mRNA turnover in eukaryotes

Question 1

What is deadenylation?

Answer 1

The shortening of the poly(A) tail of mRNAs
-by deadenylases (adenylate-specific exonucleases)
-defines functional lifetime of mRNA (∴ inversely related to translational efficiency)

Question 2

How can the decrease of mRNA poly(A) tails be seen?

Answer 2

gel electrophoresis
-follow pool of transcripts over lifetime (can see smears getting smaller and pool depleting)

Question 3

What happens during mRNA turnover?

Answer 3

-deadenylation by specifc deadenylases
-once mRNA reaches certain length, poly(A) binding protein can no longer bind so rapid degradation is triggered
-2 mechanisms of degradation:
-removal of m7G cap by decapping protein (Dcp1/2) followed by 5’-3’ degradation by exoribonucleases (Xrn1)
or
-continuous 3’-5’ degradation by exosome complex (less common and less efficient)

Question 4

What are the rate limiting steps of mRNA turnover?

Answer 4

-deadenylation
-decapping (by Dcp1/2)

Question 5

What is the amount of mRNA dependent on?

Answer 5

-rate of synthesis
-rate of degradation (turnover)

Question 6

What is the advantage of mRNA being stable?

Answer 6

accumlates to higher levels than unstable mRNA under the same transcriptional control

Question 7

What is the advantage of mRNA being unstable?

Answer 7

unstable mRNA is more responsive to transcription rate and turnover rate than stable mRNA
-very rapid changes in levels of transcript (v. rapid degradation or production of mRNA)

Question 8

What is ARE-mediated decay?

Answer 8

decay mediated by A/U-rich elements (AREs) present in 3’ UTR
-ARE-binding proteins can stimulate or slow down decay eg. AUF1 stimulates degradation, Hur slows deadenylation
-ARE-binding proteins are regulated by alternative splicing and post-translational modifications

Question 9

How are ARE-binding proteins regulated?

Answer 9

-alternative splicing
-post-translational modifcations

Question 10

What are miRNAs?

microRNAs

Answer 10

v. short (20-30nts) non-coding RNAs which interact with mRNA transcripts to affect their stability and/or their translational ability
-pri-miRNAs are processed by endonucleases Drosha (in nucleus) and Dicer (in cytoplasm) to produce miRNAs
-miRNAs anneal to sites within target mRNAs (typically in 3’ UTR)
-miRNAs are incorporated into RNA-induced silencing complexes, which cleave target mRNAs, recrut adenylases, repress translation
-cellular miRNA profiles are cell specific and vary in dev

Question 11

How are mRNAs with early stop codons degraded?

Answer 11

via nonsense-mediated decay (NMD)
-ribosome can’t interact with 3’ mRNP domain when it interacts with an early stop codon so triggers NMD
-early stop codons generated by alternative splicing (physiological response not mut)
-stops production of truncated proteins