 mRNA TRANSPORT
Once Processed, mRNA Is Packaged and Exported from the Nucleus into the
Cytoplasm for Translation
Once it has been fully processed—capped, spliced, and polyadenylated an
mRNA is transported out of the nucleus and into the cytoplasm where it is
translated to give its protein product. Movement from the nucleus to the
cytoplasm is not a passive process. Indeed, it must be carefully regulated: the
fully processed mRNAs represent only a small proportion of the RNA found in
the nucleus, and many of the other RNAs would be detrimental to the cell if
exported. RNA export from the nucleus is an active process, and only certain
(appropriate) RNAs are selected for transport. To be selected for transport,
the RNA must have the correct collection of proteins bound to it. These will
distinguish it from other RNAs, which must be retained in the nucleus or
destroyed. Proteins that recognize exon:exon boundaries, for example,
indicate that an mRNA that has been appropriately spliced, whereas proteins
that bind introns indicate that an RNA that should be retained in the nucleus.
Once in the cytoplasm, some proteins are shed and others are taken on in
readiness for translation.
Export requires energy, and this is supplied by hydrolysis of GTP by a GTPase
protein called Ran. Like other GTPases, Ran exists in depending on whether
complexed with GTP or GDP, and two conformations the transition from one
state to the other drives movement into or out of the nucleus.
 rRNA Splicing
pre-rRNA cannot be used for protein production until splicing of the introns occurs, forming a new bond between the exons and
resulting in mature ribosomal RNA (rRNA).
In eukaryotes there are four types of rRNA. One of these is 5s rRNA, is transcribed by RNA POL-III and does not undergo
processing. The remaining three (the 5.8S, 18S and 28SrRNAs) are transcribed by RNA Polymerase 1 in the form of a pre-rRNA
(45s-rRNA) which is processed by chemical modification, cutting, end-trimming and splicing.
Cutting and end-trimming process involves the use of endonucleases (to cut the specific sites within a longer pre-rRNA) and
exonucleases (to trim back from the new ends to make the mature product).

Chemical modification of rRNA requires a class of small RNAs., 60-300 nucleotide long, called snoRNAs (small nucleolar RNAs).
snoRNAs primarily guide chemical modification of other RNAs, mainly rRNAs, tRNAs and small nuclear RNAs. There are two
main classes of snoRNAs, the C/D box snoRNAs, which is associated with the methylation and the H/ACA box snoRNAs, which
are associated with pseudouridylation. It base pair with the rRNA to create the double standard region that is recognized as a
substrate for the methylation and pseudouridylation. Chemical modification occurs within the region of complementarity.
Each chemical modification event is specified by a different snoRNA.

During processing, pre-rRNA also is extensively modified, mostly by methylation of the 2′-hydroxyl group of specific riboses and
conversion of specific uridine residues to pseudouridine. Some of the proteins in the pre-rRNPs found in nucleoli remain
associated with the mature ribosomal subunits, whereas others are restricted to the nucleolus and assist in assembly of the
subunits.
Unlike pre-rRNA genes, 5S-rRNA genes are transcribed by RNA polymerase III in the nucleoplasm outside of the nucleolus.
Without further processing, 5S RNA diffuses to the nucleolus, where it assembles with the 28S and 5.8S rRNAs and proteins into
large ribosomal subunits. When assembly of ribosomal subunits in the nucleolus is complete, they are transported through nuclear
pore complexes to the cytoplasm, where they appear first as free subunits.
 Processing of pre-rRNA in prokaryotes
involves cleavage, trimming
and methylation of 30S pre-
rRNA. There is a difference
in the organization of the
precursor rRNA in bacteria
as compared to eukaryotes.
30S pre-RNA also contains
the 5S rRNA and one or two
tRNAs between the 16S and
23S rRNA sequences.
Pre-tRNA intron
 Pre-tRNA Splicing
Involves cleavage, trimming chemical modification and splicing of Pre-tRNA. tRNAs are commonly synthesized as the precursor containing extra
sequence that are removed by combinations of endonucleolytic and exonucleolytic activities.
The processing to convert the pre-tRNA to a mature tRNA involves five steps:
1. The 5′ end of the pre-tRNA, called the 5′ leader sequence, is cleaved off by ribonuclease P (RNase P).
2. The 3′ end of the pre-tRNA is cleaved off. By endonuclease RNase E/F and exonuclease D.
3. In all eukaryote pre-tRNAs, but in only some bacterial and archaeal pre-tRNAs, a CCA sequence of nucleotides is added to the 3′ end of the pre-
tRNA after the original 3′ end is trimmed off. Some bacteria and archaea pre-tRNAs already have the CCA encoded in their transcript immediately
upstream of the 3′ cleavage site, so they don’t need to add one. The CCA at the 3′ end of the mature tRNA will be the site at which the tRNA’s
amino acid will be added.

tRNA
nucleotidyltransferase
4.Multiple nucleotides in the pre-tRNA are chemically modified, altering their nitorgen bases. On average about 12
nucleotides are modified per tRNA. The most common modifications are the conversion of adenine (A) to pseudouridine
(ψ), the conversion of adenine to inosine (I), and the conversion of uridine to dihydrouridine (D). But over 100 other
modifications can occur.
5. A significant number of eukaryotic and archaeal pre-tRNAs have introns that have to be spliced out. Introns are rarer
in bacterial pre-tRNAs, but do occur occasionally and are spliced out.
 mRNA degradation in Eukaryotes

Half life of eukaryotic mRNA is about 10-20 minutes in lower eukaryotes and may be upto several hours in case of higher
eukaryotes like mammals. All organisms require a reliable mechanism to turn genes on and off. This regulation of gene
expression underlies cellular processes ranging from the response to environmental signals to the development of multi-
cellular organisms and cell-cell communication. 

After export to the cytoplasm, mRNA is protected from degradation by a 5’ cap structure and a 3’ poly adenine tail. In the
deadenylation dependent mRNA decay pathway, the polyA tail is gradually shortened by exonucleases (deadenylase).
After the deadenylated mRNA, ultimately attracts the degradation machinery that rapidly degrades the mRNA in both in
the 5’ to 3’ direction and in the 3’ to 5’ direction. In the 3’ to 5’ degradation pathway, Exosome which is related to
Degradosome is used.
 Degradosome activation
This multi-protein complex is stimulated by a non-
coding RNA, called miRNA in Eukaryotic cells and
sRNA in bacteria. Small sequences of amino acid
are usually used to target mRNA for its
destruction. sRNA target translation-initiation
region (TIR).
Firstly, to attach sRNA to targeted mRNA
a Hfq (chaperone protein) is needed. Once the
attachment is done, if the complex Hfq-sRNA ends
on TIR, it blocks ribosome binding site (RBS)
so ribosomes can not translate, and activates
nucleases (RNase E) to eliminate mRNA.
Another possibility is ending on another region,
that makes the complex work as a finisher point of
the translation. This way, the ribosomes can do
their job of decoding, process that stops when
they arrive to the complex, where all the
destruction procedure is switched on.
Thank You