lecture 10

Question 1

canine patient is coughing, sneezing has nasal discharge, runny eyes, fever, is lethargic and is having trouble breathing
what might be the problem?
Treatments?

Answer 1

influenza infection

possible treatment is RNA CAP Snatch-and Grab inhibitors (virus try to steal 5’ cap from mRNA, if you stop this, virus is easier to detect and easier to kill)

Question 2

your patient is very anemic, she makes Bglobin RNA transcripts but the mRNA in the cytoplasm is very unstable.
What is the problem

Answer 2

a point mutation in the polyadenylation signal results in very unstable mRNA reducing the level of b-globin protein- this results in B-thalassemia

1st AAUAAA is not cut currently have to use next AAUAAA =, mRNA has really long tail -unstable

Question 3

Your patient is a golden retriever showing exercise intolerance, a stilted gait and muscle atrophy
what is the disease and cause of this problem

Answer 3

canine muscular dystrophy cause by a single base change that changes the 3’ consensus splice site of exon 6

Question 4

your patient has osteoscarcoma. during evaluation of RNA-seq analysis you find that the c-fos transcript is elevated. however its transcription rate is normal.
why is the c-fos mRNA level high?

Answer 4

deletion of a destabilizing RNA sequence (AUUUA) in the 3’ untranslated region of the c-fos transcript

c-fos usually has AUUUA which tells mRNA to be broken down after use. if these AUUUA are deleted c-fos is never broken down and keeps telling cell to divide

Question 5

Lupus like condition

Answer 5

autoantibodies against SNRPs (things that help slicing of RNA to take out introns)

Question 6

Three steps of RNA transcript

Answer 6

CAP
cleavage/polyadenylation
splicing

Question 7

gene has intron and exons, what part leaves the cell?

Answer 7

introns get taken out

exons leave the cell

mRNA gets 5’ and 3’ polyA tails

Question 8

Splice RNA means

Answer 8

genes have introns and exons

introns get taken out

mRNA gets 5’ and 3’ polyA tails

Question 9

5’ cap

Answer 9

protects RNA from degradation by 5’ ribonucleases

some viruses will steal/ or add their own 5’ cap

RNA guanylaltransferase + 5’ triphophatase
RNA guanylaltransferase
RNA guanine-7 methlytransferase

RNGTT and RNMT

these enzymes live on the phosphorylated CTD (carboxy terminal domains) tail of RNA polymerase II and will add 5’ cap as RNA is being made

Question 10

how does polyadenylation help

Answer 10

process of adding poly A 3’ end to mRNA

RNA polymerase doesn’t know where to stop

• poly A tails defines the 3’ end
• protects the 3’ end of the mRNA from exonucleases
• important for translation
• development-involved in masking mRNAs in occytes to keep them from being translated

Question 11

polyadenylation

Answer 11

1. a trimer of 3 proteins called cleavage/polyadenylation specificity factor (CPSF) binds to an AAUAAA sequence toward the end of the RNA transcript
2. CstF (cleavage stimulatory Factor) binds downstream of the AAuAAA at at GU rich area
3. cleavage factor 1 and 2( CF1 and CF2) and poly A polymerase (PAP) bind to RNA
4. RNA is cleaved about 10-15 nucleotides downstream of the AAUAAA
5. PAP adds about 200 A residues with the help of PolyA binding protein (PABP)

All these proteins needed are binded to CTD of RNA polymerase

Question 12

polyadenylation short hand

Answer 12

1. CPSF binds to AAUAAA
2. CstF binds to GU rich area downstream
3. CF1, CF2 and PAP bind to RNA
4. RNA is cleaved downstream of AAUAAA
5. PAP with the help of PABP adds 200 As

Question 13

general splicing
first two nucleotides of intron are ___
last two nucleotides of intron are___

Answer 13

GU

AG

Question 14

splicing

Answer 14

GU is cut and makes loop and binds to branching site which is usually 15-20 basepairs up from AG
(lariat structure) 5’ to 2’ linkage

then AG is spliced

and two exons are stitched together with ligase

process involves the spliceosomes, which is very large and contains RNA and protein complexes called SNRPS

Question 15

splicing in more detail snRNAs

Answer 15

SNRPs (spliceosomes)
help RNA splicing

U1 binds to GA (5’ splice site) and helps cutting at that spot

U2 binds to branching site and helps RNA fold

U4/U5/U6 hold RNA in place so that exon1/ intron can be broken and then helps looping into the lariat

finally breakage at the intron/exon 2 boundary and ligation of the RNA exons together with release of the lariat structure completes the reaction

Question 16

what is special about tetrahymena

Answer 16

can self splice

Question 17

what is alternate processing of exons

Answer 17

introns and exons are cut in weird order, skipped or rearranged

example: immunoglobulin location
if cell has S exon then immunoglobulin is secreted from cell (later in development when there is a bunch of CstF and can bind to S site)

if M1 M2 then immunoglobulin is membrane bound (early in development when small amounts of CstF)

Question 18

mRNA transport

Answer 18

mRNA leaves nulceus

Question 19

NMD

Answer 19

(type of quality control for mRNA before it leaves the cell)
nonsense mediated decay
will find and destroy mRNA that are too short. (have premature stop codon)

Question 20

non-stop decay

Answer 20

(type of quality control for mRNA before it leaves the cell)

will find and destroy mRNA with no termination

Question 21

your patient is a 14 week old male corgi exhibiting failure to thrive, diarrhea and vomiting. The patient has very low serum immunoglobulin
what disease and what is the cause?

Answer 21

canine X-linked SCID caused by a single nucleotide change resulting in a premature stop codon in the IL-2 receptor gene

Question 22

RNA mix up is rare but what is one example

Answer 22

trypanosomes

will edit mRNA in the cytoplasm.

trans-splicing- mixing exons between two mRNA molecules

AUUUA causes mRNA to break down

Question 23

*** describe in detail cleavage/polyadenylation reactions

Answer 23

when mRNA has a AAuAAA sequence, CPSF and CstF move from the CTD to the new mRNA. CPSF binds to the AAUAAA sequence, while CstF binds to a GU downstream. Cleavage factors 1 and 2 then act to cleave the mRNA near the Cstf. PAP then acts to initiate/produce a polyA tail of 200 into the end of the mRNA. In this way the mRNA is cleaved and then a poly-A tail is added.

1. CPSF binds to AAUAAA
2. CstF binds to GU rich area downstream
3. CF1, CF2 and PAP bind to RNA
4. RNA is cleaved downstream of AAUAAA
5. PAP with the help of PABP adds 200 As