The role of RNA degradation in gene expression

Question 1

What are the concepts associated with RNA degradation? (3)

Answer 1

• Functional RNAs are processed from larger transcripts
• A significant fraction of newly synthesised RNA is degraded i.e. non-functional
• Physiological RNAs are subjected to quality control

Question 2

How much of pre-mRNA is degraded during its processing?

Answer 2

Approx 85%

Question 3

What is pulse-chase analysis?

Answer 3

• Add isotopic label (e.g. radioactive uridine) which is incorporated into newly synthesised RNA in growing cells for an amount of time
• Then add excess normal uridine
• Can identify the RNAs that were made during that timeframe and can track their processing

Question 4

What is the difference between RNA polymerase I and II? (2)

Answer 4

• RNA pol I transcribes rRNA
• RNA pol II transcribes mRNA

Question 5

Why do you get stronger signals for rRNA than mRNA in pulse-chase labelling? (3)

Answer 5

• Similar proportion of cellular transcription is done by RNA pol I and II
• Pol I is mostly just rRNA but pol II does a whole range of cellular mRNAs so the signal for these will be more diffuse
• Also rRNA is more stable than mRNA

Question 6

How does rRNA differ from hnRNA (pre-mRNA)?

Answer 6

rRNA has a higher GC content and is more stable

Question 7

What is a tiled microarray?

Answer 7

Microarray with overlapping probes rather than one probe per gene so you can follow the entire sequence of the genome

Question 8

What was shown by transcriptome studies? (3)

Answer 8

• A large proportion of RNAs are intronic/intergenic
• Protein-coding sequences constitute ~2% of the genome
• RNAs are found in the nucleus and the cytoplasm

Question 9

What is pervasive transcription?

Answer 9

Transcription throughout the genome rather than just in protein-coding regions

Question 10

What is the importance of bidirectional transcription? (2)

Answer 10

• Bi-directional transcription from areas of chromatin that are depleted in nucleosomes (RNA polymerase promoter and terminator regions) producing non-coding RNAs e.g. enhancer RNAs (eRNAs)
• Bidirectional transcription occurs from most RNA polymerase II promoters

Question 11

What are the 2 types of non-coding RNAs produced by bidirectional transcription? (2)

Answer 11

• Short RNAs associated with paused polymerases at the promoter which aren’t competent for elongation
• Long non-coding RNAs that are released by transcription termination and capped etc

Question 12

What suppresses antisense transcription? (5)

Answer 12

• Factors that influence chromatin structure
• Histone methyltransferase Set2
• Histone deacetylase Rpd3
• Protein Ssu72 which promotes gene looping between 5’ and 3’ ends
• Transcription of many long non-coding RNAs is coupled to degradation

Question 13

What are ribonucleases doing in the cell? (3)

Answer 13

• Processing pre-mRNA
• Suppressing expression of non-coding RNAs
• Turnover of functional RNAs

Question 14

How is quality control involved in RNA degradation? (3)

Answer 14

• 50% tRNA and 10% pre-rRNA is degraded by RNA quality control
• Each step of mRNA synthesis is subject to quality control
• Mutations of the exosome complex cause accumulation of tRNAs, pre-rRNAs and unspliced pre-mRNAs

Question 15

Why might tRNAs be degraded?

Answer 15

tRNAs undergo a lot of post translational modifications and if these don’t happen correctly the tRNA is degraded

Question 16

How nucleases determine which RNAs need to be processed and which need to be degraded? (2)

Answer 16

• Proteins associated with nascent transcripts recruit RNases for both processing and decay
• Degradation is the default and transcripts acquire specific features that protect them from degradation i.e. m7g cap, polyA tail

Question 17

What do histone mRNAs have instead of a polyA tail? (2)

Answer 17

• Stem loop structure which binds to stem loop binding protein (SLBP)
• Performs same 3’ protective function as polyA

Question 18

What is the kinetic proofreading model of quality control? (4)

Answer 18

• Differentiate correct and incorrect RNA complexes
• Unlikely to be a purely competitive equilibrium model between the correct and incorrect complexes because this would be very inefficient
• Kinetic proofreading involves an energy cost which traps the bound substrate, if it is correctly bound it is processed, if incorrect it is dissociated or degraded
• Ribosome assembly and pre-mRNA splicing pathways have multiple associated GTPases and/or ATPases that might drive coupled kinetic proofreading reactions
• Steps arranged in series provide greater efficiency overall

Question 19

How important are RNA quality control pathways? (3)

Answer 19

• RNA turnover is widespread suggesting that RNA production is error prone
• Loss of RNA quality control can cause disease
• Widespread degradation of transcripts provides a substrate for genetic evolution because a functional RNA could be generated from those being degraded

Question 20

What is caused by mutation of critical rRNA nucleotides in E.coli vs yeast? (2)

Answer 20

• Dominant negative in E.coli because no repair mechanism so cell dies
• Recessive in yeast because they have cytoplasmic ribosome surveillance pathway that eradicates non-functional ribosomes

Question 21

What disease can be caused by loss of RNA quality control? (2)

Answer 21

• Toxic accumulation of RNAs containing repeats overwhelm the RNA quality control system
• Causes ALS