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Biochem 385 Unit 3 > Lecture 33: RNA Processessing > Flashcards

Lecture 33: RNA Processessing Flashcards

1
Q

spliced functional RNA products

A

generated after cleavage

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2
Q

Processing of mRNA transcripts

A

5’ capping (protect from degredation)
intron splicing (take out non coding regions)
polyadenylation (protect from degredation on 3’ end)
(also cleavage)

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3
Q

how can multiple protein be derived from 1 gene

A

alternative intron splcing

use of differential polyadenylation sites

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4
Q

Micro RNAs

A 
short double stranded RNA molecs
interferring RNA molecs (RNAi)
target mRNA degredation or to interfere w/ translation
to decrease expression 
important for gene regulation
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5
Q

where does RNA processing happen?

A

in nucleus
after transcription
BEFORE exportation of mRNA to cytoplasm for translation

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6
Q

what is nascent RNA

A

pre RNA

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7
Q

pre rna has…

A 
both exons (coding) and introns(non-coding)
need cleavage and religation to get mature RNA product
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8
Q

rRNA and tRNA modifcations

A

modification at ribonucleotide bases
to produce a variety of methylated products
structural roles
modified bases required for function

unlike mRNA!!! its just a sequence, a message to go to a protein

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9
Q

S

A

spedbergs
sedimentation properties
the larger the number, the more sedimentary it will be when ceterfuged

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10
Q

rRNA processing

A

in nucleolus

make mature 60S (large subunit) and 40S (small subunit) ribosomal complexes before exportation to cytoplasm

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11
Q

modification of rRNA and tRNA

A

bases modified at specific sites BEFORE processing
produces variety of methylated products

needed for function

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12
Q

45S rRNA contains…

A

28S, 18S, and 5.8S genes

portions that eventually encode the mature rRNA
rRNA modfication happens in nucleolus

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13
Q

tRNA processing

A

remove intron and add 3’CCA sequence
remove 5’ and 3’ sequences and an intron near anticodon sequence
base modification

adopts cloverleaf structure
3D: L structure
all have basically same shape

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14
Q

to get amino acid attachment by a tRNA synthase enzyme we need…

A

prescence of specific modifed bases
anticodon sequence
addition of CCA to 3’

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15
Q

review figures on slide 6

A

review figures on slide 6

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16
Q

processing of mRNA transcript

A

5’ gets gaunine cap
introns removed
add 80-250 adenine residues to 3’ end

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17
Q

Ovalbumin gene: mRNA processessing

A

77000 basepairs
one of first genes studied for processing
primary transcript is huge, extra RNA at 3’ end
add cap, splice, cleave, polyadenylate
end up w/ mature RNA of 1800 nucleotides long

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18
Q

Proccessing seems wasteful: Why do it??

A

so that we can get many different proteins out of just one gene
differential splicing

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19
Q

mRNA Processing: CTD

A

Carboxy terminal domain of RNA Pol II has 50 repeats of serine rich amino acid sequence that some become phosphorylated (after initiation of RNA synth)

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20
Q

phosphorylated CTD region….

A 
binding site for enzymes needed for 
5' capping
intron splicing
3' polyadenylation 
so things can happen right away
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21
Q

What is the advantage of mRNA processing factors binding to the phosphorylated CTD?

A

they’re ready to work as its being made

makes things a lot faster

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22
Q

5’ Capping: how

A

cap synthing complex binds to CTD
catalyzes addition of GTP to 5’ NTP (4 step rxn; methylation of guanine base and ribose sugar of 5’ NTP (Adds cap)

5’ cap synth complex dissociates and is replaced by the cap binding complex

this dissociates when RNA Pol II disengages from DNA templaet

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23
Q

why cap?

A

to stop other enzymes from degrading the mRNA

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24
Q

Self Splicing reactions

A

intron has sequence that lets it adopt a structure that lets it catalyze the reaction to splice

25
Q

what enzyme catalyzes RNA splicing?

A

ribozymes

26
Q

What is splicing?

A

removal of introns

27
Q

group 1 introns

A

a few particular sequence must be conserved in order for the right orientation to facilitate this reactio
intron is removed in rxn catalyzed by GTP mediated nucleophilic attack on RNA sequence in the intron
bind to intron
new 3’ end, make new attack, cleave
exons covalently joined again

28
Q

splice junctions

A

junctions between exons and introns

things need to be here for splicing

29
Q

Group 2 introns

A

a few particular sequence must be conserved in order for the right orientation to facilitate this reaction
fungi
nucleophile for reaction is hydroxyl group fron adenosine in the intron attacks

lariat structure formed (HOWS ITS DIFFERNFT FROM GROUP 1)
formation of 2’-5’ phosphodiester linkage
lariat structure gets released at the end

happens only once per mRNA

30
Q

true or false: most introns are released by self splicing

A

False

31
Q

most introns are removed by…

A

action of small nuclear RNA (snRNA) within small ribonucleoproteins (snRNPs)

32
Q

5 snRNAs contained in snRNPs

A 
U1
U2
U4
U5
U6

snRNP happens when snRNAs associate with proteins

33
Q

What do snRNAs do?

A

mediate splicing reaction using sequences at the 5’ and 3’ splice junctions and w/in the intron

34
Q

U1

A

binds to 5’ splice site

35
Q

U2

A

binds to branch site and forms part of catalytic center

36
Q

U4

A

binds to 5’ splice site and then 3’ splice site

37
Q

U5

A

masks catalytic activity of U6

38
Q

U6

A

catalyzes splicing

39
Q

what recognizes 5’ splice junction?

A

U1 snRNP

40
Q

what recognizes intron branch site?

A

U2 snRNP

41
Q

what binds to U1 and U2 to form almost active splicesome complex?

A

U4/U6 snRNP and U5 snRPN

42
Q

what activates the inactive splicesome complex?

Then what happens?

A

ATP mediated conformational changes
this results in formation of active U2, U5, U6 splicesosome (U1 and U4 leave)

lariat is formed, cleavage occurs, the intron gets released

43
Q

What are RNA catalyzed cleavage steps and lariat formation similar to?

A

mechanism for self cleavage by group II intronts

44
Q

Final step of splicing

A

splicesome and intron complex dissociate

5’ and 3’ junctions of 2 adjacent exons are ligated together

45
Q

How do snRNPs know the difference between an exon and intron sequence? i.e. what makes splicing so precise?

A

Base pairing! (recognition by U1 and U2)

there IS structire to mRNA

46
Q

Polyadenylation

A

cleavage signal at 3’ end of gene is transcribed, this marks transcript for cleavage by endonuclease

transcript is released from DNA template

polyA tail added by enzyme poly(A) polymerase

47
Q

what is splicesome associated with?

A

CTD of RNA Pol II

so they are ready to act on intron as soon as it has been synthed

this all hapens while the RNA is synthed

48
Q

why would the cell do all these modifications?

A

we have a lot more proteins than we have genes

we can do alternate RNA processing to get this diversity

49
Q

Ways to get diversity

A

alternate RNA processing

a little bit from things like antibodies and alternate start site

50
Q

2 primary mechanisms of alternate RNA processing

A 
differential RNA splicing
alternate Poly (A) sites
51
Q

how to get alternate RNA processing

A

skip splice sites
combine splice sites
regulatory splicesomes ignroe splice sites (tissue specific expression)

when we also consider alternative poly (A) sites, there are huge numbers of possible mRNA transcripts

could be random or due to the fact that different tissues recognize/use different things

52
Q

Defective spliceing and human disease

A

point mutation in intron

of beta globin gene has been linked to decreaed hemoglobin (thalassemia) due to degredation of an abnromaly spliced mRNA

53
Q

what kind of mutations cause defects in alternative splicing

A

point mutations

deletions

54
Q

micro RNAs

A

noncoding RNAs in cells that are complementary to regions of “target” mRNA transcripts

22 nucleotides long and double stranded

partially processed in nucleus and then cleaved by Dicer enzyme in cytoplasm

55
Q

Micro RNAs and function

A

interferring RNAs
incorporated into RISC protein complexs
to mediate mRNA cleavage or translational repression

56
Q

How might miRNA be useful to eukaryotic cells? (Hint: protection and regulation)

A

degrade RNA viruses and modulate expression of regulatory proteins

down regulate mRNAs

57
Q

how MiRNAs work

A 
make premiRNA in nucleus, export
recognized by DIcer
Direcer makes duplex from hairpin
unwind duplex with helicase
load with RISK, its MATURE!
target either near perfect or partial complementarity
58
Q

partial complementarity of miRNA and RNA

A

repress translation from the RNA

59
Q

near perfect complementarity of miRNA and RNA

A

cleave the targeted RNA

Biochem 385 Unit 3 (11 decks)

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