Gene Regulation 6 - Regulation of eukaryotic gene expression - post transcriptional and post translational regulation

Question 1

Learning Outcomes

Answer 1

Students will be able to
▪ describe mRNA processing events and their outcomes and how these impact on mRNA
export and translation initiation.
▪ describe an mRNA degradation pathway.
▪ state what miRNAs are and how they can impact of gene expression.
▪ understand that PTMs can impact on protein function.
▪ describe the role of a proteasome in protein degradation.

Question 2

Comparing a Gene and Its Transcript

Answer 2

Question 3

Pre-mRNA processing - overview

Answer 3

• Primary transcript = pre-mRNA
  undergoes extensive processing to
  produce the mature, functional RNA
  – 5’cap = 7-methyl guanosine linked
  by 3 Phosphates
  – Splicing - removal of introns
  – Poly A tail (±200), polyA
  polymerase

Answer 4

Question 5

Co-transcriptional processing: 1) Capping

Answer 5

During transcription:
* ~ 25 nt of the mRNA 5’-end are synthesised.
* Capping factors (proteins) attaches a CAP
structure to the 5’-end of the mRNA.
* CAP: unusual structure containing 5’ to 5’
triphosphate bridge between the CAP and the 5’-
end of the mRNA.
* CAP functions:
– signals correct 5’-end of the mRNA ➔ required
for:
* Export of mRNA from the nucleus
* Initiation of translation
– Protection from 5’ degradation by RNases

Answer 6

Question 7

Human genes can vary greatly in size
and numbers of exons and introns

Answer 7

• Average mammalian intron ± 2000 nt, average exon ±200 nt
  – Human β-globin gene encodes for one of the protein subunits of the oxygen carrying
  protein haemoglobin - 3 exons, 2000 bp = 2 kbp
  – Human Factor VIII gene encodes blood clotting protein – 26 exons , 200000 bp = 200
  kbp
  – Human Dystrophin gene encodes for a skeletal muscle protein, largest known human
  gene, 2200000 bp = 2200 kbp (0.1 of the human genome), 79 exons, takes 16 hours to
  be transcribed, mutations are associated with Duchenne muscular dystrophy

Answer 8

Question 9

Co-transcriptional processing: 2) Splicing

Answer 9

• Splicing removes introns as ‘lariat’ structures
  – VERY precise
• Requires splicing sequences
  – intron/exon boundary sequences
  – sequences in the intron
• Splicing is carried out by interaction of
  spliceosomes ‘snurps’ with splicing sequences
  on the pre-mRNA.
  – Spliceosomes are complexes of proteins &
  small nuclear RNAs (snRNA).
  – snRNAs hybridise with splicing sequences

Answer 10

Answer 11

Question 12

Why do genes have introns ?

Answer 12

Intron maintenance in the genome = extra cost and energy
Is there a selective advantage justifying this?
▪ Drosophila melanogaster has fewer genes than Caenorhabditis elegans BUT: the protein
number does not reflect the lower gene number
WHY? - Alternative splicing
▪ Results in a number of different mRNAs from one gene ➔ increases diversity of proteins and
functionality
~ 95% of human genes with more than one exon are alternatively spliced
~ 20,000 protein coding genes (~2% of human genome) ➔ >80,000 proteins
▪ THUS one gene one protein is not always true, one gene can result in many proteins
▪ Many splicing events are rare, tissue specific, developmentally regulated

Answer 13

Question 14

Co-transcriptional processing: 3) Polyadenylation

Answer 14

Cleavage and polyadenylation specificity factor (CPSF)
▪ cleaves 3’-end of the pre-mRNA from the mRNA still
being synthesized
Poly-A-polymerase (PAP)
* Recognises poly A signal on the mRNA (AAUAAA or
AUUAAA)
* binds about 10 nucleotides behind the signal and
adds many hundreds adenines (one at a time)
Poly-A-binding proteins (Pab)
* assemble on poly-A tail and determines final poly-A
tail length (unknown mechanism), protects mRNA 3’-
endCPSF
Polyadenylation protects the mRNA’s 3’ end from
degradation by RNase

Question 15

mRNA export to the cytosol

Answer 15

➢ Proteins bound to mRNA after processing are required
for export to the cytosol
* CAP binding protein
* exon – exon junctions proteins
* Poly A binding proteins
➢ These proteins interact with export proteins that allow
mRNA export through the nuclear pore complex to the
cytosol.
➢ In the cytosol, some of the proteins are exchanged for
cytosolic variants before the mRNAs associates with
ribosomes.
* Example Cap binding protein is exchanged for eIF4

Answer 16

Question 17

Translational Control is often exerted at the
initiation of translation stage

Answer 17

• 5’ cap binds to eIFs = eukaryotic
  initiation factors.
• Eukaryotes KOZAK consensus
  sequence ACCAUGG (not shown)
  around start codon is important
  in binding small ribosomal
  subunit.
  – In prokaryotes: Shine-Dalgarno
  sequence
• Ribosome SSU assembles & slides
  along mRNA finding translation
  start codon AUG.
• Loop between 3’ and 5’ mRNA
  ends forms = Checkpoint for
  intact mRNA ends.
• 5’ and 3’-UTRs sequences specify
  efficiency of translation.

Answer 18

Answer 18

Question 19

mRNA half-life and degradation

Answer 19

• Eukaryotic mRNA half life: several minutes to over 24 hours
• mRNA sequences determine half life and mRNA degradation pathway
  – Example: 3’-UTRs carries information to specify mRNA half-life
• Degradation begins with poly-A tail shortening by a 3’ to 5’ exonuclease
  – The 3’ to 5’ degradation can continue to degrade the whole mRNA
• Decapping enzyme removes the 5’-cap
• Degradation of mRNA in 5’ to 3’ direction by 5’ to 3’ exonuclease

Answer 19

Question 20

miRNA mediated RNA silencing and post-transcriptional
regulation of gene expression

Answer 20

• miRNAs ‘micro RNAs’: small noncoding RNA molecules (~ 22 nt)
• Encoded by nuclear DNA in plants
  and animals and by viral DNA in
  certain viruses
• Incorporated into a protein
  complex (RISC)
• Function via base-pairing with
  mRNA molecules:
  ➢ extensive sequence match
  to mRNA: mRNA cleavage
  ➢ less extensive sequence
  match with mRNA:
  translational repression

Answer 21

Question 22

Post-translational modification (PTM) of proteins

Answer 22

• Covalent modification of amino acids
  or N- or C-terminus of a protein after
  its biosynthesis
• Modifications are done by enzymes,
  e.g. phosphorylation by kinases -
  most common PTM.
• PTMs diversify and specify protein
  function by:
  – promoting protein folding
  – improving protein stability
  – regulating protein activity,
  – targeting protein to the cell
  membrane

Answer 22

Question 23

Proteasome degrades short-lived and misfolded proteins

Answer 23

• Proteins to be degraded are marked by covalent addition of several proteins named ubiquitin
  ➔ polyubiquitin
• Proteasome is a multiprotein complex:
  – The proteasome stopper region recognises polyubiquitin marked proteins
  – The marked proteins is unfolded by the stopper and channelled into the central cylinder of
  the proteasome.
  – Inside the cylinder are several proteases that degrade the marked protein

Answer 23

Question 24

The Phenotype of a cell, tissue or organism
is determined by the Genotype BUT…..

Answer 24

• Genotype determines the phenotype ➔ the phenotype is the end product of gene expression
• BUT – there it is a long way from a genotype to a phenotype with many opportunities to
  influence gene expression

Answer 25

Question 26

Can you …

Answer 26

▪ … name and explain the workings and outcomes of mRNA processing mechanisms in eukaryotes?
▪ … can you explain alternative splicing and how this demonstrates that one gene one protein is often not
true?
▪ … can you name the main proteins involved in the export of a completely processed mRNA from the
nucleus to the cytosol?
▪ … can you describe how mRNA sequence motifs and proteins control the initiation of translation?
▪ … describe how mRNA degradation works and how it contributes to regulation of gene expression?
▪ … explain the basic workings of miRNAs in the regulation of gene expression?
▪ … give an overview of post-translational protein modifications and give examples oi their outcomes?
▪ … give an overview of the ubiquitin mediated degradation of proteins as an example of the regulation of
gene expression