POST TRANSCRIPTIONAL MODIFICATIONS IN EUKARYOTES

1. PTM

2. Messenger RNAs Undergo 5 Capping
and Addition of a 3 Tail
• In prokaryotes, most primary mRNA
transcripts are translated without further
modification. Indeed, protein synthesis usually
begins before transcription is complete. In
eukaryotes, however, mRNAs are synthesized
in the cell nucleus, whereas translation occurs
in the cytosol. Eukaryotic mRNA transcripts
can therefore undergo extensive
posttranscriptional processing while still in the
nucleus.

3. Eukaryotic mRNAs Have 5 Caps
• Eukaryotic mRNAs have a cap structure
consisting of a 7-methylguanosine (m7G)
residue joined to the transcript’s initial (5’)
nucleotide via a 5’–5’ triphosphate bridge.
• The cap, which adds to the growing transcript
when it is ~30 nt long, identifies the
eukaryotic translation start site

4. • Capping involves several enzymatic reactions:
(1) the removal of the leading phosphate
group from the mRNA’s 5’ terminal
triphosphate group by an RNA
triphosphatase; (2) the guanylylation of the
mRNA by capping enzyme, which requires
GTP and yields the 5’–5’ triphosphate bridge
and PPi ; and (3) the methylation of guanine
by guanine-7-methyltransferase, in which the
methyl group is supplied by S-adenosylmethionine
(SAM).

5. • In addition, the cap may be O2’-methylated at
the first and second nucleotides of the
transcript by a SAM-requiring 2’- O-methyltransferase.
Capping enzyme binds to
RNAP II’s phosphorylated CTD, and hence it
appears that capping marks the completion of
RNAP II’s switch from transcription initiation to
elongation. Capped mRNAs are resistant to 5’-
exonucleolytic degradation.

6. FIG. 26-20 Structure of the 5¿ cap
of eukaryotic mRNAs. A cap may
be O2’-methylated at the
transcript’s leading nucleoside
(the predominant cap in
multicellular organisms), at its
first two nucleosides, or at
neither of those positions (the
predominant cap in unicellular
eukaryotes). If the first nucleoside
is adenosine (it is usually a urine),
it may also be N6-methylated.

7. Eukaryotic mRNAs Have Poly(A) Tails
• Mature eukaryotic mRNAs, however, have
well-defined 3’ ends terminating in poly(A)
tails of ~250 nt (~80 nt in yeast). The poly(A)
tails are enzymatically appended to the
primary transcripts in two reactions:

8. • A transcript is cleaved 15 to 25 nt past a highly
conserved AAUAAA sequence and less than 50 nt
before a less-conserved U-rich or G + U–rich
sequence. The precision of this cleavage reaction
has apparently eliminated the need for accurate
transcription termination.
• Nevertheless, the identity of the endonuclease
that cleaves the RNA is uncertain although
cleavage factors I and II (CFI and CFII) are
required for this process.

9. • The poly(A) tail is subsequently generated from
ATP through the stepwise action of poly(A)
polymerase (PAP), a template-independent RNA
polymerase that elongates an mRNA primer with
a free 3’-OH group. PAP is activated by cleavage
and polyadenylation specificity factor (CPSF)
when the latter protein recognizes the AAUAAA
sequence.
• Once the poly(A) tail has grown to ~10 residues,
the AAUAAA sequence is no longer required for
further chain elongation

10. • CPSF binds to the phosphorylated RNAP II
CTD; deleting the CTD inhibits polyadenylation
• poly(A) binding protein II (PAB II) determine
length of poly A tail.
• cleaved transcript is polyadenylated before it
can dissociate and be digested by cellular
nucleases

11. Splicing Removes Introns from
Eukaryotic Genes
• The primary transcripts, also called pre-mRNAs
or heterogeneous nuclear RNAs
(hnRNAs), are variable in length and are
much larger (~2000 to > 20,000 nt)
• intervening sequences (introns),
• flanking expressed sequences (exons)

12. • In humans, the number of introns in a gene
varies from none to 364 (in the ~2400-kb gene
encoding the 34,350-residue muscle protein
titin, the largest known gene and single-chain
protein, with intron lengths ranging from ~65
to ~800,000 nt (in the gene encoding the
muscle protein dystrophin; and averaging
~3500 nt (exons, in contrast, have lengths that
average ~150 nt and range up to 17,106 nt in
the gene encoding titin).

13. • The introns from corresponding genes in two
vertebrate species rarely vary in number and
position, but often differ extensively in length and
sequence so as to bear little resemblance to one
another.
• The production of a translation-competent
eukaryotic mRNA begins with the transcription of
the entire gene, including its introns (Fig. 26-22).
Capping occurs soon after initiation, and splicing
commences during the elongation phase of
transcription. The mature mRNA emerges only
after splicing is complete and the RNA has been
polyadenylated. The mRNA is then transported to
the cytosol, where the ribosomes are located, for
translation into protein.

15. Exons Are Spliced in a Two-Stage
Reaction
• GU at the intron’s 5’ boundary and an
invariant AG at its 3’ boundary.
• These sequences are necessary and sufficient
to define a splice junction
• The splicing reaction occurs via two
transesterification reactions

16. • A 2’,5’-phosphodiester bond forms between an
intron adenosine residue and the intron’s 5’-
terminal phosphate group. The 5’ exon is thereby
released and the intron assumes a novel lariat
structure (so called because of its shape). The
adenosine at the lariat branch point is typically
located in a conserved sequence 20 to 50
residues upstream of the 3’splice site. Mutations
that change this branch point A residue abolish
splicing at that site.

18. • The 5’ exon’s free 3 ’ OH group displaces the 3 ’
end of the intron, forming a phosphodiester bond
with the 5 ’ -terminal phosphate of the 3 ’ exon
and yielding the spliced product. The intron is
thereby eliminated in its lariat form with a free 3 ’
-OH group. Mutations that alter the conserved
AG at the 3 ’ splice junction block this second
step, although they do not interfere with lariat
formation. The lariat is eventually debranched
(linearized) and, in vivo, is rapidly degraded.

19. • Exon skipping
• This occurs, at least in part, because splicing
occurs cotranscriptionally
• Splicing Is Mediated by snRNPs in the
Spliceosome
• The eukaryotic nucleus contains numerous copies
of highly conserved 60- to 300-nt RNAs called
small nuclear RNAs (snRNAs), which form
protein complexes termed small nuclear
ribonucleoproteins (snRNPs

20. • U1-snRNA (which is so named because of its
high uridine content), is partially
complementary to the consensus sequence of
5’splice junctions. Apparently, U1-snRNP
recognizes the 5 ’splice junction. Other snRNPs
that participate in splicing are U2-snRNP, U4–
U6-snRNP (in which the U4- and U6-snRNAs
associate via base pairing), and U5-snRNP.

21. • Splicing takes place in an ~60S particle dubbed
the spliceosome. The spliceosome brings
together a pre-mRNA, the snRNPs, and a
variety of premRNA binding proteins. The
spliceosome, which consists of 5 RNAs and
~150 polypeptides, is comparable in size and
complexity to the ribosome

22. • All four snRNPs involved in mRNA splicing
contain the same so-called snRNP core
protein, which consists of seven Sm proteins,
named B, D1, D2, D3, E, F, and G proteins.
These proteins collectively bind to a
conserved AAUUUGUGG sequence known as
the Sm RNA motif, which occurs in U1-, U2-,
U4-, and U5-snRNAs.

23. • Mammalian U1-snRNP consists of U1-snRNA and
ten proteins, namely, the seven Sm proteins that
are common to all U-snRNPs as well as three that
are specific to U1-snRNP: U1-70K, U1-A, and U1-
C.
• The predicted secondary structure of the 165-nt
U1-snRNA contains five double-helical stems,
four of which come together at a four-way
junction.
• U1-70K and U1-A bind directly to RNA stem–
loops (SL) 1 and 2, respectively, whereas U1-C is
bound by other proteins

25. • Fortuitously, the 5’ ends of two neighboring
U1-snRNAs in the crystal form a double-helical
segment, which serves as a model for how the
5 ’ end of U1-snRNA base-pairs with the 5 ’
splice site of the pre-mRNA in the
spliceosome. The zinc finger domain of U1-C
interacts with this double helix and
presumably stabilizes it. This is consistent with
the observation that mutants of U1-C in this
region cannot initiate spliceosome formation.

26. Alternative mRNA Splicing Yields
Multiple Proteins from a Single Gene
• The expression of numerous cellular genes is
modulated by the selection of alternative
splice sites. Thus, genes containing multiple
exons may give rise to transcripts containing
mutually exclusive exons. In effect, certain
exons in one type of cell may be introns in
another. For example, a single rat gene
encodes seven tissue-specific variants of the
muscle protein -tropomyosin through the
selection of alternative splice sites