Processing of RNA

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• Most genes have final gene products that are proteins.

• They are first transcribed to give messenger RNA

(mRNA) and this is then translated to give protein.

• In a smaller but significant number of cases, the RNA

itself is the final gene product.

• In either case, the RNA molecule that is the initial

result of transcription, the primary transcript, may be
chemically altered before fulfilling its role.

• This is referred to as RNA processing and may

sometimes be extremely complex.

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• Almost all RNA molecules are processed in some way. The
major exception is bacterial mRNA, although even in this
case a few of these mRNA molecules are processed.

• Not surprisingly, RNA processing is more complicated in

eukaryotic cells, where it mostly occurs inside the nucleus
before the RNA molecule is released into the cytoplasm.

• In some cases, such as mRNA, modification of RNA is largely

regulatory in effect.

• In other cases, especially ribosomal RNA (rRNA) and

transfer RNA (tRNA), the modifications improve the
performance of the RNA in its final role.

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 RNA Is Processed in Several Ways
• RNA is made by RNA polymerase, using a DNA template, in the process
known as transcription.

• Sometimes the RNA molecule is ready to function immediately after it has

been transcribed [e.g., most bacterial messenger RNAs (mRNAs)].

• However, in many cases, the RNA needs further processing before it is

functional.

• In these cases, the original RNA molecule, before any further processing
occurs, is known as the primary transcript.

• For specific classes of RNA, the precursor (i.e., primary transcript) may be
referred to as pre-mRNA, pre-transfer RNA (tRNA), etc.

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• All classes of RNA are subject to processing by base
modification and cleavage.

• In addition, eukaryotic mRNA undergoes capping and

tailing as well as splicing.

• Base modifications occur primarily in tRNA and

ribosomal RNA (rRNA), and occur after the RNA is
transcribed.

• These modifications are essential for their proper

function in protein translation .
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• Certain RNA molecules such as prokaryotic and eukaryotic
rRNA are modified by cleavage; that is, the RNA is made as
a longer precursor that is trimmed to the correct length.

• In other related cases, several RNA molecules are included

in the same primary transcript, which is then cleaved into
several parts.

• In eukaryotes, the primary transcript for mRNA contains

segments called introns or intervening sequences that are
not used to encode the final protein product.

• Splicing involves the removal of these introns and rejoining

of the ends to create a streamlined mRNA with an
uninterrupted coding sequence that is translated into a
protein.
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• Most mRNA processing relies on typical enzymes
consisting of proteins to catalyze the reaction.

• However, as shown later, more complex RNA

processing involves other RNA molecules.

• These RNAs are involved both in sequence

recognition and in the actual chemical reactions
of cutting and splicing.

• Such RNA enzymes are known as ribozymes. In

fact, certain introns are self-splicing; that is, they
cut themselves out in a reaction that does not
require any protein components
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• As will be discussed in Protein Synthesis, the
formation of peptide bonds during protein
synthesis is actually catalyzed by rRNA, not a
protein.

• The involvement of RNA in such fundamental

processes as protein synthesis and RNA
processing has led to the idea that ribozymes
were more common in early life.

• Indeed the “RNA world” hypothesis suggests that

the original enzymes were all RNA and that
protein only assumed this role later in evolution.
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 Coding and Noncoding RNA
• Although most genes are transcribed to give
the mRNA that encodes proteins, this mRNA is
only a small fraction of the total RNA.

• RNA may be divided into coding RNA (i.e.,

mRNA) and noncoding RNA, which includes
tRNA, rRNA, and a variety of other RNA
molecules that function directly as RNA and
are not translated into protein.
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• Although there are many different molecules of mRNA,
each is only present in relatively few copies.

• In Escherichia coli (E. coli) there is an average of 3-4

copies each of about 400 different mRNAs.

• In contrast, there are many copies of rRNA and tRNA.

For example, E. coli contains 10-20 thousand
ribosomes, each possessing one copy of each rRNA.

• rRNA thus accounts for about 80% of the total RNA and
tRNA for 14%-15%. The mRNA only makes up 4%-5% by
weight of the RNA.

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• rRNA and tRNA are found in all living cells. The
other types of noncoding RNA vary from
organism to organism.

• For example, bacteria contain assorted small

regulatory RNAs

• Eukaryotes possess small nuclear RNA (snRNA),

small nucleolar RNA (snoRNA), and small
cytoplasmic RNA (scRNA) molecules. snRNA and
snoRNA take part in processing other RNA
molecules in the eukaryotic nucleus.
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• An increasing number of small regulatory RNA molecules
are being found in eukaryotes and, to a lesser extent, in
prokaryotes.

• In eukaryotes, two notable classes are siRNA (short

interfering RNA), involved in RNA interference, and miRNA
(microRNA), short RNA molecules involved in regulating
gene expression.

• Although most regulatory RNA is short (less than 200

nucleotides), an increasing number of “long noncoding
RNA” or lncRNA are coming to light.

• These have a variety of roles in the regulation of eukaryotic

genes.

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 Processing of Ribosomal and Transfer RNA

• The three rRNA molecules of bacteria are

transcribed together to give a single pre-rRNA.

• This contains 16S rRNA, 23S rRNA, and 5S

rRNA joined by linker regions.

• In bacteria, this pre-rRNA transcript also

includes some tRNAs.
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15
• The mature rRNAs are made by cleavage of the
precursor by ribonucleases.

• After this cleavage, the ends are trimmed by several

exonucleases.

• In eukaryotes, there are four rRNAs. The 5S rRNA is

made separately and does not need processing.

• The other three (18S rRNA, 28S rRNA, and 5.8S rRNA)
are made as a single pre-rRNA and processed much as
in bacteria.

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• tRNAs are transcribed as longer precursors that also need
processing.

• Some tRNAs are made singly; others are transcribed together; and
in bacteria, some are included in the pre-rRNA transcript.

• The 5’ end of bacterial tRNA is trimmed by ribonuclease P.

• This enzyme is of note because it is a ribozyme.

• Ribonuclease P consists of both an RNA molecule and a protein, but

the catalytic activity is due to the RNA.

• The protein component merely modulates the activity of the RNA.

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18
• The acceptor stem of tRNA, to which the amino acid
will be attached, always ends in CCA.

• Curiously, most organisms do not encode this final CCA

in the DNA.

• Instead the final CCA is added by an enzyme known as

CCA-adding enzyme.

• E. coli is one of the minority of organisms where the

final CCA is encoded in the genome.

• Nonetheless, E. coli does possess CCA-adding enzyme

in order to repair damaged tRNA.

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 Eukaryotic Messenger RNA Contains a Cap
and a Tail

• In eukaryotic cells, transcription of genes to

give mRNA is much more complex than in
prokaryotes.

• First, eukaryotic genes are inside the nucleus,

not free in the cytoplasm.

• Second, most eukaryotic genes are

interrupted by segments of noncoding DNA,
the introns.
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• The RNA molecule resulting from transcription of a
eukaryotic gene is known as the primary transcript.
This transcript is not yet mRNA because it still needs
to be processed.

• The primary transcript is trapped inside the nucleus

until it is completely processed.

• First, there is the addition of a cap structure to the

front and a tail of many adenines to the rear of the
RNA molecule.

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• Next, the introns are removed by a process
known as splicing.

• This involves cutting out the introns and

joining the ends of the exons to generate an
RNA molecule that has only the exons; that is,
it contains an uninterrupted coding sequence.

• After the processing is complete, the mRNA

exits the nucleus to be translated by
ribosomes
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24
 Capping Is the First Step in Maturation of Eukaryotic
mRNA

• Before leaving the nucleus, RNA molecules

destined to become mRNA have a cap added
to their 5’ ends and a tail added to their 3’
ends.

• This occurs inside the nucleus and before

splicing.

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• Shortly after transcription starts, the 5’ end of the
growing RNA molecule is capped by the addition of a
guanosine triphosphate (GTP) residue.

• This is added in a backward orientation relative to the

rest of the nucleotides in the RNA.

• After addition of the GTP, the guanine base has a

methyl group attached at the 7 position.

• Further methyl groups may be added to the ribose

sugars of the first one or two nucleosides of the
original mRNA in some higher eukaryotes.

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 A Poly(A) Tail Is Added to Eukaryotic mRNA

• After being capped, the growing RNA has a

poly(A) tail added.

• Transcripts destined to become mRNA have a tail

recognition sequence—AAUAAA—close to the 3’
end.

• The RNA polymerase that is making the RNA

molecule continues past this point.

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• A specific endonuclease recognizes this
sequence and cuts the growing RNA molecule
10-30 bases downstream

• The tail consists of 100- 200 adenine residues.

• The presence of poly(A) tail allows the

isolation of eukaryotic mRNA by tail trapping.

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 Introns Are Removed From RNA by Splicing

• The third step in processing pre-mRNA is to splice out

the introns.

• Splicing must be accurate to within a single base since

a mistake would throw the whole coding sequence out
of register and totally scramble the protein sequences.

• There are several classes of introns . The most frequent

class of intron in eukaryotic nuclear genes is the GT-AG
(or GU-AG in RNA ) group of introns.

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• The splicing machinery is known as the
spliceosome and consists of several proteins
and some specialized, small RNA molecules
found only in the nucleus.

• Each snRNA plus its protein partners forms a

snRNP or “snurp.”

• There are five snRNPs—numbered from U1 to

U6
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• The snRNAs of the snurps recognize three sites on the pre-
mRNA. These are the 5’ and 3’ splice sites and the branch
site.

• The vast majority of introns begin with GU and end in AG.

• Recognition is due to base pairing between the snRNA and

the primary transcript.

• The protein part of the snurp supervises the cutting and

joining reactions.

• In the middle of the intron is a special adenine residue used

as a branch site during splicing

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37
• .

38
 Different Classes of Intron Show Different
Splicing Mechanisms
• There are several classes of introns.

• The GT-AG (or GU-AG) introns described earlier are by

far the most frequent in eukaryotic nuclear genes.

• The AT-AC (or AU-AC) introns are extremely similar to

the GT-AG introns except for their different intron
boundary sequences.

• They are processed in an almost identical manner, by a

different, but closely related, set of splicing factors.

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40
• Group I introns are self-splicing. The RNA itself provides the
catalytic activity and thus acts as an RNA enzyme or
ribozyme.

• No proteins are required for splicing.

• Folding of the RNA into a series of base-paired stem and

loop structures is needed for ribozyme activity.

• The 3D structure is folded so as to bring the two splice sites

together and to strain the bonds that will be broken.

• Group I introns include those in the rRNA of lower

eukaryotes, such as the single-celled, ciliated freshwater
protozoan, Tetrahymena. However, most are found in genes
of mitochondria and chloroplasts.
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42
• Group II introns are found in the organelles of
fungi and plants and occasional examples
occur in prokaryotes.

• Group III introns are found in organelles.

• Both classes are also self-splicing

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 Alternative Splicing Produces Multiple Forms of RNA

• Although any particular splice junction must be

made with total precision, eukaryotic cells can
sometimes choose to use different splice sites
within the same gene.

• Typically, different cell types within the same

animal use alternative splicing.

• This allows a single original DNA sequence to be

used to make several different proteins that have
distinct but overlapping functions.
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 Alternative Promoter Selection

• Alternative promoter selection occurs when two

alternative promoters are available.

• The choice of which promoter to use depends on

cell-type specific transcription factors.

• As Fig. 12.14 shows, two alternative transcripts

result in two different mRNAs.

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46
 Alternative Splicing by Exon Cassette Selection

• Alternative splicing by exon cassette selection involves a genuine

choice between actual splicing sites.

• Depending on the choice made, a particular exon may or may not

appear in the final product.

• Here, the primary transcripts are identical.

• Alternative splicing depends on both positive and negative

recognition sequences—the splicing enhancers and splicing
silencers.

• These are located near the different possible splice sites.

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48
• For example, exon cassette selection occurs in the gene for
the skeletal muscle protein troponin T.

• In the rat this gene has 18 exons. Of these, 11 are always

used.

• Five (exons 4 through 8) may be used in any combination

(including none used) and the final two (exons 17 and 18)
are mutually exclusive, and one or the other must be
chosen.

• This gives a theoretical mind-boggling 64 possible final

mRNAs.

• The result is that muscle tissue contains multiple forms of

this structural protein.
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 Degradation of mRNA
• mRNA molecules are relatively short-lived and in bacteria,
the half-life is generally only a few minutes.

• Molecules of mRNA that are not bound to ribosomes are

especially vulnerable to degradation.

• Bacteria contain multiple ribonucleases that are involved

both in processing the precursors to tRNA and rRNA and in
the degradation of mRNA.

• These ribonucleases can often substitute for one another at

least to some extent and so mutants that have lost only one
ribonuclease are usually still viable.
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• Bacterial mRNA is degraded in two stages.

• First, an endonuclease, usually ribonuclease E,

cleaves regions that are unprotected by
ribosomes. Next, exonucleases that move in a
3’-to-5’ direction degrade the fragments.

• Note that overall degradation moves in a 5’-

to-3’ direction due to the initial endonuclease
following the ribosomes.
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52
• Degradation of mRNA follows a different route in
eukaryotes such as yeast.

• First, the poly(A) tail and then the cap must be

removed before nuclease digestion of the body of the
RNA.

• Once the poly(A) tail is shortened to 10-20 bases the

poly (A)-binding protein (PABP) is released.

• Only after the PABP has gone can the cap be removed.

• Once the cap has also gone, an exonuclease degrades

the mRNA in the 5’-to-3’ direction.

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 YouTube videos
• https://www.youtube.com/watch?v=YjWuVrzvZYA&ab_
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• https://www.youtube.com/watch?v=FVuAwBGw_pQ&
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• https://www.youtube.com/watch?v=Dp_b9elTxdc&ab_
channel=Biologyanimationvideos

• https://www.youtube.com/watch?v=O0wouZGmXwI&a
b_channel=BiotechReview

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