1. RNA Processing Capping Polyadenylation Introns vs exons Splicing
Genomic vs cDNA
Ribosomal RNA processing
t-RNA processing

2. Prokaryotes vs Eukaryotes
PRO: All three classes of RNA are synthesized by one
polymerase.
EU: There are 3 major RNA polymerases. Pol 1
synthesizes rRNA; Pol 2 synthesizes mRNA; Pol 3
synthesizes tRNA.
PRO: mRNA undergoes hardly any posttranscriptional
processing. It is translated as it is synthesized.
EU: mRNA is capped, polyadenylated, spliced
PRO: mRNA contains no introns
EU: mRNA contains intervening sequences (introns)
that are removed during processing

3. mRNA Capping RULE: Capping the 5’ end of mRNAs serves 2 purposes.
Added Guanine
mRNA Capping
CH3
RULE: Capping the 5’ end
of mRNAs serves 2 purposes.
First, the cap protects the mRNA
from 5’-exonuclease activity.
Second, the cap interacts with
eIF-2, a translation initiation
factor required to position the
mRNA on the ribosome
5’-5’ triphosphate
CH3
Gppp + pppApNpNp…
CH3
GpppApNpNp… + pp + p

4. Polyadenylation In eukaryotes, mRNA is polyadenylated by
an enzyme system
that cuts the RNA 30 bs
downstream from an
AAUAAA, then
adds A to the 3’ end
at the cleavage site
Thus, poly A is not
coded in the DNA, but
is added after
transcription

5. Dialogue Q: How should one picture a typical mammalian gene?
A: Mammalian genes have both introns and exons.
Only the exons encode information that will appear
in a protein.
Q: What are introns?
A: Introns appear in unprocessed mRNA. The term is
a shortened version of the words “intervening sequences”
Q: How do exons differ from introns?
A: One could say that the typical mammalian gene has
7 or 8 exons in a length of about 15 kb. The exons are
short, ( bp) whereas introns are large (>1000 bp)

6. Q: What happens to introns?
A: During nuclear processing, the introns are spliced
out and exons are joined together in a linear continuum
Q: How is this accomplished?
A: Cells have mechanism that recognize introns. The
most common is a spliceosome that recognizes the
boundaries of intron-exon junctions and knows were to
cleave and splice
Q: What is involved in the recognition?
A: Small RNA molecules that work with spliceosomes,
The RNA hybridizes to the residues at the splice junctions
and tells the enzyme where to cut.

7. Stages in the Life of a Typical mRNA
DNA
Transcription via RNA Pol II
Primary Transcript
Capping and polyadenylation
Capped-polyadenylated mRNA
Splicing
Mature mRNA

8. Chick Ovalbumin Introns form loops that help with their
excision and splicing
Intron I is generally
quite large in mammalian
mRNAs
1,872 nucleotides (24.3%)
represented as exons

9. Dialogue on mRNA Splicing
What can you tell me about splicing?
Ans: Splicing occurs in two steps. First the junction at the
5’-end of the intron is broken. This is called the 5’-splice site
Then what happens?
Ans: The -OH on the 3’ end of the liberated exon becomes a
nucleophile and attacks the 3’-splice site of the intron. This is
the second step. The two exons are now joined.
What happens to the intron?
Ans: The intron is set free. Because a 2’-OH on an
adenosine caused the initial cleavage, there is a loop in the
intron (called a lariat)

10. Does this happen automatically
Ans: Sometimes. Some RNAs are capable of self-splicing.
Most of the time the splicing occurs through a large complex
called a spliceosome.
What is a spliceosome?
Ans: A spliceosome is a giant 50-60S particle composed of
splicing proteins, pre-mRNAs and small nuclear RNA proteins
or “SNURPS”.
What do the snRNPs do?
Ans: The 5’-end of some of the snRNPs complements bases
at the splice junctions. The snRNPs probably locate the
exon-intron boundaries which tend to be constant for all
eukaryote mRNAs. There are about 6 of them U1-U6. Not
all functions are known.

11. Adenosine A U1-snRNP O OH O OH2’ Exon 2 Exon 1 pApA pGpU pApG pGp C
Intron
U1-snRNP
pApA
pGpU
pApG O
pGp OH
pGpU
pApG O
Intron with lariat
-OH3’
pApA
pGp
Spliced exons
Exon 1
Exon 2

12. Ribosomal RNA Q: Is ribosomal RNA processed the same way as mRNA?
A: No
Q: How is it different?
A: In bacteria, r-RNA is not spliced, it is only cut. All processing is done with a special class of RNAases
Q: What about eukaryotes?
A: Eukaryotes employ basically the same mechanism,
but they also can engage in self-splicing
Q: What is self-splicing?
A: Self-splicing implies that a pre-rRNA can carry
out its own splicing without the need of a spliceosome.

13. Q: Does this mean the RNA is acting as its own
nuclease?
A: Yes. It is called a ribozyme in recognition of its
catalytic activity.
Q: How does self-splicing work?
A: In self-splicing 3’-OH group on the donor exon is
primed by an attack by GMP, GDP, or GTP.
Q: Then what?
A: As before, the freed -OH attacks the phosphate at the
3’ end and forms a new linkages that joins to two segments

14. Bacteria rRNA 16S 23S 5S Ribosomal RNA Primary transcript
III P F E
Ribosomal RNA Primary transcript
Primary processing
1700
2920
200
150
300 5’ 3’
RNase:
16S
23S 5S 4S 4S
M16
M23 M5 D
Pre 16S rRNA
Pre 23S rRNA
Pre-5S
rRNA 5’ 3’
RNase:
Secondary processing
16S rRNA
23S rRNA
1541
2904
120
tRNA(s)
5S rRNA 5’ 3’
Number
of bases

15. Stages in the Life of a Eukaryotic Ribosomal RNA
DNA (300 randomly repeated copies of rRNA genes in the genome
that are transcribed via RNA pol I and processed
in the nucleolus) 5’ 3’
18S
5.8S
28S
45S RNA
spacers
Primary Transcript
Heavily methylated
Methylation at 110 sites
Methylated Transcript
RNase III,
RNase P
tRNA and 5S sequences
are not part of the 45S transcript

16. Self-splicing pre-rRNA
Any guanine nucleotide (GMP, GDP,GTP) sets it off

17. Processing t-RNA
Cut
Spliced
out

19. Amino acid
attachment site
Note: mature t-RNA has
the highest number
of odd bases
methyladenosine
2’-methylguanosine
See p345
in Strategies
dihydrouridine
isopentenyladenosine
Anticodon loop 
pseudouridine