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CMCB Fall > #23 RNA Transcription and Processing > Flashcards

#23 RNA Transcription and Processing Flashcards

1
Q

Learning objectives

A
  1. compare and contrast the differences between bacterial transcription and euk transcription by RNA poly II

2 Describe 5’ caping, pre-mRNA splicing and 3’ end formation and how the CTD of Pol II mediates these steps that rpocess pre-mRNA into mature mRNA

  1. Describe the roels of teh 5 snRNPs and splicing factors in splice site slection and in the two phosphoryl transfer reactions
  2. Describe how alternate splicing creates gene diversity and causes disease
  3. Describe how the moleuclar basis by which alternative splicing caues one type of Spinal muscular atropy and one type of Duchenne Muscular dystrophy
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2
Q

What fraction of all diseases affect splicing?

A

1/5 of all sdisease alleles

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3
Q

What is spinal muscular atrophy?

A

Autosomal-recessive disease with progressive degeneration of alpha-motor neurons in the spinal cord, resulting in msucle atrophy and preature death

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4
Q

What makes bacterial transcripts different from Eukaryotic transcripts?

A
  1. 5’ and 3’ ends are unmodified
  2. Sometimes mRNA are polycistronic- one message contains several coding genes-
    Polycistronic genes often share a common regulation and function (enzymes that code for differet steps int eh synthesis of a metabolite
  3. other times mRNAs are monoscistronic–one message contains one coding sequence
  4. Transcription and translation can occur simulatneously, b/c both processes occur int eh bacterial cytoplasm (no nuclear membrane)
  5. Non-splicesosomal splciing (catalyzed by catalytic introns) does occur in bacteir but it is unusual
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5
Q

Describe Euakryotic transcripts

A

Mesages are transcribed as pre-mRNAs by Pol II and are processed to become mature mRNAs
1. Attaching a cap structure to 5’ end

  1. ading poly A tail to 3’ end of mRNA
  2. Excising introns to join exons in the coding region -through process caleld splicing and is directed by a alrge RNA protein comple xcalled splicesoome
  3. mRNAs are almost exclusively monocistronic
  4. Transcription occurs in the nucleus but translation occurs it the cytoplasm
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6
Q

What is the 5’ cap of mRNA (chemical name)

A

7-methylguanosine

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7
Q

How is 5’ it attached to RNA?

A

7-methylguanosin is attached to 5’ end of RNA in a head to head manner

5’ sugar position of both the 7-methylguanosine and the first nucleotide of the mRNA connect to opposite ends of triphospahte

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8
Q

what is the purpsoe of the cap?

A

helps initate mRNA translation and stabilizes this RNA by inhibiting degradation pathways

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9
Q

How does the 5’ Cap help distinguish its identigy of RNA?

A

b/c RNA Pol II is the only one wtih 5’ cap. Products form Pol I and POl III don’t have 5’ caps

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10
Q

What is Poly A Tail?

A

approxi 200 adenin nucleotides is added to 3’ en of most eukaryotic mRNA.

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11
Q

What are exceptions that don’t have poly A tail?

A

Histones and Interferons

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12
Q

Is Poly A tail transcribed?

A

No, it is synthesized by poly A polymeraise (PAP) in iteratieve and sequential fashion using ATP

There is no template

C

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13
Q

What lies between 5’ cap and 3’ poly A tail

A

exons and introns

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14
Q

What disease do mutations in Factor VIII cause?

A

leads to common bleedign disorder hemophilia A

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15
Q

Put in order of lenght of transcription ffrom shortest to longest (duchenne muscular dystrophy gene, apoliporotein b ,tRNA)

A

tRNA (no introns), apolipoprotein B (LDL), and dystorphin

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16
Q

What percent of dystrophen are introns?

A

99.4%

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17
Q

How many introns and exons does a typical gene have?

A

8 introns and 9 exons

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18
Q

What is the spliceosome made up of?

A

5 small nuclear RNA-protein complexes called snRNPs (snurps)

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19
Q

What does each snRNP contain?

A

one small nuclear RNA (snRNA) and many proteins

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20
Q

What are snRNPs named after?

A

snRNA components

U1, U2, U4, U5, U6

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21
Q

How does systemic lupuus erythematosus SLE affect snRNPs?

A

SLE is an often fatal inflammatory autoimmune disease

Patients develop antibodies against their own proetins, including snRNPs

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22
Q

What is the spliceosome?

A

directs eukaryotic pre-mRNA splicing

made of 5 small nulear RNA-protein complexes called snRNPs

Each snRNP contains one small nuclear RNA and many proteins

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23
Q

U3

A

U3 is essential for eukaryotic ribosome biogenesis and does not contribute to splicing

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24
Q

What are the two steps of pre-mRNA splicing?

A
  1. 2’OH of branch point A attacks 5’ splcie site to create a 2’-5’ lariat intermediate
  2. 3’ OH f 5’ exon attacks the 3’ splice site to join the 5’ and teh 3’ exons
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25
Q

Where does pre-mRNA splicing occur?

A

complex assembly of 5 RNAs and about 200 proteins called teh spliceosome

5

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26
Q

Desribe first reaction of pre-mRNA reaction

A

involves nucelophilic attack of 2’OH orup of branch point adenosine in teh intron on teh 5’ splice site

This reaction epxoses free 3 end of the 5 exon and circular RNA structure caleld a lariat

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27
Q

Are sequenceing at and flanking the 5’ splice site, the branch point and the 3’ splice site are conserved

A

yep

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28
Q

There are 4 nucleotides that are invariant

A
  1. GU at the 5’ end of the intron and the

AG at the 3’ end of the intron

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29
Q

What funciton is sequence conservations

A

helps RNAs in teh U1, U2 and U4 snRNPs recgonize the 5’ splci site, the brach point and the 3 splice site

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30
Q

What kind of interactions are made in teh splicing reaction>

A

RNA-RNA and/or RNA-rotein rearrangments

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31
Q

Does splicing require ATP?

A

Yes, it reaquires ATP hyrdolysis

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32
Q

What does U1 snRNP do (1st step of spliceosome splicig)

A

U1 snRNP is improtant for selction of 5’ splice site

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33
Q

2nd step of splicesosome

A
  1. Branch point binding protein (BBP) and U2 Auxiliary factor (U2AF) recruit U2 snRNP and are simultaneously displaced
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34
Q

3rd step of splicesosome

A
  1. U4, U5 and U6 complex are recruited and cause dissociation of U1
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35
Q

4th step of spliceseosome

A
  1. U4, which is base-paired to U6 and regulates it, dissociates from the U4-6 complex.
36
Q

5th step of splicesoeoe

A
  1. U6 base pairs with U2 (and brings 5 along with it).
37
Q

6th step

A
  1. U5 stabilizes the movement of the splice site so that the 5’ and 3’ exon are next to each other (juxtaposed).
38
Q

7th step

A
  1. Second splicing step occurs and ligated exons are released with snRNPs attached.
39
Q

All splicesosome steps

A
  1. U1 attaches to 5’ splice site, selecting it.
  2. Branch point binding protein (BBP) and U2 Auxiliary factor (U2AF) recruit U2 snRNP and are simultaneously displaced
  3. U4, U5 and U6 complex are recruited and cause dissociation of U1
  4. U4, which is base-paired to U6 and regulates it, dissociates from the U4-6 complex.
  5. U6 base pairs with U2 (and brings 5 along with it).
  6. U5 stabilizes the movement of the splice site so that the 5’ and 3’ exon are next to each other (juxtaposed).
  7. Second splicing step occurs and ligated exons are released with snRNPs attached.
40
Q

What is U2 snRNP function

A

selection of branch point

41
Q

What happens when U4/U5/U6 is recruited?

A

U1 snRNP dissociates

42
Q

function of U4 snRNP

A

regulates the action of U6 snRNP: the U4:U6 base pairs ensures that the U6 snRNA is in a catalytically inactive state

43
Q

What is fucniton of U2 and U6 snRNPs

A

contribute to catalysis in both the first and second steps of splicing

44
Q

What does U5 snRNP?

A

helps juxtapose the 3’ exon next to the 5’ exon

45
Q

What are ribozymes?

A

Catalyitic RNAs.

Bacteriana d organelles of eurkaryotes have some pre-RNA that self splice

either group I introns or group II introns

46
Q

What catalyzes teh reaction so ribozyme splicing?

A

The introns themselves

47
Q

Describe the mechanism of Group I Intorns (catalytic intron)

A

use the 3’-OH of G, GMP, GDP, or GTP to attack the 3’ splice site, and then that liberated end (on the 5’ side) attacks the 3’ splice site to join the two exons and liberate the intron.

48
Q

Describe the mehcnaims of Group II self-splicing intron sequences

A

branch point adenosine 2’-OH attacks teh 5’ splice site

the liberated 3’OH of the 5’ exon then attacks the 3’ splice site to join the 5’ and 3’ exons and simultaneously liberate teh lariat RNA

49
Q

What does Pol II do?

What does it phosphorylate?

A

liek an RNA factory, b/c it nt only transcribes teh pre-mRNA, it also, via phorphorylation of its C-terminal domain (CTD), recruiets the enzymes needed for
5’ capping,

3’ adenylation,

50
Q

Why do you need to phosphorylate C-terminal domain (CTD)

A

to recruit roteins needed for 5’ capping, 3’ polyadenylation, and splicing

51
Q

What is CTD made of?

A

heptad repeats with two serines in positions 2 and 5

52
Q

What do changes in phosphoryation of CTD provide?

A

a code that recruites either 5’ capping, splicing, or polyadenylation machinery during transcription

53
Q

What happens when there is Phosphorylation at Ser 5? what phosphorylats it?

A

This is the first phosphoryaltion at signal phosphorylation at Ser5, recruites capping factors, which in turn attaches a cap on the 5’ termini of the growing transcript. Ser5 is phosphorylated by kinases associated with elongating Pol II

54
Q

WHat happens when Ser2 is phosphorylated?

A

the 2nd signal phosphorylation at Ser2 and Ser5, recruits splicing factors

As a resutl, splciing occurs duing transcritpion

55
Q

Wen ahppens when Ser 5 is dephosphoryalted and there is phosphorylation at Ser 2 only>

A

recruits 3’ end processing enzymes, which in turn polyadenylates te 3’ end of transcrip (adds a 3’ poly (A) tail)

56
Q

What is first signal from Pol II on transcription?

A

Signal to recruit 5’ Cap formation

57
Q

What is 2nd signal of Pol II (when BOTH ser 2 and ser 5 are phosphorylated)

A

to recruit splicing factors

58
Q

What is 3rd signal when ONLY Ser2 is phosphorylated?

A

recruit Poly A Tail formation

59
Q

What does phosphorylated CTD (phosphate at Ser2 not at Ser5 ) attract?

A

cleavage stimulation factor (CstF) and a cleavage and Polyadenylation specificyt factor (CPSF)

60
Q

What is a Cstf

A

Cleavage stimuation factor

61
Q

What is a CPSF

A

cleavage Polyadenylation specificy factor

62
Q

what so Cstf and CPSF bind to>

A

polyadenylation sigal sequence AAUAAA once it emerges from POl II

63
Q

What is the result of CstF and CPSF binding to AAUAAA?

A

additiaonl factors are recruited and RNA is cleaved 10-30 nucleotdies downstream

64
Q

waht happens ahver RNA is cleaved>

A

Poly A Polymerase (PAP) is recruited to add abaout 200 nucleotides to the 3’ termini

Growing poly A tail in turn recruites poly A bidning protien (PABP)

evntually Pol II transcription terminates

65
Q

Any transcription taht occurs after cleavage is rapidly degraded bc this RNA transcirp lacks what two important protective features

A

5’ cap and poly A tail

66
Q

What is the signifance of AAUAA

A

CTD, CstF and CPSF cleave site 10-30 nts downstream

67
Q

What is PABP

A

Poly A Binding Protein

Pol II transcription terminates

68
Q

wha thappens to any trancription that occurs after claveage?

A

it is rapidly degraded b/c this RNA transcrip lacks two important protective feaure: a 5’ cap and poly A tail

69
Q

Once PABP coats the entire 3’ poly (A) tail,

A

CPSF protein dissociates form teh polyadenylation signal sequence AAUAAAA

70
Q

What are the different kinds of alternate splicing?

A
  1. Intron Retention
  2. Exon Skipping
  3. Alternate 3’ splice site selection
  4. Alternate 5’ splice site selection
71
Q

What % of all human genes are spliced in more than one way ?

A

75% of all human genes

72
Q

What is Spinal Muscle Atrophy (SMA)

A

degenerative motor neruon disease

auto recessive

l

73
Q

What causes SMA

A
  1. homozygous mutation of Survival of motor nuron gene SMN1- distrupting its esxpression
74
Q

What are SMN proteins>

A

essential b/c they are important in biogeneiss of snRNPs which are responsible for splicing

75
Q

Diff between SMN1 and SMN2

A

SMN10 functinoal protein

SMN2- C–>T SMN2 mutations,

silent in respect to translation, but causes skipping of exon 7 b.c it replaces exonic splicing enhancer (ESE) with an exonic splicing silencer (ESS)

76
Q

What is an example of an ESE (exonic splicingn enchancer)

A

splicing activaors, such as those rih in serine and arginine (SR proteins) ind to ESE elements to promote splicing

77
Q

example of splciing repressors

A

heterongoenous nuclear ribonuclear rteins (hnRNPs) bind to ESSs to silence splicing

78
Q

Waht is the mutation in SMN2

A

freakquen skipping of exon 7 ; has a C–>T mutation

ESE (exonic splicing enhancer) to ESS (exonic splicing silencer)

79
Q

What i the serverity of this disease correlation with protein

A

Severity of SMA inversely correlates with amt of funcitonaing SMn protein (less protein results in more severe form of disease)

80
Q

what is DMD (Duchenne’s muscular dystropy?

A

65% genomi mutaiton while a number of them are aberrant splicing

81
Q

Example of DMD mutation

A

mutation of T to A in exon 31’ caues mild form of disease ==> creates mild termform;

Premature termination codon (PTC) and introduces an exon splicing slencer that inds to hnRNP A1, resulting in partial skillping of exon 31

82
Q

What needs to be done for large moleucles to corss nuclear pore?

A

receptors are needed to gain passage through this pore complex

Some proteins associated with teh mature message are shed before trnasprot, other ind upon entry

83
Q

how is the mature message circularized

A

by bridging interactison fo eIF4G

84
Q

Wha does eIF4G bind to?

A

poly A binding protein (PABP) annd eIF4E

85
Q

What is the Balbiani Ring?

A

abundant and large mRNA

CMCB Fall (17 decks)

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