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Flashcards in Eukaryotic RNA Processing Deck (31):
1

Actinomycin D

Inhibitor of Transcription

-slips into the major groove between the DNA strands (intercalates), preventing the unwinding of the DNA template strands

-inhibits initiation and elongation

-effective in both prokaryotes and eukaryotes

2

In bacterial mRNA, the 5' end would have the structure:

5' PPP

b/c mRNA (bacterial) has no processing and it means the 1st base is a triphosphate and it won't be cleaves

3

What is hnRNA?

heterogenous nuclear RNA --> precursor of mRNA in eukaryotic cells

4

Post-transcriptional modification in mRNA (eukaryotes)

5' cap
3' poly A tail
Splicing to remove introns

5

Where does post-transcriptional modification happen in eukaryotes?

Nucleus, to allow for transport into cytoplasm

6

mRNA cap

GTP added backwards to 5' end to form 5'-5' triphosphate linkage thru Guanylyl Transferase

7

How many phosphates released in making the mRNA cap?

Release of gamma phosphate from RNA and release of PPi from GTP

8

Function of Poly-A tail-3' end

related to transcription termination
Protects 3' end from degradation
Stabilization of mRNA

9

Function of AAUAAA in poly A tail formation

Functions as a signal for dephosphylation and endonuclease to cleave upstream 3' --> poly A polymerase using ATP adds around 200 A's --> poly A binding proteins bind for protection of 3' end and stabilizes mRNA

10

What does splicing require?

Involves snRNA + proteins= snRNPs
U1,2,4,5,6

2'-5' phosphodiester bonds at branch point

11

Consensus Sequences on Exon/Intron borders

All introns begin with GU and end with AG

12

What is the branch point

An A located in a pyrimidine-rich sequence approx 50 bases from the 3' end of the intron

13

Role of snRPs

Involved in mRNA precursor splicing
contains one small snRNA and several proteins

14

U1

Binds to 5' splice site (there can be mismatched sequences in this bindings, but there is a conservative sequence at which U1 binds to)

15

U2

Binds the branch site and forms part of the catalytic center

16

U5

Binds the 3' splice site, loops over to the 5' site

17

U4

Masks the catalytic activity of U6, until ready to splice

18

U6

Catalyzes splicing- it is a ribozyme

19

Why does U1 snRNP bind the 5' splice site?

Bc it is complimentary to the sequence on the 5' end

20

Pathway for Spliceosome Assembly

1) U1 binds to 5' end of intron
2) U2 binds to branch site
3) U4,5,6 complex comes in and brings exon1/intron (U1 area) junction closer to the branch site (A) thru looping --> creation of spliceosome
--> structured so that A is close to the 3' end of exon 1
4) U1/U4 leave

21

Actual splicing pathway

1) U2 and U6 have complementary sequences and can base pair- they form the catalytic site

U6 catalyzes:
1) attack of 2' OH on A to the 5' splice site
2)Newly formed 3' OH on exon 1 attacks the 3' splice site on the other exon still attached to the intron
3) Exons are joined
4) Intron is released in the form of a lariat

22

Key Points in Splicing

Highly regulated process- introns removed in order unique to each mRNA

Alternative splicing

Mutations in splice sites are involved in many diseases processes

23

RNA binding proteins

Facilitate alternative splicing
Also important as many genes are alternatively spliced in malignant cells

Blocks snRPs from binding so those exons are excluded from specific tissues

24

SR

Serine/arginine protein (positive-favors splicing)- binds on exons

25

hnRNPS

heterogeneous nuclear ribonucleoprotein (negative- inhibits splicing)
binds both on exons and introns or both

26

ESE, ISE

exon splicing enhancer- binds SR
intron splicing enhancer- binds SR

27

ESS, ISS

exon splicing silencer- binds hnRNP
intron splicing silencer- binds hnRNP

28

beta-thalassemia major

one base mutation in the beta globin gene (G to A)
causes low beta globin protein and severe anemia

Single point mutation of G --> A in an intron of the beta-globin gene results in mis-splicing..creates a new 3' splice site --> protein cut short and not functional

29

Variant exons

V5 forms in many metastatic cancers--> facilitates movement of cancer cells

30

Inclusion of variant exon

SAM binds ESS site --> hnRNP cannot bind --> SR binds --> U2 binds --> splicing occurs --> exon will be included in that variant protein

31

Exclusion of variant exon

hnRNP + silencer --> prevents binding of SR proteins and blocks E2 from binding --> prevents splicing of variant exon --> variant exon expressed in final product