Welcome Each of You to My Molecular Biology Class

1
Welcome Each of You to My Molecular Biology Class
2
Molecular Biology of the Gene, 5/E --- Watson et
al. (2004)

• Part I Chemistry and Genetics
• Part II Maintenance of the Genome
• Part III Expression of the Genome
• Part IV Regulation
• Part V Methods

4/3/05
3

• Ch 12 Mechanisms of Transcription
• Ch 13 RNA Splicing
• Ch 14 Translation
• Ch 15 The Genetic code

4/3/05
4

• Molecular Biology Course

• CHAPTER 13
• RNA Splicing

5
Primary transcript
Figure 13-1
6

• Most of the eukaryotic genes are mosaic (???),
  consisting of intervening sequences separating
  the coding sequence
• Exons (???) the coding sequences
• Introns (???) the intervening sequences
• RNA splicing the process by which introns are
  removed from the pre-mRNA.
• Alternative splicing (????) some pre-mRNAs can
  be spliced in more than one way , generating
  alternative mRNAs. 60 of the human genes are
  spliced in this manner.

7
CHAPTER 13 RNA Splicing

• Topic 1 THE CHEMISTRY OF RNA SPLICING

8
Sequences within the RNA Determine Where Splicing
Occurs
The chemistry of RNA splicing

• The borders between introns and exons are marked
  by specific nucleotide sequences within the
  pre-mRNAs.

9
The consensus sequences for human
Figure 13-2
10

• 5splice site (5????) the exon-intron boundary
  at the 5 end of the intron
• 3 splice site (3????) the exon-intron boundary
  at the 3 end of the intron
• Branch point site (????) an A close to the 3
  end of the intron, which is followed by a
  polypyrimidine tract (Py tract).

11
The intron is removed in a Form Called a Lariat
(???) as the Flanking Exons are joined
The chemistry of RNA splicing

• Two successive transesterification
• Step 1 The OH of the conserved A at the branch
  site attacks the phosphoryl group of the
  conserved G in the 5 splice site. As a result,
  the 5 exon is released and the 5-end of the
  intron forms a three-way junction structure.

12
Figure 13-3
Three-way junction
13
The structure of three-way function
Figure 13-4
14

• Step 2 The OH of the 5 exon attacks the
  phosphoryl group at the 3 splice site. As a
  consequence, the 5 and 3 exons are joined and
  the intron is liberated in the shape of a lariat.

15
Figure 13-3
16
Exons from different RNA molecules can be fused
by Trans-splicing
The chemistry of RNA splicing

• Trans-splicing the process in which two exons
  carried on different RNA molecules can be spliced
  together.

17
Trans-splicing
Figure 13-5
Not a lariat
18
CHAPTER 13 RNA Splicing

• Topic 2
• THE SPLICESOME MACHINERY

19
RNA splicing is carried out by a large complex
called spliceosome
The spliceosome machinery

• The above described splicing of introns from
  pre-mRNA are mediated by the spliceosome.
• The spliceosome comprises about 150 proteins and
  5 snRNAs (?).
• Many functions of the spliceosome are carried out
  by its RNA components.

20

• The five RNAs (U1, U2, U4, U5, and U6, 100-300
  nt) are called small nuclear RNAs (snRNAs).
• The complexes of snRNA and proteins are called
  small nuclear ribonuclear proteins (snRNP,
  pronounces snurps).
• The spliceosome is the largest snRNP, and the
  exact makeup differs at different stages of the
  splicing reaction

21

• Three roles of snRNPs in splicing
• 1. Recognizing the 5 splice site and the branch
  site.
• 2. Bringing those sites together.
• 3. Catalyzing (or helping to catalyze) the RNA
  cleavage.
• RNA-RNA, RNA-protein and protein-protein
  interactions are all important during splicing.

22
RNA-RNA interactions between different snRNPs,
and between snRNPs and pre-mRNA
Figure 13-6
23
CHAPTER 13 RNA Splicing

• Topic 3 SPLICING PATHWAYS

24
Assembly, rearrangement, and catalysis within the
spliceosome the splicing pathway (Fig. 13-8)

• Assembly step 1
• 1. U1 recognize 5 splice site.
• 2. One subunit of U2AF binds to Py tract and the
  other to the 3 splice site. The former subunits
  interacts with BBP and helps it bind to the
  branch point.
• 3. Early (E) complex is formed

Splicing pathways
25

• Assembly step 2
• 1. U2 binds to the branch site, and then A
  complex is formed.
• 2. The base-pairing between the U2 and the branch
  site is such that the branch site A is
  extruded(Figure 13-6). This A residue is
  available to react with the 5 splice site.

26
E complex
Figure 13-8
A complex
Figure 13-6b
27

• Assembly step 3
• 1. U4, U5 and U6 form the tri-snRNP Particle.
• 2. With the entry of the tri-snRNP, the A complex
  is converted into the B complex.

28
A complex
B complex
Figure 13-8
29

• Assembly step 4
• U1 leaves the complex, and U6 replaces it at the
  5 splice site.
• U4 is released from the complex, allowing U6 to
  interact with U2 (Figure 13-6c).This arrangement
  called the C complex.

30
B complex
C complex in which the catalysis has not occurred
yet
Figure 13-6c
Figure 13-8
31

• Catalysis Step 1
• Formation of the C complex produces the active
  site, with U2 and U6 RNAs being brought together
• Formation of the active site juxtaposes the 5
  splice site of the pre-mRNA and the branch site,
  allowing the branched A residue to attack the 5
  splice site to accomplish the first
  transesterfication reaction.

32

• Catalysis Step 2
• U5 snRNP helps to bring the two exons together,
  and aids the second transesterification reaction,
  in which the 3-OH of the 5 exon attacks the 3
  splice site.

• Final Step
• Release of the mRNA product and the snRNPs

33
C complex
Figure 13-8
34
splicesome-mediated splicing reactions
E complex
A complex
B complex
Figure 13-8
C complex (???complex??)
35
Self-splicing introns reveal that RNA can
catalyze RNA splicing

• Self-splicing introns the intron itself folds
  into a specific conformation within the precursor
  RNA and catalyzes the chemistry of its own
  release and the exon ligation

Splicing pathways
36
Adams et al., Nature 2004, Crystal structure of a
self-splicing group I intron with both exons
37
Practical definition for self-splicing introns
the introns that can remove themselves from
pre-RNAs in the test tube in the absence of any
proteins or other RNAs. There are two classes
of self-splicing introns, group I and group II
self-splicing introns.
38
TABLE 13-1 Three class of RNA Splicing TABLE 13-1 Three class of RNA Splicing TABLE 13-1 Three class of RNA Splicing TABLE 13-1 Three class of RNA Splicing
Class Abundance Mechanism Catalytic Machinery
Nuclear pre-mRNA Very common used for most eukaryotic genes Two transesterification reactions branch site A Major spliceosome
Group II introns Rare some eu-Karyotic genes from organelles and prokaryotes Same as pre-mRNA RNA enzyme encoded by intron (ribozyme)
Group I introns Rare nuclear rRNA in some eukaryotics, organlle genes, and a few prokaryotic genes Two transesterific-ation reactions exogenous G Same as group II introns
39

• The chemistry of group II intron splicing and RNA
  intermediates produced are the same as that of
  the nuclear pre-mRNA.

40
Figure 13-9
41
Group I introns release a linear intron rather
than a lariat

• Instead of using a branch point A, group I
  introns use a free G to attack the 5 splice
  site.
• This G is attached to the 5 end of the
  intron.The 3-OH group of the 5 exon attacks the
  5 splice site.
• The two-step transesterification reactions are
  the same as that of splicing of the group II
  intron and pre-mRNA introns.

Splicing pathways
42
G instead of A
a Lariat intron
a linear intron
Figure 13-9
43
Group I introns

1. Smaller than group II introns
2. Share a conserved secondary structure, which
   includes an internal guide sequence
   base-pairing with the 5 splice site sequence in
   the upstream exon.
3. The tertiary structure contains a binding pocket
   that will accommodate the guanine nucleotide or
   nucleoside cofactor

44
The similarity of the structures of group II
introns and U2-U6 snRNA complex formed to
process first transesterification
Figure 13-10
45
How does spliceosome find the splice sites
reliably
Splicing pathways

• Two kinds of splice-site recognition errors
• Splice sites can be skipped.
• Pseudo splice sites could be mistakenly
  recognized, particularly the 3 splice site.

46
Figure 13-12
47
Reasons for the recognition errors

• (1) The average exon is 150 nt, and the average
  intron is about 3,000 nt long (some introns are
  near 800,000 nt)
• It is quite challenging for the spliceosome to
  identify the exons within a vast ocean of the
  intronic sequences.

48
(2) The splice site consensus sequence are rather
loose. For example, only AG?G tri-nucleotides is
required for the 3 splice site, and this
consensus sequence occurs every 64 nt
theoretically.
49
Two ways to enhance the accuracy of the
splice-site selection

• 1. Because the C-terminal tail of the RNA
  polymerase II carries various splicing proteins,
  co-transcriptional loading of these proteins to
  the newly synthesized RNA ensures all the splice
  sites emerging from RNAP II are readily
  recognized, thus preventing exon skipping.

50

• 2. There is a mechanism to ensure that the
  splice sites close to exons are recognized
  preferentially. SR proteins bind to the ESEs
  (exonic splicing enhancers) present in the exons
  and promote the use of the nearby splice sites by
  recruiting the splicing machinery to those sites

51
Figure 13-13
SR proteins, bound to exonic splicing enhancers
(ESEs), interact with components of splicing
machinery, recruiting them to the nearby splice
sites.
52
SR proteins are essential for splicing

1. Ensure the accuracy and efficacy of constitutive
   splicing
2. Regulate alternative splicing
3. There are many varieties of SR proteins. Some are
   expressed preferentially in certain cell types
   and control splicing in cell-type specific
   patterns

53
Topic 4 ALTERNATIVE SPLICING
CHAPTER 13 RNA Splicing
54
Single genes can produce multiple products by
alternative splicing
Alternative splicing

• Many genes in higher eukaryotes encode RNAs that
  can be spliced in alternative ways to generate
  two or more different mRNAs and, thus, different
  protein products.

55
Drosophila DSCAM gene can be spliced in 38,000
alternative ways
Figure 13-13
56
There are five different ways to alternatively
splice a pre-mRNA
Figure 13-15
57
Alternative splicing can be either constitutive
or regulated

• Constitutive alternative splicing more than one
  product is always made from a pre-mRNA
• Regulative alternative splicing different forms
  of mRNA are produced at different time, under
  different conditions, or in different cell or
  tissue types

58
An example of constitutive alternative splicing
Splicing of the SV40 T antigen RNA
Figure 13-16
59
Alternative splicing is regulated by activators
and repressors

• The regulating sequences exonic (or intronic)
  splicing enhancers (ESE or ISE) or silencers (ESS
  and ISS). The former enhance and the latter
  repress splicing.
• Proteins that regulate splicing bind to these
  specific sites for their action

Alternative splicing
60

• SR proteins binding to enhancers act as
  activators.
• (1) One domain is the RNA-recognition motif (RRM)
• (2) The other domain is RS domain rich in
  arginine and serine. This domain mediates
  interactions between the SR proteins and proteins
  within the splicing machinery.

61

• hnRNPs binds RNA and act as repressors
• Most silencers are recognized by hnRNP (
  heterogeneous nuclear ribonucleoprotein) family.
  
• These proteins bind RNA, but lack the RS domains.
  Therefore, (1) They cannot recruit the splicing
  machinery. (2) they block the use of the specific
  splice sites that they bind.

62
Regulated alternative splicing
Figure 13-17
63
An example of repressors inhibition of splicing
by hnRNPI
Coats the RNA and makes the exons invisible to
the splicing machinery
Binds at each end of the exon and conceals (??)
it
Figure 13-18
64

• The outcome of alternative splicing
• 1. Producing multiple protein products, called
  isoforms.
• 2. Switching on and off the expression of a given
  gene. In this case, one functional protein is
  produced by a splicing pattern, and the
  non-functional proteins are resulted from other
  splicing patterns.

65
A small group of intron are spliced by minor
spliceosome
Alternative splicing

• This spliceosome works on a minority of exons,
  and those have distinct splice-site sequence.
• The chemical pathway is the same as the major
  spliceosome.

66
U11 and U12 are in places of U1 and U2,
respectively
Figure 13-19 The AT-AC spliceosome
67
Topic 5EXON SHUFFLING
CHAPTER 13 RNA Splicing
68
Exons are shuffled by recombin-ation to produce
gene encoding new proteins

• All eukaryotes have introns, and yet these
  elements are rare in bacteria. Two likely
  explanations for these situation
• 1. Introns early model introns existed in all
  organisms but have been lost from bacteria.
• 2. Intron late model introns never existed in
  bacteria but rather arose later in evolution.

Exon shuffling
69

• Why have the introns been retained in eukaryotes?

70

• 1. The need to remove introns, allows for
  alternative splicing which can generate multiple
  proteins from a single gene.
• 2. Having the coding sequence of genes divided
  into several exons allows new genes to be created
  by reshuffling exon.

71

• Three observations suggest exon shuffling
  actually occur
• 1. The borders between exons and introns within a
  gene often coincide with the boundaries between
  domains within the protein encoded by that gene.

72
For example DNA-binding protein
Figure 13-21
73

• 2. Many genes, and proteins they encode, have
  apparently arisen during evolution in part via
  exon duplication and divergence.

74

• 3. Related exons are sometimes found in unrelated
  genes.

Exons have been reused in genes encoding
different proteins
Figure 13-22
75
Topic 6RNA EDITING
CHAPTER 13 RNA Splicing
76
RNA editing is another way of changing the
sequence of an mRNA

• I. Site specific deamination
• 1. A specifically targeted C residue within mRNA
  is converted into U by the deaminase.
• 2. The process occurs only in certain tissues or
  cell types and in a regulated manner.

RNA editing
77
Figure 13-25
78
The human apolipoprotein gene
Figure 13-25
Stop code
In liver
In intestines
79

• 3. Adenosine deamination also occurs in cells.
  The enzyme ADAR (adenosine deaminase acting on
  RNA) convert A into Inosine. Insone can base-pair
  with C, and this change can alter the sequence of
  the protein.
• 4. An ion channel expressed in mammalian brains
  is the target of Adenosine deamination.

80

• II Guide RNA-directed uridine insertion or
  deletion.
• 1. This form of RNA editing is found in the
  mitochondria of trypanosomes.
• 2. Multiple Us are inserted into specific region
  of mRNAs after transcription (or US may be
  deleted).

81

• 3. The addition of Us to the message changes
  codons and reading frames, completely altering
  the meaning of the message.
• 4. Us are inserted into the message by guide RNAs
  (gRNAs) .

82
gRNAs

• Having three regions
• anchor directing the gRNAs to the region of
  mRNAs it will edit.
• editing region determining where the Us
  will be inserted
• poly-U stretch

83
Figure 13-26
84
Topic 7mRNA TRANSPORT
CHAPTER 13 RNA Splicing
85
Once processed, mRNA is packaged and exported
from the nucleus into the cytoplasm for
translation
mRNA transport

• All the fully processed mRNAs are transported to
  the cytoplasm for translation into proteins

86

• Movement from the nucleus to the cytoplasm is an
  active and carefully regulated process.
• The damaged, misprocessed and liberated introns
  are retained in the nucleus and degraded.
• A typical mature mRNA carries a collection of
  proteins that identifies it as being ready for
  transport.
• Export takes place through the nuclear pore
  complex.

87

1. Once in the cytoplasm, some proteins are
   discarded and are then imported back to the
   nucleus for another cycle of mRNA transport. Some
   proteins stay on the mRNA to facilitate
   translation.

88
Figure 13-27
89
Key points of the chapter

1. Why RNA splicing is important?
2. Chemical reaction determination of the splice
   sites, the products, trans-splicing
3. Spliceosome splicing pathway and finding the
   splice sites
4. Self-splicing introns and mechanisms
5. Alternative splicing and regulation, alternative
   spliceosome
6. Two different mechanisms of RNA editing
7. mRNA transport-a link to translation

90
HOMEWORKS
Watch the animations Enjoy the critical thinking
excises on your study CD.