5Cap addition , poly A tail and RNA editing with a note on Alternative splicing

1. RNA Processing
KRVS Chaitanya

2. Messenger RNA (mRNA) and its region
• Messenger RNA operates as the template for protein
synthesis.
• Messenger RNA encodes genetic information from DNA as a
transcript and translates the information to a ribosome and
helps assemble amino acids in their proper order.
• mRNA is directly transcribed from DNA, whereas in case of
eukaryotes, a pre-mRNA is first transcribed from DNA and
then processed to yield mature mRNA.

3. Three main regions occur in both prokaryotic and
eukaryotic mRNAs.
1. 5’ UTR:
•The 5′ untranslated region (5′ UTR; also called the
leader) is a nucleotide sequence at the 5′ end of the
mRNA that does not encode any of amino acids.
•In bacterial mRNA, this region consists of the
consensus sequence termed as the Shine-Dalgarno
sequence.

4. •During translation, Shine-Dalgarno sequence
serves as a ribosome binding site. This sequence is
found approximately seven nucleotides upstream of
the first codon that is translated into the amino acid,
also termed as start codon.
•In its 5′ untranslated region, eukaryotic mRNA has
no equivalent consensus sequence.
•Ribosomes bind to a modified 5′ end of mRNA in
eukaryotic cells.

5. 2. Protein coding region:
•The next section of mRNA is the protein-coding
region, containing the codons that describe the
protein’s amino acid sequence.
•The protein-coding region starts with a start codon
and terminates with a stop codon.

6. 3. 3’ UTR:
•The 3′ untranslated region (trailer), a nucleotide
sequence at the3′ end of the mRNA, is the last
mRNA region and not translated into protein.
•The 3′ UTR affects mRNA stability and the
translation of the protein-coding sequence of the
mRNA.

8. Post-transcriptional modification in
Eukaryotes
• Transcription and translation take place concurrently in
bacterial cells; when the 3′ end of an mRNA is undergoing
transcription, ribosomes bind near the 5′ end to the Shine-
Dalgarno sequence and begin translation.
• Since transcription and translation are coupled, before
protein synthesis, bacterial mRNA has little opportunity to be
changed.
• In contrast, in eukaryotic cells, transcription and translation
are segregated both in time and space.

9. •In the nucleus, transcription takes place while
translation takes place in the cytoplasm; this
separation offers a chance to modify eukaryotic RNA
before translating it.
•Indeed, after transcription, eukaryotic mRNA is
altered extensively.
•Changes are made to the RNA molecule’s 5′ end, the 3′
end, and protein coding portion.
•Following are the examples of Post-transcriptional
modification:

10. 1. The 5 ‘Cap Addition:
• One type of eukaryotic pre-mRNA modification is the addition of a
structure called a 5 ‘cap at its 5’end.
• At the 5’ end of the mRNA, the cap consists of an additional
nucleotide and methyl groups (CH3) at the base of one or more
nucleotides at the 5′ end of the newly inserted nucleotide and the 2′-
OH group of sugar.
• After transcription initiation, the insertion of the cap takes place
quickly.
• It is possible to represent the 5′ end of pre-mRNA as 5′-pppNpNpN, in
which a ribonucleotide is represented by the letter ‘N‘ and a
phosphate by ‘p‘.

12. • One of these phosphate groups is removed shortly
after the start of transcription and a guanine
nucleotide is added.
• A special 5′-5′ bond connects this guanine nucleotide
to the pre mRNA, which is somewhat different from the
normal 5′-3′ phosphodiester bond that binds all the
other RNA nucleotides.
• To the 5′ end, one or more methyl groups are added.

13. • The first of these methyl groups is attached to the
position 7 of the base of the terminal guanine
nucleotide making the base 7-methyl guanine.
• Next, in the second and third nucleotides, a methyl
group may be attached to the 2′ position of the sugar.
• Additional methyl groups can rarely be attached to the
bases of the second and third nucleotides of pre-
mRNA.

14. 2. The Poly A tail addition:
• The addition of 50 to 250 or more adenine nucleotides at the 3′
end, forming a poly(A) tail, is a second kind of modification to
eukaryotic mRNA.
• These nucleotides are not encoded in the DNA, but are inserted
in a process called polyadenylation following transcription.
• Many RNA polymerase II transcribed eukaryotic genes are
transcribed well past the end of the coding sequence; much of
the extra material is then cleaved at the3′ end and the poly(A)
tail is inserted.

17. •Sequences both upstream and downstream of the cleavage
site are necessary for processing the3′ end of pre-mRNA.
•Generally, downstream of the cleavage site is a sequence
rich in uracil nucleotides.
•On many mRNAs, the poly(A) tail confers stability,
increasing the time during which the mRNA remains
intact and available for translation.
•The poly(A) tail also enhances the ribosome’s attachment
to the mRNA.

18. 3. RNA splicing:
• The removal of introns by RNA splicing is the other
major type of eukaryotic pre-mRNA modification.
• Before the RNA moves to the cytoplasm, this
modification takes place in the nucleus.
• The presence of three sequences in the intron is
required for splicing.

20. •One end of the intron is referred to as the 5′ splice site,
and the other end is the 3′ splice site.
•Most introns begin with GU in pre-mRNAs and end with
AG.
•The third sequence that is necessary for splicing is
present at the branch point, which is an adenine
nucleotide that is situated 18-40 nucleotides upstream of
the 3′ splice site.

21. • Splicing occurs within a large structure called the
spliceosome, which is one of the largest and most
complex of all molecular complexes.
• Five RNA molecules (U1, U2, U4, U5, and U6) and
almost 300 proteins form the spliceosome.
• Small nuclear RNAs (snRNAs) ranging in length from
107 to 210 nucleotides are the RNA components; these
snRNAs are associated with proteins to form small
particles of ribonucleoprotein.

22. Process of RNA splicing:
• An intron is between an upstream exon (exon1) and a
downstream exon (exon 2) before splicing takes place.
• In two distinct stages, pre-mRNA is spliced.
• The pre-mRNA is cut at the 5 ‘splice site in the first
stage of splicing.
• This cut frees exon 1 from the intron, and the intron’s
5′ end connects to the branch point; that is, the intron
folds back on itself, creating a structure called a lariat.

23. • In this reaction, via a trans-esterification reaction, the
guanine nucleotide in the consensus sequence at the 5′
splice site binds with the adenine nucleotide at the
branch stage.
• To the cytoplasm, where it is translated, the mature
mRNA consisting of the exons spliced together is
exported.
• A cut is made at the3′ splice site in the second step of
RNA splicing and, simultaneously, the3′ end of exon 1
is covalently connected (spliced) to the5′ end of exon
2.

24. • It releases the intron as a lariat.
• When the bond splits at the branch stage, the intron
becomes linear and is then quickly degraded by
nuclear enzymes.
• To the cytoplasm, where it is translated, the mature
mRNA consisting of the exons spliced together is
exported.

25. Alternative processing pathways for RNA
splicing:
• In order to generate alternative forms of mRNA, a single pre-
mRNA is processed in various ways, resulting in the
development of various proteins from the same DNA
sequence.
• Alternative splicing, in which the same pre-mRNA can be
spliced in more than one way to generate multiple mRNAs
that are translated into different amino acid sequences and
thus different proteins, is one form of alternative processing.
• Another method of alternative processing involves the use of
several 3′ cleavage sites, where the pre-mRNA comprises two
or more potential cleavage and polyadenylation sites.

27. • In the same pre-mRNA transcript, both alternative
splicing and multiple 3′ cleavage sites can exist.
• In multicellular eukaryotes, alternative processing of
pre-mRNAs is common.
• Researchers predict, that more than 90% of all human
genes undergo alternate splicing.
• The type of splicing also varies between human
tissues; compared to other tissues, the human brain
and liver tissues have more alternatively spliced RNA.

28. RNA editing:
• The coding sequence of an mRNA molecule is altered after
transcription in RNA editing, so that the protein has an amino
acid sequence that varies from that of the gene encoded.
• There were substitutions in some of the mRNA nucleotides in
some nuclear genes in mammalian cells and in some
mitochondrial genes in plant cells.
• More extensive RNA editing for certain mitochondrial genes in
trypanosome parasites has been found in the mRNA.
• More than 60 percent of the sequence is determined by RNA
editing in some of these organisms’mRNAs.

31. • In RNA sequences, a variety of mechanisms can bring
about changes.
• Molecules called guide RNAs (gRNAs) play a key role
in certain situations.
• gRNAs consist of sequences that are partly
complementary to pre-edited RNA segments.
• In these sequences, the two molecules goes through
base pairing.

32. • The mRNA undergoes cleavage after the mRNA is
anchored to the gRNA and nucleotides are inserted,
removed or altered according to the gRNA template
given.
• Enzymes bring about the conversion of the base in
other cases.
• For example, in humans, a gene is transcribed into
mRNA that encodes a lipid-transporting polypeptide
called apolipoprotein-B100, which is synthesized in
liver cells and has 4563 amino acids.

33. •By editing the apolipoprotein-B100 mRNA, a
truncated version of the protein called apolipoprotein-
B48 with only 2153 amino acids is synthesized in
intestinal cells.
•A cytosine base is deaminated by an enzyme in this
editing, transforming it into uracil.
•This conversion converts a codon that specifies the
glutamine amino acid into a stop codon that
terminates translation prematurely, resulting in the
protein being shortened.

34. Thank You