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RNA Splicing

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Title: RNA Splicing


1
Chapter 13?????? ?? 200431060017
  • RNA Splicing

2
OUTLINE
  • The Chemistry of RNA Splicing
  • The Spliceosome Machinery
  • Splicing Pathways (important)
  • Alternative Splicing (important)
  • Exon Shuffling
  • RNA Editing
  • mRNA Transport

3
Key Words
  • Exon coding sequences
  • Intron intervening sequences
  • Pre-mRNA the primary transcript of DNA
  • RNA splicing process that intron are
    moved from the pre-RNA
  • Spliceosome a huge molecular machine catalyse
    RNA splicing
  • Alternative splicing some pre-mRNAs can be
    spliced in more than one way,and produce
    alternative mRNAs.

4
Topic 1 The Chemistry of RNA Splicing
  • Sequences within the RNA determine where
    splicing occurs
  • The intron is moved in a form called Lariat as
    the flanking exons are joined
  • Exons from different RNA molecules can be fused
    by trans-splicing

5
one Sequences within the RNA determine
where splicing occurs
  • The borders between
  • introns and exons are
  • marked by specific
  • nucleotide sequences
  • ( GUAG Law )
  • within the pre-mRNAs.
  • Py-tract rich in pyrimidine

6
Notice
  • GU in 5 splicing site, AG in 3 splicing site
    and A in branch point site are the most conserved
    sequences, and they are all in the intron.
  • These sequences are important for the distinguish
    between intron and exon ,remove of intron
    ,linkage of exons and delineate where splicing
    will occur.

7
Two The intron is moved in a form called Lariat
as the flanking exons are joined
  • RNA splicing consists of two successive
    transesterification reaction.

8
  • Reaction 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.

9
  • Reaction 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.

10
The structure of three-way junction
In addition to the 5 and 3 backbone linkages,a
third phosphodiester extends from the 2OH of
that A to create a three-way junction.
11
Reaction 2
  • Ensure the splicing only goes forward
  • One an increase in entropy.
  • Two the excised intron lariat is rapidly
    degraded after its removal.

12
Notice
  • In the two reactions, there is no net gain in the
    number of chemical bonds. So no energy is
    demanded by the process.
  • But, we see a large amount of ATP is consumed
    during the splicing reaction. Why? This energy is
    required to properly assemble and operate the
    splicing machinery, not for the chemistry.

13
Three Exons from different RNA molecules can be
fused by trans-splicing
  • Trans-splicing
  • the process in which two exons carried
    on different RNA molecules can be spliced
    together.
  • This process is rare .But all mRNAs in
    the nematode worm undergo trans-splicing.

14
Topic 2 The Spliceosome Machinery
  • RNA splicing is carried out by a large complex
    called spliceosome
  • Spliceosome is a complex that mediates splicing
    of introns from pre-mRNA.
  • And it comprises about 150 proteins and 5
    snRNAs .
  • Many functions of the spliceosome are
    carried out by its RNA components.

15
  • 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 (snRNPs).
  • The spliceosome is the largest snRNP, and the
    exact makeup differs at different stages of the
    splicing reaction.
  • Different snRNPs come and go at different
    times,each carrying out particular functions in
    the reaction.

16
  • 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

17
RNA-RNA interactions between different snRNPs,
and between snRNPs and pre-mRNA
Branch-point binding protein
18
Topic 3 Splicing Pathways
  • Assembly, rearrangement, and catalysis within the
    spliceosome the splicing pathway
  • Self-splicing introns reveal that RNA can
    catalyze RNA splicing
  • Group I introns release a linear intron rather
    than a lariat
  • How does spliceosome find the splice sites
    reliably

19
One Assembly, rearrangement, and catalysis
within the spliceosome the splicing pathway
  • Steps of splicing pathway
  • Assembly step one
  • 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

20
Assembly
  • Step two
  • 1.With the help of U2AF, U2 binds to the branch
    site replacing of BBP, 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 .
    This A residue is available to react with the 5
    splice site.

21
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22
Assembly
  • Step three
  • 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.

23
Assembly
  • Step four
  • 1 , U1 leaves the complex, and U6 replaces it
    at the 5 splice site.
  • 2 , U4 is released from the complex, allowing
    U6 to interact with U2 .This arrangement called
    the C complex.

24
Catalysis
  • Step one
  • 1 , Formation of the C complex produces the
    active site, with U2 and U6 RNAs being brought
    together

25
  • 2 , 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.

26
Catalysis
  • Step two
  • 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.
  • Step three
  • Release of the mRNA product and the
    snRNPs

27
(No Transcript)
28
Two Self-splicing introns reveal that RNA can
catalyze RNA splicing
  • Self-splicing introns the intron itself folds
    into a specific conformation within the pre-mRNA
    and catalyzes the chemistry of its own release
    (recall RNA enzyme ) and the exon ligation.
  • 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.
  • Two classes of self-splicing introns, group I and
    group II self-splicing introns.

29
Three classes of RNA splicing
30
Notice The chemistry of group II intron
splicing and RNA intermediates produced are the
same as that of the nuclear pre-mRNA.
31
Three 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.

32
Three classes of RNA splicing
33
Group 1 intron structure
Share a conserved secondary structure, which
includes an internal guide sequence
base-pairing with the 5 splice site sequence in
the upstream exon.
  • A complex secondary structure

34
Group 1 intron structure
The tertiary structure contains a binding
pocket that will accommodate the guanine
nucleotide or nucleoside cofactor
Bind any G-containing ribonucleotide.
35
Steps of group 1 intron splicing
  • free guanosine binds in the guanine-binding
    pocket
  • 3OH of guanosine attacks phosphate at 5end of
    intron
  • 3OH of exon 1 attack 5phosphate of exon 2
  • Intron is released

36
  • Internal guide sequence base pairs with 5end of
    intron
  • 3guanosine binds in guanine-binding pocket
  • 3OH of bound guanosine attacks phosphate to the
    right of IGS
  • Bond between 3G and 5 phosphate hydrolyzes,
    leaving inactive intron

37
The similarity of the structures of group II
introns and U2-U6 snRNA complex formed to
process first transesterification
38
Four How does spliceosome find the splice sites
reliably
  • splice-site recognition is prone to 2 kinds of
    errors
  • ?Splice sites can be skipped.
  • ?some site close in sequence but not legitimate
    splice site ,could be mistakenly recognized.
  • For example ,Pseudo splice sites could be
    mistakenly recognized and pair with component at
    5site, particularly the 3 splice site.

39
Error produced by mistakes in splice-site
selection
40
Two ways to enhance the accuracy of the
splice-site selection
  • 1 , the C-terminal tail of the RNA polymerase II
    carries various splicing proteins.
  • when a 5splice site is encountered in the
    newly synthesized RNA, those components are
    transferred from the Pol II C-terminal tail onto
    the RNA .

41
  • Once in place ,the 5splice site components are
    poised to interact with those that bind to the
    next 3splice siteto be synthesized. Thus ,the
    correct 3splice site can be recognized before
    any competing sites further downstream have been
    transcribed.
  • This co-transcriptional loading process greatly
    diminishes the likelihood of exon skipping.

42
  • 2 , a second mechanism guides against the use of
    incorrect sites by ensuring that splice sites
    close to exons are recognized preferentially.
  • SR proteins bind to sequences called exonic
    splicing enhancers (ESEs) within the exons.
  • SR bound to ESE interacts with components of the
    splicing machinery ,recruiting them to the nearby
    splicing sites. In this way , the machinery binds
    more efficiently to the nearby sites than to
    incorrect sites not close to exons.

43
SR protein recruits spliceosome components to the
5and 3 splice sites
SR proteins bind to ESEs ,recruit U2AF and
U1snRNP to the downstream 5and upstream 3splice
sites respectively. This initiates the assembly
of the splice machinery on the correct sites and
splice can proceed as outlined earlier.
44
SR proteins function
  • Ensure the accuracy and efficiency of
    constitutive splicing.
  • Regulate alternative splicing.
  • They come in many varieties ,controlled by
    physiological signals and constitutively active.
    And some express preferential in certain cell
    types and control splicing in cell-type specific
    patterns.

45
Topic 4 Alternative splicing
  • Single gene can produce multiple products by
    alternative splicing
  • Alternative splicing is regulated by activators
    and repressors
  • A small group of introns are spliced by an
    spliceosome composed of a different set of snRNPs

46
One Single gene 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
    ,different protein products.

47
  • As we know ,exons are not skipped and splice
    sites not ignored, why does alternative splicing
    occur so often ?
  • Answer some splice sites are used only some of
    the time ,leading to the production of different
    versions of the RNA from different transcripts of
    the same gene.

48
Five ways to splice an RNA
49
  • Alternative splicing can be either constitutive
    or regulated.
  • Constitutive more than one product from the
    same gene
  • Regulated different products are generated at
    different times, under different conditions , or
    in different cell or tissue type.

50
Constitutive alternative splicing
high level SF2/ASF
Splicing of the monkey SV40 T antigen RNA.
51
Two Alternative splicing is regulated by
activators and repressors
  • Proteins that regulate splicing bind to specific
    sites called exonic (or intronic ) splicing
    enhancers or silencers.
  • Protein specific sequence can guide elements of
    spliceosome to exons.

52
  • SR protein family is large and diverse, has
    specific roles in regulated alternative splicing
    by directing the splicing machinery to different
    splice sites under different conditions.
  • The presence or activity of a given SR can
    determine whether a particular splice site is
    used in particular cell type ,or at a particular
    stage of development.

53
Regulated alternative splicing
54
  • RNA-recognition motif RRM
  • RS domain rich in Arg and

Binding RNA
RS protein
Ser. Found at C-terminal end of protein ,mediates
interactions between SR and the pro. within the
splicing machinery, recruiting them to a nearby
splice site.
55
An example of repressors inhibition of splicing
by hnRNPI
  • Repressor are hnRNPs . They can bind RNAs
    sliencer and repress the use of those sites.
  • They dont have RS domain ,so cant recruit
    spliceosome. By binding sites, this blocking can
    repress splice.

Coat exon entirely to avoid splicing
Conceal exon as a loop
56
  • The second way alternative splicing can be used
    as an on/off switch is by regulating the use of
    an intron, which, when retained in the mRNA ,the
    mRNA will be never transported out of nucleous
    and be never translated.

57
Three A small group of introns are spliced by
an spliceosome composed of a different set of
snRNPs
  • Higher eukaryotes use the major splicing
    machinery we have discussed before, and some
    pre-mRNAs are spliced by a low-aboundance form of
    spliceosome.
  • The rare form contains some components common to
    major spliceosome but other unique. U11 and U12
    have the same roles in the splicing reaction as
    U1 and U2, but they recognize distinct sequences.
    U4 and U6 share the same names but their snRNPs
    are distinct. U5 is identical.

58
  • The minor spliceosome recognizes rarely occurring
    introns having consensus sequences distinct from
    the sequences of most pre-mRNA. This intron
    contains 5 AT and 3AC ( AT-AC low ), so the
    new form is known as the AT-ACspliceosome.
  • Later ,we discover that it also recognizes GT-AG.

59
  • The ability of the snRNAs and splice site
    sequences to base-pair is conserved, not any
    sequences within either.
  • Although the different splice sites and branch
    site ,the two forms of spliceosome share the same
    chemical pathway to remove introns.

60
Topic 5 Exon Shuffling
  • Exons are shuffled by recombination to produce
    genes encoding new proteins.
  • All eukaryotes have introns which rare in the
    bacteria. Two model
  • intron early model all organism have
    introns, and bacteria lost them.
  • intron late model introns were inserted
    into genes by a transposon-like machanism

61
  • Why introns can exist in eukaryotes??
  • First the presence of introns and the need to
    remove them ,allow for alternative splicing which
    can generate multiple proteins from a gene.
  • Second having the coding sequence of genes
    divided into several exons allows new gene to be
    created by reshuffling exons.

62
Three observations
  • 1. The borders between exons and introns within a
    gene often coincide with the boundaries between
    domains within the protein encoded by that gene.

63
  • 2. Many genes, and proteins they encode, have
    apparently arisen during evolution in part via
    exon duplication and divergence.
  • 3.Related exons are sometimes found in unrelated
    genes.

?Exons have been reused in genes encoding
different proteins
64
Topic 6 RNA editing
  • RNA editing is another way of changing the
    sequence of an mRNA.
  • Two mechanisms mediate editing
  • Site-specific deamination
  • Guide RNA-directed uridine insertion or
    deletion

65
Site-specific deamination
  • The process occurs only in certain tissues or
    cell types and in a regulated manner.

66
RNA editing by deamination of human
apolipoprotein gene
67
  • 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.
  • ? An ion channel expressed in mammalian brains is
    the target of Adenosine deamination.

68
Guide RNA-directed uridine insertion or deletion.
directing the gRNAs to the region of mRNAs it
will edit
determining where the Us will be inserted
69
Topic 7 mRNA transport
  • Once processed (capped, intron-free and
    polyadenylated ), mRNA is packaged and exported
    from the nucleus into the cytoplasm for
    translation.

70
How are RNA selection and transport achieved?
  • RNA associates with proteins as soon as
    transcription Initially proteins involved in
    capping, then splicing factors, and finally the
    proteins that mediate polyadenylation .
  • Some proteins are replaced at various steps
    during the processing path ,but some such as SR
    are not, moreover ,additional proteins join.

71
  • As a result, a typical mature mRNA carries a
    collection of proteins that identifies it as
    being mRNA destined for transport.
  • Others not only lack the particular signature
    collection required for transport, also have
    their collection that block transport.

72
Examples
  • The excised introns carry hnRNPs which mark such
    an RNA for nuclear retention and destruction.
  • Mature mRNA carry residual SR proteins , even
    another group of proteins binding specifically to
    exon-exon junctions.
  • Conclusion the set of proteins, not any
    individual kind of protein, marks RNAs for either
    export or retention in the nucleus.

73
  • Nuclear pore complex a specific structure in
    the nuclear membrane. Small molecules under 50Kd
    can pass through it unaided. And large
    molecules and complexes require active transport.
  • mRNAs and their associated proteins can actively
    transport through NPC

74
Transport of mRNAs out of the nucleus
GTPase
NPC
75
  • The end !!
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