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Alternative Splicing:

Many eukaryotic pre-mRNAs can be spliced in more than one way, leading to two or
more alternative mRNAs that encode different proteins.

It can make the difference between:

a) a secreted or a membrane-bound protein

b) activity and inactivity.

First example of alternative splicing:

Discovered by Leroy Hood and colleagues in the mouse immunoglobulin u heavy-chain

gene, in 1980.

The u heavy chain exists in two forms:

i.

a secreted form (us)

ii.

a membrane-bound form (um).

The difference in the two proteins lies at the carboxyl terminus, where the membranebound form has a hydrophobic region that anchors it to the membrane, and the secreted
form lacks this membrane anchor.

Fig: Alternative splicing pattern in the mouse immunoglobulin heavy-chain gene.

The S exon (pink) encodes the signal peptide
The V exons (orange) encode the variable region of the protein.
The C exons (blue) encode the constant region of the protein.
Near the end of the fourth constant exon (Cm4) lies the coding region (yellow) for the secreted
terminus of the ms protein.

Another example is the sex determination system in Drosophila.

Sex in the fruit fly is determined by a pathway that includes alternative splicing of the
pre- mRNAs from three different genes:

a) Sex lethal (Sxl)

b) transformer (tra)
c) Double sex (dsx)

These genes function in a cascade as follows:

Female-specific splicing of Sxl transcripts gives an active product that reinforces femalespecific splicing of Sxl transcripts and also causes female-specific splicing of tra
transcripts, which leads to an active tra product.

The active tra product, together with the product of another gene, tra-2, causes femalespecific splicing of transcripts of the dsx gene. This female-specific dsx product
inactivates male-specific genes and therefore leads to female development.

By contrast, male-specific splicing of Sxl transcripts gives an inactive product because it

includes an exon with a stop codon. This permits default (male-specific) splicing of
tra transcripts, which again leads to an inactive product because of the inclusion of an
exon with a stop codon.

With no tra product, the developing cells splice the dsx transcripts according to the
default, male-specific pattern, yielding a product that inactivates female-specific genes
and therefore leads to development of a male.

Figure: Alternative splicing cascade in Drosophila sex determination

Control of alternative splicing:

Depends on:

a) Splicing factors
OR
a) Proteins that bind sequences known as
exonic splicing enhancers (ESEs)
exonic splicing silencers (ESSs)

These sequences presumably bind protein factors that are produced in certain cell types,
or at certain stages in a cells life, or in response to external agents, such as hormones.
Such binding can then presumably either activate or repress splicing at nearby splice
sites.

Self-Splicing RNAs:

Group I Introns: initiating event in group I splicing is attack by an independent guanine

nucleotide e.g., 26S rRNA precursors of Tetrahymena can splice themselves without any
protein.

Group II Introns: the initiating event in group II splicing involves intramolecular attack
by an A residue in the intron to form a lariat.

Group I Introns:

Splicing reaction begins with an attack by a guanine nucleotide on the 5-splice site,
adding the G to the 5-end of the intron, and releasing the first exon.

In the second step, the first exon attacks the 3 splice site, ligating the two exons together,
and releasing the linear intron.

The intron cyclizes twice, losing nucleotides each time, then linearizes for the last time.

Fig: Self-splicing of Tetrahymena rRNA precursor

Group II Introns:

RNAs containing group II introns self splice by a pathway that uses an A-branched lariat
intermediate, just like the spliceosomal lariats.

The secondary structures of the splicing complexes involving spliceosomal systems and
group II introns are also strikingly similar.

CAPPING AND POLYADENYLATION:

Messenger RNAs are subject to further processing:

i.

Capping (5 end)

ii.

Polyadenylation (3 end)

Capping:

The 5' end of the RNA transcript contains a free triphosphate group since it was the first
incorporated nucleotide in the chain. The capping process replaces the triphosphate group
with another structure called the "cap".

Caps are made in steps:

1. An RNA triphosphatase removes the terminal phosphate from a pre-mRNA.

2. A guanylyl transferase adds the capping GMP (from GTP).
3. Two methyltransferases methylate the N7 of the capping guanosine and the 2-O-methyl
group of the penultimate nucleotide.

These events occur early in the transcription process, before the chain length reaches 30
nt.

Figure: Sequence of events in capping

Functions of Caps

Caps appear to serve at least four functions.

1) They protect mRNAs from degradation (The cap is joined to the rest of the mRNA
through a triphosphate linkage found nowhere else in the RNA, which may protect from
nucleases attack)
2) They enhance the translatability of mRNAs (eukaryotic mRNA gains access to the
ribosome for translation via a cap-binding protein that recognizes the cap)
3) They enhance the transport of mRNAs from the nucleus into the cytoplasm.
4) They enhance the efficiency of splicing of mRNAs.

POLYADENYLATION: a long chain of AMP residues about 250 nt long called poly(A)
at 3 end.

only in hnRNA and mRNA, added posttranscriptionally by poly(A) polymerase

Functions of Poly(A):

Different evidences show different functions:

1) it helps protect mRNAs from degradation.

2) it stimulates translation of mRNAs to which it is attached (One of the proteins that binds
to a eukaryotic mRNA during translation is poly(A)-binding protein I, (PAB I). Binding
to this protein seems to boost the efficiency with which an mRNA is translated)
3) poly(A) plays a role in splicing
4) poly(A) plays a role transport of mRNA out of the nucleus.

Process of polyadenylation:

Transcription of eukaryotic genes extends beyond the polyadenylation site. Then the
transcript is cleaved and polyadenylated at the 3-end created by the cleavage.

Polyadenylation signals:

An efficient mammalian polyadenylation signal consists of an AAUAAA motif about 20

nt upstream of a polyadenylation site in a pre-mRNA, followed 23 or 24 bp later by a
GU-rich motif, followed immediately by a U-rich motif. Many variations on this theme
occur in nature, which results in variations in effciency of polyadenylation.

cleavage of the premRNA requires several proteins

initiation of polyadenylation depends on AAUAAA signal two proteins participate in the
initiation process:
a) poly(A) polymerase

b) CPSF (cleavage and polyadenylation specificity factor), which binds to the AAUAAA
motif.

Once the poly(A) reaches about 10 nt in length, further polyadenylation becomes

independent of the AAUAAA signal

Elongation requires a specificity factor called poly(A)-binding protein II (PAB II).

This protein binds to a preinitiated oligo(A) and aids poly(A) polymerase in elongating
the poly(A) up to 250 nt or more.

PAB II acts independently of the AAUAAA motif.

R AND T RNA PROCESSING

Ribosomal RNA Processing:

The rRNA genes of both eukaryotes and bacteria are transcribed as larger precursors that
must be processed (cut into pieces) to yield rRNAs of mature size.
Several different rRNA molecules are embedded in a long precursor, and each of these
must be cut out.

Eukaryotic rRNA Processing:

trimming:rRNAs and tRNAs first appear as precursors that sometimes need splicing,
but they also have excess nucleotides at their ends, or even between regions that will
become separate mature RNA sequences. These excess regions must also be removed by
trimming.

rRNA genes in eukaryotes are repeated several hundred times and clustered together in
the nucleolus of the cell.

Fig: Map of a portion of the newt (amphibian) rRNA precursor gene cluster, showing the
alternating rRNA genes (orange) and nontranscribed spacers (NTS, green).

Example:

Mammalian RNA polymerase I makes a 45S rRNA precursor, which contains the 28S,
18S, and 5.8S rRNAs, embedded between transcribed spacer RNA regions.

The processing of the precursor (Fig) takes place as follow:

Figure: Processing scheme of 45S human

(HeLa) rRNA precursor.

Step 1: The 59-end of the 45S precursor RNA is removed,

yielding the 41S precursor.

Step 2: The 41S precursor is cut into twoparts, the 20S

precursor of the 18S rRNA, and the 32S precursor of

the 5.8S and 28S rRNAs.

Step 3: The 39-end of the 20S precursor is removed,

yielding the mature 18S rRNA.

Step 4: The 32S precursor is cut to liberate the 5.8S and

28S rRNAs.

Step 5: The 5.8S and 28S rRNAs associate by base-pairing.

The rRNA-processing steps occur in the nucleolus by a class of small nucleolar

RNAs (snoRNAs), associated with proteins in small nucleolar ribonucleoproteins,
(snoRNPs).

BACTERIAL RRNA PROCESSING:

The bacterium E. coli has seven rrn operons that contain rRNA genes.

An example is rrnD, which has three tRNA genes in addition to the three rRNA genes.

Transcription of the operon yields a 30S precursor, which must be cut up to release the
three rRNAs and three tRNAs.

The rRNAs are released from their precursors by RNase III and RNase E.

Figure: Structure of the E. coli rrnD operon. This operon is typical of the rRNA-encoding
operons of E. coli in that it includes regions that code for tRNAs (red), as well as rRNA-coding
regions (orange), embedded in transcribed spacers (yellow). As usual with bacterial operons, this
one is transcribed to produce a long composite RNA. This RNA is then processed by enzymes,
including RNase III, to yield mature products.

Transfer RNA Processing:

Transfer RNAs are made in all cells as overly long precursors that must be processed by
removing RNA at both ends.

In the nuclei of eukaryotes, these precursors contain a single tRNA.

In bacteria, a precursor may contain one or more tRNAs, and sometimes a mixture of
rRNAs and tRNAs.

The tRNA processing schemes in eukaryotes and bacteria are so similar.

Cutting Apart Polycistronic Precursors: (in bacteria)

RNase III cuts the precursor up into fragments with just one tRNA each.

Forming Mature 5-Ends:

After cutting bacterial tRNA still contains extra nucleotides at both 5- and 3-ends.

Maturation of the 5-end of a bacterial or eukaryotic tRNA involves a single cut just at
the point that will be the 5-end of the mature tRNA, as shown in Fig. The enzyme that
catalyzes this cleavage is RNase P.

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Figure: RNase P action. RNase P makes a cut at the site

that will become the mature 5-end of a tRNA. Thus, this
enzyme is all that is needed to form mature 59-ends.

Forming Mature 3-Ends:

To remove nucleotides from the 3-ends of tRNAs in bacteria 6- RNases are required.

In eukaryotes, a single enzyme, tRNA 3-processing endoribonuclease (3-tRNase),

processes the 3-end of a pre-tRNA.

RNA editing:

Some mRNAs are edited before translation.

RNA editing can involve the addition, deletion, or alteration of nucleotides in the
RNA in a manner that affects the meaning of the transcript when it is translated.

Addition or deletion of nucleotides has been most commonly observed in RNAs

originating from the mitochondrial and chloroplast genomes of eukaryotes.

The reactions require a special class of RNA molecules encoded by these same
organelles, with sequences complementary to the edited mRNAs. These guide RNAs act
as templates for the editing process.

Example:

The initial transcripts of the genes that encode cytochrome oxidase subunit II in some
protist mitochondria provide an example of editing by insertion.

These transcripts do not correspond precisely to the sequence needed at the carboxyl
terminus of the protein product.

A posttranscriptional editing process inserts four U residues that shift the translational
reading frame of the transcript.

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FIG:RNA editing of the transcript of the cytochrome oxidase subunit II gene from
Trypanosoma brucei mitochondria.
A special class of guide RNAs, complementary to the edited product, act as templates for the
editing process. Note the presence of two GUU base pairs, signified by a blue dot to indicate
non- Watson-Crick pairing.

RNA editing by alteration of nucleotides most commonly involves the enzymatic

deamination of adenosine or cytidine residues, forming inosine or uridine, respectively.

Deamination reactions involve deaminase enzymes.

Example of RNA editing by deamination occurs in the gene for the apolipoprotein B
component of low-density lipoprotein in vertebrates.

Two forms encoded by mRNA from the gene for apoB-100.

1. apoB-100: synthesized in liver

2. apoB-48: synthesized in intestine

An APOBEC (apoB mRNA editing catalytic peptide) cytidine deaminase found only in
the intestine binds to the mRNA at the codon for amino acid residue 2,153 (CAA Gln)
and converts the C to a U, to create the termination codon UAA.

The apoB-48 produced in the intestine from this modified mRNA is simply an
abbreviated form (corresponding to the amino-terminal half) of apoB-100.

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FIG: RNA editing of the transcript of the gene for the apoB-100 component of LDL.
Deamination, which occurs only in the intestine, converts a specific cytidine to uridine,
changing a Gln codon to a stop codon and producing a truncated protein.
POST-TRANSCRIPTIONAL CONTROL OF GENE EXPRESSION:

mRNA Stability:

The most prevalent form for control of gene expression is by blocking transcription, but
is not the only way.

Another important posttranscriptional control of gene expression is control of mRNA

stability.

Example: The response of mammary gland tissue to the hormone prolactin.

In response to hormone mammary gland produce the milk protein casein.

In cultured mammary gland tissue the number of casein mRNA molecules increases
about 20-fold in 24 h following the hormone treatment.

But this does not mean the rate of casein mRNA synthesis has increased 20-fold. In fact it
only increases about two- to threefold, and the rest of the increase in casein mRNA level
depends on an approximately 20-fold increase in stability of the casein mRNA.

mRNA Decay:

A. Most mRNAs undergo decay by the deadenylation-dependent pathway. The poly(A)

tail is removed by a deadenylase activity. Following deadenylation, two mechanisms can
degrade the mRNA:
1. Decapping followed by 5-3' decay or 3-5' decay. In the decapping pathway, decapping
at the 5' end of the mRNA transcript leaves the mRNA susceptible to decay by the 5-3'
exoribonuclease.
2. Alternatively, the deadenylated mRNA can be degraded in the 3-5' direction by the
exosome, with the remaining cap structure being hydrolysed by the scavenger-decapping
enzyme.
B. In Saccharomyces cerevisiae, deadenylation-independent pathways require recruitment of
the decapping machinery Following decapping, the mRNA is degraded by 5-3'
exoribonuclease.
C. Endonuclease-mediated mRNA decay initiates with internal cleavage of the mRNA,
which generates two fragments each with one unprotected end. The fragments are
degraded by XRN1 and the exosome.
RNA INTERFERENCE:

RNA interference (RNAi) occurs when a cell encounters dsRNA from a virus, aberrant
transcripts from repetitive sequences in the genome such as transposons, or a transgene
(or experimentally added dsRNA), and results in destruction of the mRNA corresponding
to the trigger dsRNA.

How transgene?

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Placing transgenes into various organisms sometimes had the opposite of the desired
effect. Instead of turning on the transgene, organisms
sometimes turned off, not only the transgene, but the
normal
cellular copy of the gene as well. One of the first examples
was
an attempt to intensify the purple color of a petunia.

This phenomenon was called by several names:

cosuppression and post-transcriptional gene silencing (PTGS)

in plants

RNA interference (RNAi) in animals and fruit fly

quelling in fungi.

Experiments have shown that:

antisense RNA, which is complementary to mRNA, base-pair to the mRNA and inhibit
its translation.
sense RNA worked just as well as antisense RNA in blocking expression of a particular
gene.
double-stranded RNA (dsRNA) worked much better than either sense or antisense RNA.
So, the main reason sense and antisense RNAs worked appears to be that they were
contaminated with (or produced) small amounts of dsRNA, and the dsRNA actually did
the most to block gene expression.

Mechanism of RNAi: (in Drosophila)

Endogenous triggers of RNAi pathway include:

foreign DNA or double-stranded RNA (dsRNA) of viral origin

aberrant transcripts from repetitive sequences in the genome such as transposons

pre-microRNA (miRNA: Naturally expressed small RNAs that interact with components
shared by the RNA-induced silencing complex (RISC)

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Fig: Endogenous triggers of RNAi pathway

short hairpin RNA

(shRNA)

Steps:

1. The trigger dsRNA is degraded into 2123nt fragments (siRNAs) by an RNase III-like
enzyme called Dicer (cytoplasmic).
2. The doublestranded siRNA, with Dicer and the Dicer-associated protein R2D2 recruit
(Argonaute2) Ago2 to form a pre-RISC complex
3. pre-RISC complex can separate the siRNA into its two component strands:
a) the guide strand, which will base-pair with the target mRNA in the RNA induced
silencing complex (RISC) and guide cleavage of the mRNA
b) the passenger strand, which will be discarded. (Ago2 cleaves the passenger strand,
which then falls off the pre-RISC complex)
4. The guide strand of the siRNA then base-pairs with the target mRNA in the active site of
Ago2, which is an RNase H-like enzyme, also known as slicer (endoribonuclease).
5. Slicer cleaves the target mRNA in the middle of the region of its base-pairing with the
siRNA.
6. In an ATP-dependent step, the cleaved mRNA is ejected from the RISC, which can then
accept a new molecule of mRNA to be degraded.

https://www.youtube.com/watch?v=cK-OGB1_ELE

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Figure: A simplified model for RNAi.

(a) Dicer (yellow) recognizes and binds to a double-stranded RNA (red and blue), then cleaves
the RNA into siRNAs about 2123 nt long (depicted here as 10 nt long, for simplicity), with 2-nt
39-overhangs. The ends of the central siRNA are labeled to illustrate the 3 overhangs.
(b) One of the siRNA strands (red) associates with RISC (orange) and base-pairs to a target
mRNA (blue).
(c) The siRNA strand in the RISC complex serves as a guide RNA to direct the cleavage of
the target mRNA in the middle of the sequence opposite the siRNA.
AMPLIFICATION OF siRNA:

In certain organisms, including C. elegans, siRNA is amplified during RNAi

Indeed, Dicer does create many molecules of siRNA out of the trigger dsRNA, but it
cant explain how Just a few molecules of dsRNA can set in motion a process that totally
silences a gene, not only in one cell, but in a whole organism.

Fire and colleagues solved this puzzle by showing that C. elegans cells employ an
enzyme: RNA-directed RNA polymerase (RdRP) that uses antisense siRNAs as
primers to make many copies of siRNA.

Figure: Amplification of siRNA.

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(a) Dicer chops up trigger dsRNA to make

(b) The antisense strands of siRNA hybridize
(c) RdRP uses the siRNA antisense strands
as template to make long antisense

siRNA.
to target mRNA.
as primers and target mRNA
strands.

(d) The product of step (c) is new trigger

dsRNA.
(e) Dicer chops up the new trigger dsRNA to make
more siRNA, which can start a new round of
priming and siRNA amplification.

MicroRNAs:

Another class of small RNAs called

microRNAs
(miRNAs) are 18-25-nt RNAs produced naturally in plant and animal cells by cleavage
from a larger, stem-loop precursor.

Different sets of miRNAs expressed in different cell types and tissues and multiple roles
for miRNAs in plant and animal development and in many other biological
processes. Aberrant miRNA expression are implicated in disease states.

In animals, these miRNAs then base-pair (though imperfectly) with specific mRNAs and
silence gene expression primarily by blocking translation of those mRNAs. Many
mechanisms are suggested e.g.,

cotranslation peptide degradation

less efficient ribosomal loading and thus miRNA-mediated repression at the translation
initiation step
increased premature termination (ribosomal drop-off)
impaired elongation

In plants, miRNAs base-pair perfectly (or almost so) with mRNAs and direct the
cleavage of those mRNAs.

There are three important distinctions between the actions of siRNAs and miRNAs in
animals:

1. Mechanism of silencing: The siRNAs silence genes by inducing degradation of the

target mRNAs, while the miRNAs tend to silence genes by interfering with protein

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synthesis. However, if base pairing between an animal miRNA and its target mRNA is
perfect or near perfect, the miRNA can cause cleavage of the target mRNA.
2. Source of production: The siRNAs are formed by Dicer action on double stranded
RNAs that usually are foreign to the cell, or derive from transposons (cellular products).
On the other hand, the miRNAs are formed by Dicer action on the double-stranded part
of a stem-loop RNA that is a normal cellular product.
3. Base Pairing: The siRNAs base-pair perfectly with the target mRNAs, whereas the
miRNAs usually base-pair imperfectly with their target mRNAs (exceptions are there)
Silencing with both kinds of small RNA, siRNA and miRNA, depends on a RISC
complex.

Fig: A simplified model for the RNAi pathway

RNAi in experiments and therapeutics:

RNAi can be triggered experimentally by exogenous introduction of dsRNA or constructs

which express shRNAs.

RNAi is used in

functional genomics (systematic analysis of loss-of-function phenotypes induced by

RNAi triggers)
developing therapies for the treatment of viral infection, heritable disorders, and many types of
cancers (in vivo inactivation of gene products linked to human disease progression and
pathology).