Processing of

Eukaryotic RNA

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Processing of tRNA
 All the three types of eukaryotic transcriptional
products are processed
 tRNA precursors are converted into mature tRNAs
by a series of alterations
 Cleavage of a 5’ leader sequence,
 Splicing to remove an intron,
 Replacement of the 3’ terminal UU by CCA
 Modification of several bases
 A series of enzymes act on RNA chain or its
constituent bases to achieve the final product

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 Processing of rRNA
 RNA Pol-I makes a single precursor rRNA of (45S in mammals)
 First the precursor undergo extensive modifications on
 Bases

 Ribose

 These changes are directed by small nucleolar ribonuclear proteins

(snoRNPs)

 Each snoRNP contains one snoRNA and many proteins

 Finally the precursor splits into three RNA components
 18S for small subunit of ribosome

 28S and 5.8 S for large subunit of ribosome

 The processing takes place in the nucleolus

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Processing of mRNA
 The most extensively modified transcription
product is the product of RNA polymerase II
 The majority of this RNA will be processed to
mRNA
 The primary transcript is some time called as
pre-mRNA
 Cap is added at 5’

 Poly A tail added at 3’ end

 Splicing of pre-mRNA

 Editing of RNA

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Addition of 5’ Cap
 The 5’ end of pre-mRNA contains A or G
 After synthesis the 5’ end of nascent
RNA is immediately modified, First a,
phosphate is released by hydrolysis
 5’-5’ triphosphate linkage is formed
between a GTP and 5’ end of RNA, called
Cap
 Methylation at Cap 0, Cap 1 and Cap 2

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Addition of Poly A tail
 Poly(A) is added at 3’ end by poly(A)
polymerase after the cleavage of 3’
end at a specific sequence (AAUAAA),
recognized by an endonulease
 The modification may increase the
half-life of the mRNA, helps in
effective transport and translation

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 RNA Editing
 RNA editing changes the protein
encoded by mRNA
 The sequence content of some mRNAs
is altered after transcription
 RNA editing is the term for a change in
the base sequence of RNA after
transcription by processes other than
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RNA Editing-Example #1
 Apolipoprotein B (apo B) plays an important
role in the transportation of triglycerols and
cholesterol
 Apo B exist in two forms
 Apo B-100, 4563 aa long

 Apo B-48, 2152 aa of N-terminal of full protein

 The larger form, synthesized by the liver,

participates in the transport of lipids
synthesized in our body cell
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 These two forms are synthesized by a newly
reported mechanism of changing the nucleotide
sequence of mRNA after transcription
 A specific cytidine residue of mRNA is
deaminated to uridine, which changes the codon
at residue 2153 from CAA (Gln) to UAA (stop)
 The deaminase that catalyzes this reaction is
present in the small intestine but not in the liver,
and is expressed only at certain developmental
stage

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RNA Editing-Example #2
 A glutamate binding receptor in the vertebrate
central nervous system affects cation channel
 RNA editing changes a single glutamate codon (CAG)
to arginine (CGG) in this receptor
 Substitution of Gln by Arg in the receptor prevents
Ca2+, but not Na+ from flowing through the channel
 RNA editing is likely much more common than was

previously thought
 De-amination, that induces complex DNA-repair

mechanism has been used for generating molecular

diversity at the RNA and hence protein level

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RNA Editing-Example #3
 In trypanosomes a different kind of RNA
editing markedly changes several
mitochondrial mRNAs.
 Nearly half the uridine residues in these
mRNAs are inserted by RNA editing
 A guide RNA molecule identifies the
sequence to be modified and
 A poly-U tail on the guide donates uridine
residues to the mRNA undergoing editing

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Splicing of Pre-mRNA
 Most genes in higher eukaryotes are composed of exons
and introns
 Introns must be spliced and exons linked to form the final
mRNA in a process called Splicing
 Comparison of intron sequences shows some highly
conserved sequence from yeast to mammals, begin with GU
and end with AG
 The conserved sequences make three important sites called
 5’ splice site

 3’ splice site

 Branch site

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 In some forms of Thalassemia, defective
synthesis of hemoglobin results from a
mutation leading to creation of new 3’ splicing
site
 Mutations affecting splice sites have been
estimated to cause 15% of all genetic diseases

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Mechanism of Splicing
 Splicing is very complex process which
involves many small RNAs and proteins
 These RNAs and proteins forms a large
complex called spliceosome.
 Splicing consists of two trans-esterification
reactions

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 Splicing begins with attack by 2’-OH of an adenylate
residue in the branch site
 This results in cleavage of the phosphodiester bond
between exon 1 and the 5’ end of the intron
 A 2’,5’-phosphodi-ester bond is formed between ribose
of the adenylate and the 5’ terminal phosphate of the
intron, this reaction is a transesterification.
 This branch residue is also joined to two other
nucleotides by normal 3’, 5’-phosphodiester bonds.
Hence a branch is generated at this site and a “lariat
intermediate” is formed
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 The 3’-OH terminus of exon 1 then attacks the
phosphodiester bond between the intron and exon 2.
 Exon 1 and 2 become joined, and the intron is released
in the form of a lariat
 This again is a trans-esterification
 Splicing is thus accomplished by two trans-esterification
reaction rather than by hydrolysis and then ligation.
 The number of phosphodiester bonds stays the same
during these steps, which is crucial because it allows
the splicing reaction itself to proceed without an energy
source such as ATP or GTP

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 Role of snRNAs
 Small nuclear RNAs in spliceosome catalyze the splicing
of mRNA precursors
 The nucleus contains many types of small RNA molecules
containing fewer than 300 nt, referred to as snRNAs
(small nuclear RNAs)
 A few of them designated U1, U2, U4, U5 and U6 are
essential for splicing mRNA precursors
 The secondary structures of these RNAs are highly
conserved in organisms ranging form yeast to humans
 These small RNAs are associated with specific proteins to
from complexes termed snRNPs or “snurps” (small
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 Spliceosome

s
Spliceosomes are large (60S), dynamic assemblies
composed of
 snRNPs

 other proteins called splicing factors

 the mRNA precursors being processed

 Splicing begins with the recognition and pairing of

the 5’ splice site of intron in pre-mRNA by U1
snRNP by highly conserved hexa-nucleotides
sequence
 U2 snRNP then binds the branch site by base-
pairing of its highly conserved
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 Pre-assembled U4-U5 –U6 complex joins U1
and U2 complex & mRNA precursor to form a
complete spliceosome, coupled with ATP
hydrolysis
 In this assembly U5 interacts with exon
sequences in the 5’ splice site and
subsequently with the 3’ exon

 U6 disengages from U4 and base-pairs with

U2, displacing U1 from the spliceosome by

interacting with the 5’end of the intron

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 The U2-U6 helix is indispensable for splicing,
suggesting that U2 and U6 snRNPs probably form
the catalytic centre of the spliceosome
 U4 serves as an inhibitor that masks U6 until the
specific splice sites are aligned
 These rearrangements results in the first
transesterification reaction, generating the lariat
intermediate and a cleaved 5’ exon
 Further rearrangements of RNA in the spliceosome
facilitate the second transesterification.

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 The 5’ exon is aligned with 3’ exon such that the 3’
hydroxyl group of the 5’ exon is positioned to
nucleophilically attack the 3’ splice site to generate
the spliced product.
 U2, U5 and U6 bound to the excised lariat intron are
released to complete the splicing reaction
 Many steps in splicing process require ATP
hydrolysis.
 RNA molecules play key roles in directing the
alignment of splice sites and in carrying out catalysis

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Alternate Splicing
 Some pre-mRNA molecules can be spliced in
alternative ways to yield different mRNAs
 Alternative splicing is a widespread mechanism for
generating protein diversity
 The differential inclusion of exons into a mature
RNA, alternative splicing may be regulated to
produce distinct forms of a protein for specific
tissues or developmental stages
 Alternative splicing provides a powerful mechanism
for expanding the versatility of genomic sequences

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Self-Splicing
 The discovery of catalytic RNA was revealing in
regard to both mechanism and evolution
 RNAs form a surprisingly versatile class of molecules
 In Tetrahymena (a ciliated protozoan), a 414-nt
intron is removed form a 6.4-kb precursor to yield
the mature 26S rRNA
 Thomas Cech and his coworkers established that the
RNA spliced itself to precisely excise the 414-nt
intron in the absence of any other protein, and
indeed have highly specific catalytic activity

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 The self-splicing reaction requires an added guanosine
nucleotide as cofactor
 G binds to the RNA and then attacks the 5’ splice site to form
a phosphodiester bond with the 5’ end of the intron.
 This transesterification reaction generates a 3’-OH group at
the end of the upstream exon. This newly attached 3’-OH
group then attacks the 3’ splice site.
 This second transesterification reaction joins the tow exons
and leads to the release of the 414-nt intron
 mRNA precursors in the mitochondria of yeast and fungi also
undergo self-splicing, as do some RNA precursors in the
chloroplasts of unicellular organisms such as Chlamydomonas

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 Self-splicing reaction can be classified according to the nature of the
unit that attacks the upstream splice site.
 Group I self-splicing is mediated by a guanosine cofactor, as in
Tetrahymena.
 Group II attacking moiety is the 2’-OH group of a specific adenylate of
the intron
 Group I and Group II self-splicing resembles spliceosome-catalyzed
splicing in two respect
 First, in initial step, a ribose hydroxyl group attacks the 5’ splice site,
the newly formed 3’-OH group of the upstream exon then attacks the 3’
splice site to form a phosphodiester bond with the downstream exon
 Second, both reactions are transesterification in which the phosphate
moieties at each splice site are retained in the products, the number of
phosphodiester bonds stays constant

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