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

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2
Q

RNA splicing

A

the process of excising the introns from RNA and connecting the exons into an continuous mRNA

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3
Q

RNA is modified

A

in the nucleus by

  • additions to the 5’ and 4’ ends
  • by splicing to remove the introns
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4
Q

RNA splicing and modification

A
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5
Q

The 5 end of eukaryotic mRNA is

A

capped

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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
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7
Q

5’ cap

A

7-methylguanosine

  • add CH3 at C7 (methylated guanine)
  • 5’ to 5’ phosphotriester linkage
  • protects unstable RNA (stops degradation)
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8
Q

The 5’ capping process takes place

A

during trancription

• may be important for release from pausing of transcription

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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
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10
Q

3 forms of capping

A
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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
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12
Q

Capping enzyme

A

RNA triphosphatase and guanylytransferase activities are present in the same protein

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13
Q

Addition of the 5’ cap

PICTURE

A
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14
Q

Addition of the 5’ cap

basic steps

A
  1. remove 1 P (from the 3 at the 5’ end)
  2. transfers guanosne to 5’ of RNA, 5’-5’ phosphodiester link

(^both in capping enzyme)

  1. add methyl to guanosine
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15
Q

Capping enzyme is recruited by

A

the CTD of RNA polymerase II

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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
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17
Q

Capping enzyme is recruited by the CTD of RNA polymerase II

PICTURE

A
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18
Q

RNA splicing occurs

A

during and/or after transcription

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19
Q

Splicing makes

A

transcript diversity

diversity in protein function in splicing the transcript

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20
Q

Nuclear splice sites are

A

short sequences

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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)
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22
Q

Splice sites

A

sequences immediately surrounding the exon-intron boundaries

sequence motifs, consensus in intron that allows splicing

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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

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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

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25
Q

GU-AG

A

U2-type introns

98% of human introns

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26
Q

Exon-intron boundaries

A
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27
Q

U12-type introns (0.1% of human introns)

A
  • 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

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28
Q

Splice sites are read in

A

pairs

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29
Q

Splice sites are read in pairs

A
  • 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
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30
Q

Pre-mRNA splicing proceeds through a

A

lariat

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31
Q

Pre-mRNA splicing proceeds through a lariat

A
  • 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
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32
Q

Pre-mRNA proceeds through a lariat

PICTURE

A
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33
Q

Pre-mRNA splicing proceeds through a lariat

2’

A
  • 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
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34
Q

Lariat formation

2’

PICTURE

A
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35
Q

% compositioin

A
  • only 25% of genome is exon+intron
  • 1% exon
  • 24% intron
  • ~25% transcribed
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36
Q

… are required for splicing

A

snRNAs

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37
Q

snRNAs are required for splicing

A
  • small cytoplasmic RNAs - scRNA, scyrps
  • small nuclear RNA - snRNA, snurps
  • small nucleolar RNA - snoRNA
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38
Q

Small cytoplasmic RA

(scRNA, scyrps)

A

RNAs that are present in the cytoplasm

(sometimes found in the nucleus also)

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39
Q

Small nuclear RNA

(snRNA, snurps)

A
  • 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

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40
Q

Small nucleolar RNA

(snoRNA)

A

a small nuclear RNA that is localized in the nucleolus

splice tRNAs, other rRNA

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41
Q

Each snRNA is present in its own

A

small riboucleoprotein particle

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42
Q

The 5 snRNPs involved in splicing are

A
  • U1
  • U2
  • U5
  • U4
  • U6
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43
Q

Together with some additional proteins the snRNPs form

A

the spliceosome

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44
Q

Spliceosome

weight, snRNPs

A
  • ~12 MDa
  • 5 snRNPs account for almost half of the mass
  • 141 proteins + 5 RNAs - sequentially
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45
Q

Splicing factor

A

a protein component of the spliceosome that is not part of one of the snRNPs

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46
Q

Spliceosome complex assembles

A

sequentially onthe pre-mRNA and passes through several “pre-splicing complexes” before forming the final,a ctive complex

47
Q

All of the snRNPs except U6 contain

A

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
Q

Commitment of pre-mRNA to the splicing pathway

A

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
Q

Commitment of pre-mRNA to the splicing pathway

SF1, U2AF

PICTURE

A
50
Q

Commitment of pre-mRNA to the splicing pathway

SR proteins

PICTURE

A
51
Q

The spliceosome assembly pathway

A
  • 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
Q

The spliceosome assembly pathway

steps

A
  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
Q

The spliceosome assembly pathway

PICTURE

A
54
Q

Commitment

A

E –> A

• lose BBP and U2AF splicing factor and recruit U2 snRNP

55
Q

Splicing utilizes a series of

A

base-pairing reactions between snRNAs and splice sites on the pre-mRNA

• RNA-RNA interactions are crucial

56
Q

Base-pairing reactions between snRNAs and splice sites on the pre-mRNA

PICTURE

A
57
Q

Splice sites are read in pairs

A
  • 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
Q

Intron and Exon definition

A
  • 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
Q

SR

A

splicing regulator

60
Q

An alternative spliceosome uses different snRNPs to process the minor class of introns

A
  • 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
Q

Autocatalytic introns

A
  • 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
Q

Splicing is temorally and functionally coupled with

A

multiple steps in gene expression

63
Q

Splicing is temporally and functionally coupled with multiple steps in gene expression

A
  • 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
Q

Alternative splicing in multicellular eukaryotes

A

alternative splicing is a rule, rather than an exception, in multicellular eukaryotes

65
Q

Alternative splicing is a rule rather than an exception in multicellular eukaryotes

A
  • 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
Q

Alternative splicing in mutlicelluar eukaryotes

PICTURE

A
67
Q

Alternative splicing of the CaMKIIδ gene

A

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
Q

CaMKIIδ splicing

PICTURE

A
69
Q

Splicing can be regulated by

A

exonic and intronic splicing enhancers and silencers

70
Q

Splicing can be regulated by exonic and intronic splicing enhancers and silencers

A
  • 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
Q

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

A
  • 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
Q

RNA is modified in the nucleus by additions to the 5’ and 3’ ends

A
  • poly(A) modification is intrinsically linked to transcriptional termination
  • mRNA is exported
73
Q

Transcription termination involves 3 steps

A
  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
Q

Transcription termination involves 3 steps

A
75
Q

The 3’ ends of mRNAs are generated by

A

• cleavage and polyadenylatioin

76
Q

The 3’ ends of mRNAs are generated by cleavage and polyadenylation

A
  • 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
Q

The 3’ ends of mRNAs are generated by cleavage and polyadenylation

PICTURE

A
78
Q

Machinery required for cleavage only

A
  • 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
Q

CPSF

A

binds to polyA signal AAUAAA

80
Q

PAP

A

adds the poly(A) tail

is recruited by CPSF

81
Q

Symplekin

A
  • 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
Q

Polyadenylation/cleavage machinery

PICTURE

A
83
Q

The specificity factor and poly(A) polymerase add

A

~200 A residues processively to the 3’ end

84
Q

The poly(A) tail controls mRNA

A

stability

and influences translation

85
Q

Cytoplasmic polyadenylation plays a role in

A

Xenopus embryonic development

86
Q

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
A
87
Q

calcitonin and IgM pre-mRNAs undergo alternative splicing and polyadenylation

A
  • different polyadenylation in different tissues
  • not at level of splicing but polyadenylation
88
Q

The 3’ mRNA end processing is critical for temination of transcription

A
  • 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
Q

Xrn2

PICTURE

A
90
Q

The 3’ end formation of histone mRNA requires U7 snRNA

A
  • 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
Q

Transcription by Pol I and Pol III uses

A

specific terminators to end transcription

92
Q

transcription by Pol I and Pol III uses terminators to end transcription

A
  • 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
Q

Transcription by Pol I and Pol III usees specific terminators to end transcription

Pol I

A

2 discrete termination sites are recognized by a DNA-binding protein (TTF1 in mouse) and cleavage mediated by the endonuclease Rnt1

94
Q

Transcription by Pol I and Pol III uses specific terminators to end transcription

Pol III

A

defined terminator sequence in the DNA molecule (oligo dT) signals release of RNA polymerase

95
Q

RNA pol II

A

protein

96
Q

RNA pol I and III

A

tRNA

mRNA

snRNA

97
Q

Transcription by Pol I and Pol III uses specific terminators to end transcription

PICTURE

A
98
Q

mRNA splicing and export

A

are coupled processes

99
Q

mRNA splicing and export are coupled processes

A
  • 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
Q

mRNA splicing and export are coupled processes

REF1

(RNA export factor 1)

A

recruited by splicing factor (UAP56)

targets mRNA

101
Q

mRNA splicing and export are coupled processes

exon junction complex (EJC)

A

a protein complex that assembles at exon-exon junctions during splicing and assists in RNA transport, localization, and degradation

102
Q

mRNA splicing and export are coupled processes

mRNA export factors dissocaite after export.

factors associated in … remain bound

A

factors associated in nonsense mediated decay (NMD) remain bound

103
Q

Nonsense-mediated mRNA decay (NMD)

A

a pathway that degrades an mRNA that has a nonsense mutation prior to the last exon

104
Q

EJC is usually ripped off by the ribosome

A

during the first round of translation

105
Q

A premature stop codon means

A
  • the ribosome dissociates
  • EJC remains which then recruits additional proteins such as Upf which recruits a decapping enzyme resulting in mRNA degradation
106
Q

Upf

A
  • recruited by EJC
  • recruits a decapping enzyme
  • resulting in mRNA degradation
107
Q

EJC and Upf

PICTURE

A
108
Q

tRNA splicing involves

A

cutting and rejoining in separate reactions

109
Q

tRNA splicing involves cutting and rejoining in separate reactiosn

A
  • 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
Q

tRNA splicing involves cutting and rejoining in separate reactions

PICTURE

introns help form clover leaf structure of mature

A
111
Q

tRNA splicing

SPLICING

A
112
Q

tRNA splicing involves cutting and rejoining in separate reactions

5’

A
  • 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
Q

tRNA splicing

phosphorylation and ligation

PICTURE

A