6. RNA degradation

Question 1

Why do RNAs need to be degraded?

Answer 1

RNAs need to be degraded:
- homeostasis: transcription bi-products need to be removed, resources recycled
- quality control: if not made correctly
- gene regulation: protein production not constant - highly regulated mRNAs - short half-lives

Question 2

What is the basic mechanism of RNA degradation?

Answer 2

Basic RNA (mRNA, ncRNA) degradation:
- exonucleases: from the ends 5’->3’ / 3’->5’
- endonucleases: internal degradation

Question 3

Explain exonucleases

Answer 3

Exonucleases - degrade RNAs from the ends - in nucleus + cytoplasm:
- nucleus: Xrn2 5’->3’ - degrades mRNA after cleaved off of RNA pol II (uncapped), pre-mRNA (incorrect splicing/ termination - need to decap first)

• cytoplasm: Xrn1 5’->3’ - mRNA degradatoin after translation - turnover (decapping first)

Question 4

What is the exosome?

Answer 4

Exosome - a protein complex of proteins + nucleases:
- works in nucleus + cytoplasm
- different co-factors determine its function
- involved in processing (quality control)
- invollved in degradation (turnover)

Question 5

What is the quality control process in the nucleus?

Answer 5

RNA quality control in the nucleus:
- capping 5’
- cleavage
- polyA
- splicing
- editing
- nuclear export
Most RNA degraded in nucleus
Introns need to be degraded

Question 6

Explain nuclear degradation processes based on incorrect mRNA production processes

Answer 6

Incorrect:
- capping -> exonuclease (Xrn2/Rat1)
- elongation -> hairpin structure
- splicing -> debranch (Dbr1) -> hairpin
- termination -> exonuclease
- export out of nucleus -> exonucleases form both ends

Question 7

What processes involve mRNA processing?

Answer 7

• 5’ capping
• 3’ polyA addition
• splicing

Question 8

Explain cytoplasmic RNA degradation

Answer 8

Ribosome bound mRNA:
- pre-mature STOP codon - non-sense mediated decay (NMD)
- no STOP codon -> translating into polyA - non-stop decay (NSD)
- stalled ribosome - no-go decay (NGD)
- normal turnover: 3’->5’ / 5’->3’ decay by exonucleases (Xrn1, Caf1-Ccr4-NOT, Dcp1-Dcp2)

Question 9

Explain non-sense mediated decay (NMD)

Answer 9

In translation - in cytoplasm: NMD when premature STOP codon / EJC (exon-junction complexes) not displaced:
- recruit Upf proteins - coordinate de-capping + de-adenylation
- mRNAs degraded by Xrn1 (cytoplasm) + exosome

Question 10

What are the origins of premature STOP codon in NMD?

Answer 10

NMD induced by premature stop codon:
- improper splicing (intron retained / frameshift)
- mutation

Question 11

What are the consequences of premature STOP codon?

Answer 11

Consequences of premature STOP codon:
- non-functional protein
- loss of regulatory region that regulates growth -> cancer
=> protein needs to be degraded by NMD before becomes functional

Question 12

What is the degradation mechanism in mRNA turnover?

Answer 12

After translation - mRNA degraded (normal turnover):
- deadenylase - removes polyA 3’
- decapping enzyme 5’
- exonuclease degrades mRNA from both ends

Question 13

How is mRNA efficiently translated?

Answer 13

Polysome / polyribosome: each mRNA translated by many ribosomes simultaneously

Question 14

What is the average half life in an mRNA?

Answer 14

10-15 mins: translated -> needs to be degraded -> short half-life to prevent protein overexpression

Question 15

What parts of mRNA influence its stability?

Answer 15

Influence mRNA stability - half life:
3’ UTR - downstream of STOP codon - encodes reg sequences for degradation - specific protein recognize - RNPs (microRNA-RISC) binding sites in 3’ UTR (many affect deadenylation)

Question 16

What are the two types of mRNA stability regulation?

Answer 16

Stabilization / de-stabilization of mRNA via 3’ UTR:
- ARE mediated stabilization: ARE sequence in 3’ UTR - deadenylation enzyme blocked
- Non-ARE mediated stabilization: UC-rich sequence bound - deadenylation enzyme blocked
- miRNA mediated de-stabilization: miRNA-RISC binds to sequence - activates deadenylation enzyme

Question 17

What are ARE sequences?

Answer 17

ARE sequence - AU-rich AUUUA / UUAUUUAUU - AUBPs bind to stabilize / destabilize mRNA -> depending leads to degradation - destabilizing leads to exonuclease recruitment

Question 18

What is a example of RNB that regulates mRNA stability?

Answer 18

RNB for mRNA stability -> argonaute: miRNA + RISC: miRNA binds to complimentary region on mRNA - RISC recruits exonucleases

Question 19

What are the methods used for detecting mRNA?

Answer 19

• Reporter assays: upstream / downstream gene regulation elements - clone into plasmids - test their functions in diff contexts => measure protein / gene / enzymatic levels in transfected cells - insert reporter (LacZ, GFP, luciferase)
• qRT-PCR: detect mRNA levels in different conditions (ex upon cell infection)

Question 20

At what two processes can mRNA levels be regulated?

Answer 20

• Transcription level - how much produced
• Post-transcription - degradation level - how much degraded

-> need assays to detect both levels

Question 21

How to measure promoter activity and importance?

Answer 21

Testing promoter proximal control regions of a gene:
- upstream region many copies - many version with 5’ deleted sections
- diff variants inserted into vectors with reporters -> transformed into E. coli for amplification
- purify plasmids from E. coli -> transfect cells
- reporter assays: see which 5’ mutant expressed protein

Question 22

How to measure mRNA half life?

Answer 22

Measuring mRNA half-life - reporter assay:
- block transcription in cells at one time (actinomycin / alpha-amanitin)
- purify mRNA
- detect specific mRNA levels at diff time points using Northern blot and probes
- compare specific mRNA levels at diff time points => calculate half lives

Question 23

Lecture summary

Answer 23

Question 24

Quiz 1

Answer 24

Question 25

Quiz 2

Answer 25

Question 26

Quiz 3

Answer 26