Week 6 - RNA Splicing and Processing Flashcards
1
Q
Pre-mRNA
A
the nuclear transcript that is processed by modification and splicing to give an mRNA
2
Q
RNA splicing
A
the process of excising the introns from RNA and connecting the exons into an continuous mRNA
3
Q
RNA is modified
A
in the nucleus by
- additions to the 5’ and 4’ ends
- by splicing to remove the introns
4
Q
RNA splicing and modification
A
5
Q
The 5 end of eukaryotic mRNA is
A
capped
6
Q
The 5’ end of eukaryotic mRNA is capped
A
- a 5’ cap is formed by adding a G to the terminal base of the trancript via a 5’-5’ link
- 5’ 7-methylguanosine cap
- the cap structure is recognized by protein factors to influence mRNA stability, splicing, export, and translation
- major function is to protect the mRNA from degradation
- cap is recognized by cap binding protein heterodimer (CBP20/80) to facilitate export from the nucleus
7
Q
5’ cap
A
7-methylguanosine
- add CH3 at C7 (methylated guanine)
- 5’ to 5’ phosphotriester linkage
- protects unstable RNA (stops degradation)
8
Q
The 5’ capping process takes place
A
during trancription
• may be important for release from pausing of transcription
9
Q
3 forms of (5’) capping
A
- all contain cap 0
- the 5’ cap of most mRNA is monomethylated, but some small noncoding RNAs are trimethylated
- 50% of mRNA is capped at 20 nucleotides
- at 30 nucleotides almost all mRNA is capped
10
Q
3 forms of capping
A
11
Q
… enzymes work together to add the cap
A
- 3 enzymes work together to add the cap
- RNA triphosphatatse and guanylytransferase activities are present in the same protein and called the capping enzyme
12
Q
Capping enzyme
A
RNA triphosphatase and guanylytransferase activities are present in the same protein
13
Q
Addition of the 5’ cap
PICTURE
A
14
Q
Addition of the 5’ cap
basic steps
A
- remove 1 P (from the 3 at the 5’ end)
- transfers guanosne to 5’ of RNA, 5’-5’ phosphodiester link
(^both in capping enzyme)
- add methyl to guanosine
15
Q
Capping enzyme is recruited by
A
the CTD of RNA polymerase II
16
Q
Capping enzyme is recruited by the CTD of RNA polymerase II
A
- RNA pol II CTD must be phosphorylated on Ser-5 to target a transcript for capping
- CE interacts with Ser-5 phosphorylated pol II
- capping must happen in transcription
17
Q
Capping enzyme is recruited by the CTD of RNA polymerase II
PICTURE
A
18
Q
RNA splicing occurs
A
during and/or after transcription
19
Q
Splicing makes
A
transcript diversity
diversity in protein function in splicing the transcript
20
Q
Nuclear splice sites are
A
short sequences
21
Q
Nuclear splice sites are short sequences
A
- splice sites - sequences immediately surrounding the exon-_intron_ boundaries
- the 5’ splice site at the 5’ (left) end of the intron includes the consensus sequence GU
- the 3’ splice site at the 3’ (right) end of the intron includes the consensus sequence AG
- GU-AG or U2 type introns (98% of human introns)
22
Q
Splice sites
A
sequences immediately surrounding the exon-intron boundaries
sequence motifs, consensus in intron that allows splicing
23
Q
U2-type introns (98% of human introns)
The 5’ splice site at the 5’ (left) end of the intron includes the consensus sequence
A
GU
24
Q
U2-type introns (98% of human introns)
The 3’ splice site at the 3’ (right) end of the intron includes the consensus sequence
A
AG
25
GU-AG
U2-type introns
98% of human introns
26
Exon-intron boundaries
27
U12-type introns (0.1% of human introns)
* the 5' splice site at the 5' end (left) end of the intron includes the consensus sequence **AU**
* the 3' splice site at the 3' (right) end of the intron includes the consensus sequence **AC**
•
28
Splice sites are read in
pairs
29
Splice sites are read in pairs
* splicing depends only on recognition of _pairs_ of splice sites
* all 5' splice sites are functionally equivalent, and all 3' splice sites are functionally equivalent
* splicing junctions are recognized only in the correct pairwise combinations
* need to known 5' splice site in same intron as 3' splice site
30
Pre-mRNA splicing proceeds through a
lariat
31
Pre-mRNA splicing proceeds through a lariat
* splicing requires the 5' and 3' splice sites and a branch site just upstream of the 3' splice site
* a **lariat** is formed when the intron is cleaved at the 5' (GU) splice site, and the 5' end is joined to a 2' position at the A at the **branch site** in the intron (covalent bond)
* 2 transesterification reactions - a reaction that breaks and makes chemical bonds in a coordinated transfer so that no energy is required
* the intron is released as a lariat when it is cleaved at the 3' (AG) splice site and the left and right exons are then ligated together
32
Pre-mRNA proceeds through a lariat
PICTURE
33
Pre-mRNA splicing proceeds through a lariat
2'
* 2'-hydroxyl on branching nucleotide initiates nucleophilic attack on 5' splice site
* 3'-hydroxyl generated at the 3' end of the exon during the first transesterification initiates a nucleophilic attack on the phosphodiester bond at the 3'-splice site
34
Lariat formation
2'
PICTURE
35
% compositioin
* only 25% of genome is exon+intron
* 1% exon
* 24% intron
* ~25% transcribed
36
... are required for splicing
snRNAs
37
snRNAs are required for splicing
* small cytoplasmic RNAs - scRNA, scyrps
* small nuclear RNA - snRNA, snurps
* small nucleolar RNA - snoRNA
38
Small cytoplasmic RA
| (scRNA, scyrps)
RNAs that are present in the cytoplasm
| (sometimes found in the nucleus also)
39
Small nuclear RNA
| (snRNA, snurps)
* one of many small RNA species confined to the nucleus
* several of them are invovled in splicing or other RNA processing reactions
* **snRNAs exist as ribonucleoprotein particles**
**(snRNA + several proteins) = snRNP or snurp**
• binds RNA not DNA because single-stranded --\> hyrbidize
40
Small nucleolar RNA
| (snoRNA)
a small nuclear RNA that is localized in the nucleolus
splice tRNAs, other rRNA
41
Each snRNA is present in its own
small riboucleoprotein particle
42
The 5 snRNPs involved in splicing are
* U1
* U2
* U5
* U4
* U6
43
Together with some additional proteins the snRNPs form
the spliceosome
44
Spliceosome
weight, snRNPs
* ~12 MDa
* 5 snRNPs account for almost half of the mass
* 141 proteins + 5 RNAs - sequentially
45
Splicing factor
a protein component of the spliceosome that is not part of one of the snRNPs
46
Spliceosome complex assembles
sequentially onthe pre-mRNA and passes through several "pre-splicing complexes" before forming the final,a ctive complex
47
All of the snRNPs except U6 contain
a conserved sequence that binds the **Sm proteins ** that are recognized by antibodies (**anti-SM**) generated in an autoimmune disease
* U4 and U6 are found together as a di-snRNP
* each snRNP is formed in a multistep process
48
Commitment of pre-mRNA to the splicing pathway
several non-snRNP proteins participate int he spliceosome pathways
* **splicing factor 1 (SF1)/branch point binding proten (BBP)** is particularly important
* another **U2AF (U2 snRNP auxilary factor)** binds to the polypyrimidine tract and the AG at the 3' splice site
* U1 snRNP initiaties splicing by binding to the 5' splice site by means of an RNA-RNA pairing reaction
* the commitment complex (**E complex**) contains U1 snRNP bound at the 4' splice site and the protein U2AF bound to a pyrimidine tract beteen branch site and the 3' splice site
* in cells of multicellular eukaryotes, **SR proteins (splicing regulatory proteins)** play an essential role in initiating the formation of the commitment complex
* pairing splice sites can be accomplished by **intron definition** or **exon definition**
49
Commitment of pre-mRNA to the splicing pathway
SF1, U2AF
PICTURE
50
Commitment of pre-mRNA to the splicing pathway
SR proteins
PICTURE
51
The spliceosome assembly pathway
* the commitment complex progressees to pre-spliceosome (the **A complex**) in the presence of ATP
* recruitment of U5 and U4/U6 snRNPs converts the A complex to the matrue spliceosome (the **B1 complex**)
* the B1 complex is next converted to the **B2 complex** in hich U1 snRNP is released to allow U6 snRNA to interact with the 5' splice site
* when U4 dissociates from U6 snRNP, U6 snRNA can pair with U2 snRNA to form the catalytic active stie
* both transesterification reactions take place in the activated spliceosome (the **C complex**)
* the splicing reaction is reversible at all steps
52
The spliceosome assembly pathway
steps
1. E complex - formation of commitment complex in which U1 is base paired with the 5' splice site
2. A complex - U2 addition to base pair with the branch site in the presence of ATP
3. B1 complex - joining of U4/6 and U5 tri-snRNPs
4. B2 complex - U1 and U4 replease, formation of the catalytic center in which U6 base pairs with the 5'splcie site - U6 also pbase pairs with U2 - u2 remains base paired with the branch site - U5 interacts with both exons through its loop
5. C1 complex - the first step of transesterification, 5' splice site cleaved, lariat formed
6. C2 complex - the second step of transesterification, 3' splice site cleaved, exons ligated
53
The spliceosome assembly pathway
PICTURE
54
Commitment
E --\> A
## Footnote
• lose BBP and U2AF splicing factor and recruit U2 snRNP
55
Splicing utilizes a series of
base-pairing reactions between snRNAs and splice sites on the pre-mRNA
• RNA-RNA interactions are crucial
56
Base-pairing reactions between snRNAs and splice sites on the pre-mRNA
PICTURE
57
Splice sites are read in pairs
* splicing depends only on recognition of pairs of splice sites
* all 5' splice sites are fuctionally equivalent and all 3' splice sites are functionally equivalent
* splicing junctions are recognized only in the otherwise correct pairwise combinations - how?
58
Intron and Exon definition
* SR proteins interact with one another as well as other proteins to form protein bridges that extend across the intron that is to be excised or across exons
* exons contain **exonic splicing enhancers (ESE)** that are binding sites for SR proteins
* when bound, SR proteins recruit U1 snRNPs to the downstream splice site and U2AF to teh pyrimidine tract and AG dinucleotide of the **upstream** 3'-splice site
* U2AF also recruits U2 snRNP to the branch point sequence - **cross-exon recognition**
59
SR
splicing regulator
60
An alternative spliceosome uses different snRNPs to process the minor class of introns
* an alternative splicing pathway uses another set of snRNPs that compise the U12 spliceosome
* works in a similar way to major intron splicing (GU-AG)
* the target introns are defined by longer consensus sequences at the splice junctions rather than strictly according to the GU-AG or AU-AC rules
* the major and minor spliceosomes share critical protein factors, including SR proteins
61
Autocatalytic introns
* found in organelles in bacteria - **group I and II introns**
* group II introns excise themselves from RNA by an autocatalytic splicing event (**autosplicing or self-splicing**)
* the splice junctions and mechanism of splicing of group II introns are similar to splicing of nuclear introns
* a group II intron folds into a secondary structure that generates a catalytic site resembling the structure of U6-U2-nuclear intron
(introns adopt structure to allow own splicing)
62
Splicing is temorally and functionally coupled with
multiple steps in gene expression
63
Splicing is temporally and functionally coupled with multiple steps in gene expression
* splicing can occur during or after transcription
* in general, splicing begins as a cotranscriptional process (when mRNA still being synthesized) and continues as a posttranscriptional proces
* the transcription and splicing machinery are physically and functionally integrated
* splicing is connected to mRNA export and stability control
64
Alternative splicing in multicellular eukaryotes
alternative splicing is a rule, rather than an exception, in multicellular eukaryotes
65
Alternative splicing is a rule rather than an exception in multicellular eukaryotes
* specific exons or exonic sequences may be excluded or included in the mRNA products by using **alternative splicing** sites
* alternative splicing contributes to structural and functional diversity of gene products
66
Alternative splicing in mutlicelluar eukaryotes
PICTURE
67
Alternative splicing of the CaMKIIδ gene
3 different alternative exons target the kinase to different cellular compartments
* way spliced determines where the protein kinase ends up
* 3 kinases from 1 gene at the level of splicing
68
CaMKIIδ splicing
PICTURE
69
Splicing can be regulated by
exonic and intronic splicing enhancers and silencers
70
Splicing can be regulated by exonic and intronic splicing enhancers and silencers
* alternative splicing is often associated with weak splice sites
* sequences surrounding alternative exons are often more evolutionarily conserved than sequences flanking constitutive exons
* specific exonic and intronic sequences can enhance or suppress splice site selection
71
The effect of splicing enhancers and silencers is mediated by sequence-specific RNA binding proteins, many of which may be developmentally regulated and/or expressed in a tissue-specific manner
* the Nova and Fox families of RNA binding proteins can promote or suppress site selection in a context dependent fashion
* the rate of transription can directly affect the outcome of alternative splicing
Nova and Fox families
* are RNA binding proteins
* accessory proteins decide if exon is included/excluded
* Nova binds intron upstream or exon itself = excluded
* binds downstream of intron = included
72
RNA is modified in the nucleus by additions to the 5' and **3' ends**
* poly(A) modification is intrinsically linked to transcriptional termination
* mRNA is exported
73
Transcription termination involves 3 steps
1. cleavage at the poly(A) site
2. addition of the poly(A) tail at the new 3' end
3. transcription termination downstream from the cleavage site
74
Transcription termination involves 3 steps
75
The 3' ends of mRNAs are generated by
• cleavage and polyadenylatioin
76
The 3' ends of mRNAs are generated by cleavage and polyadenylation
* the sequence AAUAAA is a signal for cleavage to generate a 3' end of mRNA that is polyadenylated
* the reaction requires a protein complex that contains a specificity factor, an endonuclease, and **poly(A) polymerase**
* the specificity factor and endonuclease cleave RNA downstream of AAUAAA
77
The 3' ends of mRNAs are generated by cleavage and polyadenylation
PICTURE
78
Machinery required for cleavage only
* CstF - binds to GU-rich sequence
* CFI and CFII - little is known about their function
* CTD - binds both CPSF and CSTF
* Ssu74 an PC4 - interact with CPSF and CstF
* PAB II - interacts with CPSF - stimulates rate of poly(A) addition and controls poly(A) tail length
79
CPSF
binds to polyA signal AAUAAA
80
PAP
adds the poly(A) tail
is recruited by CPSF
81
Symplekin
* part of a larger complex that includes CstF and CPSF
* it helps to assemble or stabilize the CstF complex and hold the entire cleavage/polyadenylation machinery together
* scaffold that holds it all together, without = no adenylation
82
Polyadenylation/cleavage machinery
PICTURE
83
The specificity factor and poly(A) polymerase add
~200 A residues processively to the 3' end
84
The poly(A) tail controls mRNA
stability
and influences translation
85
Cytoplasmic polyadenylation plays a role in
*Xenopus* embryonic development
86
Alternative poly(A) site selection
(picture)
a) in same exon
b) in different exons
* can generate different transcripts, different proteins, and diversifying protein functions
* 2 polyadenylations = can terminate transcription in 2 places
87
*calcitonin* and IgM pre-mRNAs undergo alternative splicing and polyadenylation
* different polyadenylation in different tissues
* not at level of splicing but polyadenylation
88
The 3' mRNA end processing is critical for temination of transcription
* there are various ways to end transcription by different RNA polymerases
* the mRNA 3' end formation signals termination of Pol II transcription
* the Elongation complex changes conformation upon recognizing the poly(A) site
* after cleavage a splicing factor (SF) recruits Xrn2 which digests downstream RNA until it reaches RNA pol II
* Pol II is released with help of a helicase
* elongating RNA pol II gets different conformation at polyadenylation sequence
* RNA pol II has antitermination factors that keep it attached to DNA after cleaving and loss of transcript
* nuclease Xrn2 recruited to RNA stuck on polymerase, helicase unravels RNA polymerase from DNA
89
Xrn2
PICTURE
90
The 3' end formation of histone mRNA requires U7 snRNA
* expression of histone mRNAs is replication dependent and regulated during the cell cycle
* histone mRNAs **are not** polyadenylated - their 3' ends are generated by a cleavage reaction that depends on a conserved hairpin structure in mRNA
* the cleavage reaction requires the SLBP to bind to a stem-loop structure and the U7 snRNA to pair with an adjacent single-stranded region
* the cleavage reaction is catalyzed by a factorr shared with the polyadenylation complex
91
Transcription by Pol I and Pol III uses
specific terminators to end transcription
92
transcription by Pol I and Pol III uses terminators to end transcription
* Pol I - 2 discrete termination sites are recognized by a DNA-binding protein (TTF1 in mouse) and cleavage mediated by the endonuclease Rnt1
* Pol III - defined terminator sequence in the DNA molecule (oligo dT) signals release of RNA polymerase
93
Transcription by Pol I and Pol III usees specific terminators to end transcription
Pol I
2 discrete termination sites are recognized by a DNA-binding protein (TTF1 in mouse) and cleavage mediated by the endonuclease Rnt1
94
Transcription by Pol I and Pol III uses specific terminators to end transcription
Pol III
defined terminator sequence in the DNA molecule (oligo dT) signals release of RNA polymerase
95
RNA pol II
protein
96
RNA pol I and III
tRNA
mRNA
snRNA
97
Transcription by Pol I and Pol III uses specific terminators to end transcription
PICTURE
98
mRNA splicing and export
are coupled processes
99
mRNA splicing and export are coupled processes
* **REF1 (RNA export factor 1)** - recruited by a splicing factor (UAP56) and targets mRNA
* **exon junction complex (EJC)** - a protein complex that assembles at exon-exon junctions during splicing and assists in RNA transport, localization, and degradation
* mRNA export factors dissociate after export. factors associated in **nonsense mediated decay (NMD)** remain bound
* splicing in the nucleus can incluence mRNA translatin in the cytoplasm
* **nonsense-mediated mRNA decay (NMD)** - a pathway that degrades an mRNA that has a nonsense mutation prior to the last exon
* EJC is usually tripped off by the ribosome during the first round of translation
* a premature stop codon means the ribosome dissociates and EJC remains which then recruits additional proteins such as Upf which recruits a decapping enzyme resulting in mRNA degradation
100
mRNA splicing and export are coupled processes
REF1
(RNA export factor 1)
recruited by splicing factor (UAP56)
targets mRNA
101
mRNA splicing and export are coupled processes
exon junction complex (EJC)
a protein complex that assembles at exon-exon junctions during splicing and assists in RNA transport, localization, and degradation
102
mRNA splicing and export are coupled processes
mRNA export factors dissocaite after export.
factors associated in ... remain bound
factors associated in **nonsense mediated decay (NMD)** remain bound
103
Nonsense-mediated mRNA decay (NMD)
a pathway that degrades an mRNA that has a nonsense mutation prior to the last exon
104
EJC is usually ripped off by the ribosome
during the first round of translation
105
A premature stop codon means
* the ribosome dissociates
* EJC remains which then recruits additional proteins such as Upf which recruits a decapping enzyme resulting in mRNA degradation
106
Upf
* recruited by EJC
* recruits a decapping enzyme
* resulting in mRNA degradation
107
EJC and Upf
PICTURE
108
tRNA splicing involves
cutting and rejoining in separate reactions
109
tRNA splicing involves cutting and rejoining in separate reactiosn
* tRNA splicing occurs by successive cleavage and ligation reactions
* RNA polymerase III terminates trancription in a poly(U)4 sequence embedded in a GC-rich sequence
* all tRNA introns include a sequence that is complementary to the anticodon of the tRNA
- this craetes an **alternative conformation** for the tRNA arm
**•** the intron in yeast tRNAPhe base pairs with the anticodon to change the structure of the anticodon arm
* an endonuclease cleaves the tRNA precursors at both ends of the intron
* release of the intron generates 2 half-tRNAs with unusual ends that contain 5' hydroxyl and 2'-3' cyclic phosphate and a **linear** intron
110
tRNA splicing involves cutting and rejoining in separate reactions
PICTURE
introns help form clover leaf structure of mature
111
tRNA splicing
SPLICING
112
tRNA splicing involves cutting and rejoining in separate reactions
5'
* the 5'-OH end is phosphorylated by a polynucleotide kinase
* the cyclic phosphate group is opened by phosphodiesterase to generate a 2'-phosphate terminus and 3'-OH group
* exon ends are joined by an **RNA ligase**
* the 2'-pshophate is removed by a phosphate
(2 single stranded RNAs, intron excision, form final stem loop, anticodon loop)
113
tRNA splicing
phosphorylation and ligation
PICTURE
