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MOLGEN > Chapter 19 - RNA splicing and Processing > Flashcards

Chapter 19 - RNA splicing and Processing Flashcards

1
Q

There is very little mRNA processing in prokaryotes, so the primary transcript is considered a

A

mature mRNA

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2
Q

The primary transcript of a eukaryotic gene has the same organization as the gene and is called a

A

pre-mRNA

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3
Q

A eukaryotic pre-mRNA is usually … before export as a mature mRNA

A

capped, poly-A tailed, and spliced

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4
Q

Enzymes for capping, tailing, and splicing are coupled to

A

the transcriptional apparatus

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5
Q

The capping enzymes help the transcriptional apparatus

A

clear the promoter

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6
Q

TFIIH phosphorylates CTD Ser5 residues during

A

transition to elongation

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7
Q

Capping enzymes binds to

A

Ser5-P CTD

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8
Q

Elongating RNAP II associates with enzymes that phosphorylate

A

Ser2

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9
Q

Some splicing and tailing enzymes bind the

A

Ser2/5-P CTD

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10
Q

The first nucleotide in an RNA transcript is usually a

A

purine nucleotide triphosphate

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11
Q

what is the mammalian enzyme responsible for adding the cap

A

guanylyl-transferase

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12
Q

what enzyme then adds a single methyl group at the 7’ position of the terminal guanine

A

guanine - 7 - methyltransferase

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13
Q

RNAP II pauses 30 nt downstream of initiation site and

A

waits for capping enzymes to act

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14
Q

Uncapped nascent RNA is vulnerable to attack by

A

exonucleases

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15
Q

Cap is also necessary for

A

initiation of splicing and cytoplasmic export

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16
Q

Most splice sites contain

A

short consensus sequences

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17
Q

major introns are also known as

A

U2-type
GU-AG introns

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18
Q

minor introns are also known as

A

U12-type introns

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19
Q

Differences in the 5’ and 3’ splice site consensus sequences gives the splice sites

A

directionality

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20
Q

Splice sites are recognized in a

A

pairwise manner

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21
Q

splice sites are

A

generic

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22
Q

the splice site apparatus is … in every tissue

A

the same

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23
Q

splicing is temporarily coupled to

A

transcription

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24
Q

branch point

A

Not well conserved in multicellular eukaryotes
Preferences for purines and pyrimidines at each position
Highly conserved target adenine nucleotide
Lies 18-40 nt upstream of the 3’ splice site
Identifies the nearest 3’ splice site for interactions with a 5’ splice site

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25
Q

The first step in the splicing reaction is a

A

nucleophilic attack by the 2’-OH of the adenine in the branch point on the 5’ splice site

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26
Q

Free 3’-OH from end of preceding exon attacks the

A

phosphodiester bond at the 3’ splice site

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27
Q

Transesterifies to form

A

5’ to 3’ bond between first and second exon

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28
Q

small cytoplasmic RNAs

A

tRNA, small rRNA, miRNA

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29
Q

small nuclear RNAs

A

snRNAs

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30
Q

small nucleolar RNAs

A

snoRNAs

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31
Q

snRNA

A

important component of the spliceosome

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32
Q

snoRNA

A

involved in processing of rRNA

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33
Q

spliceosome

A

A large complex of proteins and small RNAs that comprise the splicing apparatus

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34
Q

splicesome contents

A

Contains 5 snRNAs and 41 snRNA associated proteins
Spliceosome also contains 70 proteins called splicing factors
Also contains 30 other proteins that act as an interface to other steps of gene expression

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35
Q

splicing factors

A

Associated with spliceosome assembly, RNA transcript binding, and RNA active site assembly

36
Q

The spliceosome forms on the pre-RNA via several presplicing complexes of

A

snRNPs
small ribonucleoproteins
snRNA and proteins

37
Q

Several splicing reactions require the RNAs of the snRNPs to base pair with

A

the RNA transcript being spliced

38
Q

Spliceosome is released immediately following

A

final ligation of the exons

39
Q

spliceosome must … at each intron

A

reassemble

40
Q

Recognition of the splicing consensus sequences requires both

A

RNA and proteins

41
Q

The first step in splicing is binding of

A

the U1 snRNP to the 5’ splice site

42
Q

Binding of U1 to the RNA is stabilized by two proteins

A

Multimeric U2AF binds to a polypyrimidine tract located between the branch point and 3’ splice site
Branch point binding protein (BBP/SF1) interacts with the branch point

43
Q

E complex
commitment complex

A

U1+BBP/SF1+U2AF

44
Q

SR proteins

A

Family of splicing factors that contain RNA- and protein-binding motifs
Function in spliceosome assembly and as splicing initiators in multicellular eukaryotes
Play a key role in maintaining splicing accuracy in multicellular eukaryotes where splice site consensus is weak

45
Q

intron splice site recognition

A

5’ and 3’ splice sites are simultaneously recognized by components of E complex
Sequential deposit of U1 and then U2AF as nascent mRNA emerges from RNAP II
Used for splicing of small, single-intron genes in unicellular eukaryotes

46
Q

exon splice site recognition

A

Takes advantage of presence of small exons of a consistent size
Introns are long and variable in multicellular eukaryotes
Many sequences in introns resemble true splice sites
The paired recognition of splice sites flanking an intron is generally quite inefficient
U2AF binds to the 3’ splice site
U1 binds to the 5’ splice site at the beginning of the next intron
Bridges the exon
Sequential deposit of U2AF and then U1 as nascent mRNA emerges from RNAP II
Complexes are switched to link across the introns

47
Q

spliceosome assembly

A
  1. U2 snRNP binds to the branch point
  2. A tri-snRNP complex composed of U5 and U4/U6 binds the A complex to form the B1 complex
  3. U1 and U4 are released resulting in formation of the B2 complex
  4. Several RNA rearrangements occur in the B2 complex to form the C1 complex
  5. The U5 snRNP positions U2 and U6 in the C2 complex for the second transesterification reaction between the flanking exons
  6. The snRNPs remain attached to the lariat, but are quickly released as the lariat dissociates and is degraded
48
Q

U2 snRNP binds to the branch point

A

Facilitated by base pairing between U2 snRNA and branch point consensus
Displaces BBP/SF1 and U2AF
Requires ATP hydrolysis
Forms the A complex

49
Q

B1 complex

A

First complex considered a true spliceosome because it contains all components needed for splicing

50
Q

Upon release of U1 and U4 …

A

U6 pairs more extensively with U2
U4 sequesters the U6 snRNA until it is needed
U2 is already paired with the branch point
U6 now pairs with intronic sequence downstream of the 5’ splice site
U2-U6 pairing brings 5’ splice site in close contact with branch point
Assisted by interactions between U5 and upstream exon

51
Q

The first transesterification reaction between the 5’ splice site and branch point to form the lariat occurs in

A

the C1 complex

52
Q

Splicing seems very inefficient because it requires, per splicing event

A

100+ proteins
Five snRNA molecules
Hydrolysis of eight ATP molecules
Reassembly of the entire active site

53
Q

Inefficiency may be a consequence of

A

overexpansion of ancient self-splicing mechanisms

54
Q

Some aspects of complexity are actually needed such as

A

ATP hydrolysis

55
Q

ATP hydrolysis reactions are used to break specific RNA-RNA base pairs

A

Breaking of specific base pairs is required to make others that are specifically required for the sequential assembly of the spliceosome
If the initial correct base pairs do not form, then ATP hydrolysis will not occur, and spliceosome assembly will not proceed

56
Q

ATP hydrolysis is also used for kinetic proofreading

A

Correct base pairing is stronger than incorrect pairing
Incorrect pairing will dissociate more quickly than correct
ATP-mediated rearrangements that result in incorrect pairing will be less stable and are less likely to incorrectly proceed to the next stage of assembly than rearrangements that are correct

57
Q

ATP hydrolysis is used for

A

kinetic proofreading and to break specific RNA-RNA base pairs

58
Q

exon-junction complex

A

EJC
Deposited onto each exon-exon junction following splicing
Directly recruits RNA-binding proteins associated with nuclear export

59
Q

nonsense-mediated decay

A

The EJC is also involved in proofreading of mutant mRNA transcripts in the cytoplasm
EJCs are usually displaced from the mRNA by the ribosome during the first translation event
If a nonsense mutation has occurred, the ribosome will not remove the EJCs
EJCs will recruit decapping enzymes that result in degradation of the mRNA

60
Q

self spliced introns

A

group 1 and group 2
Require no external proteins or nucleotides to catalyze splicing in vitro
Proteins are required for folding in vivo

61
Q

group I and Group II introns

A

Found in fungal and plant organelles, some bacteria, and the nucleus of some simple eukaryotes

62
Q

Group II introns are excised via the same mechanism as

A

nuclear pre-mRNAs

63
Q

RNA world hypothesis evidence

A

Existence of self-splicing RNAs and ribozymes
Ribose can be spontaneously produced from formaldehyde in abiotic conditions
Deoxyribose is not readily produced in this manner
Deoxyribose is produced from ribose in the cell using a protein enzyme, which suggests a later origin

64
Q

why did we move away from RNA world

A

Proteins have a greater range of potential enzymatic reactions
Long double stranded DNA is a more stable hereditary material
Also more readily repaired

65
Q

Over 90% of mammalian genes are

A

alternatively spliced

66
Q

Effects of alternative splicing

A

Omit or include some coding sequences
Create alternative reading frames

67
Q

Alternative splicing is often associated with

A

weak splice sites that are easily modulated

68
Q

Specific exonic and intronic sequences can enhance or suppress splice site selection via interactions with

A

trans-acting alternative splicing regulators

69
Q

The effect of splicing enhancers and silencers are mediated by

A

sequence-specific RNA binding proteins

70
Q

The rate of transcription can also directly affect the outcome of

A

alternative splicing

71
Q

Functions of Poly(A) tail

A

Protect from 3’ to 5’ exonucleases
Facilitates nuclear export
Cap stability

72
Q

Tail addition

A

RNAP II does not terminate at specific sites
Sequences in the mRNA are recognized as targets for an endonucleolytic cut
Poly(A) tail is added at the 3’ end of the cut site
RNAP II continues after the cleavage
5’ end liberated after cleavage signals transcription termination

73
Q

The cleavage/polyadenylation site is usually flanked by

A

conserved sequences

74
Q

Cleavage stimulatory factor (CstF)

A

recognizes the upstream AAUAAA

75
Q

Cleavage and polyadenylation specific factor (CPSF)

A

recognizes the downstream U/GU-rich element

76
Q

CPSF and CstF

A

will recruit other protein factors that cleave the RNA and produce a 3’ end

77
Q

Poly(A) polymerase (PAP)

A

binds the free 3’ end of the RNA

78
Q

PAP activity

A

PAP has specific activity at the AAUAAA site when combined with other tailing factors
PAP first adds a short oligo(A) sequence at the 3’ end
Activity dependent on CPSF and CstF

79
Q

Nuclear poly(A) binding protein (PABP)

A

binds the poly(A) tail
Upon binding of PABP, PAP extends the poly(A) tail to its full length of approximately 200 nt
A cytoplasmic form of PABP participate in translation

80
Q

The final length of the tail is determined by

A

a feedback mechanism between the cooperative binding of PABPs and PAP

81
Q

RNAP II continues transcription for hundreds of nucleotides after

A

RNA is cleaved

82
Q

two factors lead to RNAP II termination

A
  1. allosteric changes
  2. Exonuclease torpedo
83
Q

allosteric changes

A

Binding of cleavage factors and subsequent RNA cleavage leads to a conformational change in RNAP II
Conformational change makes the enzyme less processive and more likely to dissociate from the DNA

84
Q

exonuclease torpedo

A

RNA cleavage produces an uncapped 5’ RNA end which is eventually bound by a 5’3’ exonuclease
Exonuclease is carried on RNAP II?
The exonuclease degrades the RNA 5’3’
When the exonuclease reaches RNAP II it destroy the RNA-DNA hybrid
RNAP II dissociates

85
Q

RNAP III termination

A

looks for a discrete poly-T sequence in the template strand

86
Q

RNAP I termination requires

A

Accessory terminator proteins that recognize one of two terminator sequences
Cleavage of the nascent RNA

MOLGEN (28 decks)

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