L5, Post Transcriptional Modifications of Gene Expression

Question 1

+ At what points does the spiceosome recognise exon-intro boundaries and points within introns?

Answer 1

• cis elements present at exon-intron boundaries
• branch point sequence and polypyrimidine tracts within introns

Question 2

+ Types of splicing regulator:

Answer 2

• heterogenous nuclear ribonucleoproteins (hnRNPs)
• serine-arginine-rich proteins (SR proteins)
• Auxiliary proteins
  These groups comprise exonic and intronic splicing silencers and enhancers

Question 3

Key benefits of PTMs:

Answer 3

• Precursor RNAs are modified to make mature, functional RNA
• RNA processing has three main benefits; contributes to regulation of gene activity, diversity (via alternative splicing) and quality control (since defective/uncapped mRNAs are detected and degraded)

Question 4

What key processes occur during PTMs?

Answer 4

Coupled processes:

• Cleavage
• Splicing
• 5’ capping
• Polyadenylation

Question 5

Why is the CTD of RNA PII useful in PTM?

Answer 5

• Allows 5’ capping and 3’ polyadenylation to be coupled
• Acts to recruit the machinery (i.e. processing factors) dependent upon its phosphorylation pattern at a given point -> capping then splicing then polyadenylation and cleavage factors

Question 6

Phosphorylation status of CTD of RNAPII at different stages:

Answer 6

• Partially phosphorylated on transcription initiation -> recruits capping enzyme
• Elongation leads to further phosphorylation, recruiting splicing machinery
• This also leads to recruitment of the cleavage and polyadenylation complex

Question 7

What is used to cap the 5’ end?

Answer 7

• Capped by a7-methylguanine nucleotide via and unusual 5’-5’ triphosphate linkage
• The guanine in this cap is then methylated at N7
• Further methylation in complex eukaryotes leads to further level of regulation

Question 8

Outline the 3 step capping process:

Answer 8

• RNA triphosphate removes terminal phosphate at 5’ end (PPP -> PP)
• Guanyl transferase uses GTP to attack a GMP to end in a 5’-5’ triphosphate linkage (PP -> GPPP)
• The guanine is methylated by a guanine-7-methyl transferase (GPPP -> mGPPP)

Question 9

Contrast: enzymes for the 3 steps of 5’ capping in yeast vs mammals

Answer 9

• Yeast: all 3 steps are carried out by separate enzymes
• Mammals/c.elegans: First two reactions are carried out by the same enzyme

Question 10

How is 3’ polyadenylation carried out?

Answer 10

• Cleavage after consensus sequence (by XRN2)
• Polyadenylation and specificity factors associate, then poly A polymerase (PAP) adds the A tail

Question 11

Sequences associated with polyA sites

Answer 11

• Consensus; AAUAA
• CA is the site of cleavage, which should lie between this consensus sequence and a U or GU-rich region
• Entire code spans around 70 nucleotides

Question 12

How can polyadenylation regulate gene expression?

Answer 12

• Some mRNAs have multiple polyadenylation sites
• Use of certain sites will retain or exclude areas inbetween
• e.g. cyclin D1 3’UTR inclusion due to use of the distal polyadenylation site will alter level of gene expression

Question 13

What common mechanism is used in all splicing events?

Answer 13

• Transesterification reactions

Question 14

What 3 ways can an intron be removed?

Answer 14

• Removed by proteins
• By RNPs
• By themselves

Question 15

What two ways can splicing confer specificity?

Answer 15

• Alternative splicing
• Exon shuffling

Question 16

What is the spliceosome made up of?

Answer 16

• 5x snRNPs: U1, 2, 5, 4, 6
• Each has a snRNA of about 100-300 nts (sn = small nuclear)
• Additional proteins also involved

Question 17

How is the spliceosome assembled?

Answer 17

• snRNAs form base pairs with pre-mRNA (recognition)
• (1) 5’ splice site recognised by U1
• (2) Rest of splicing apparatus binds, sometimes displacing other factors, including branch-point binding protein; BPP
• (3) pre-mRNA rearranges, transesterification occurs and lariat forms
• (4) Two exons brought together, transesterification occurs again

Question 18

What feature typically defines the ends of introns

Answer 18

• 5’-GU
• 3’-AG

Question 19

Why might some spliceosome components remain on transcript after splicing?

Answer 19

• If they have roles in subsequent processes
• e.g. EJC

Question 20

How does the spliceosome recognised the correct splice sites? What is to be avoided?

Answer 20

• Since the defining intron sequences are simplistic they can occur by chance elsewhere -> cryptic splice sites
• Should also be binding the correct true splice site out of a few
• Mechanism: Exon definition

Question 21

Exon definition process:

Answer 21

• 5’ and 3’ ends of an exon are brought together by interactions between U1 and U2 complex
• Mutations at a 5’ splice site would result in exon exclusion since the exon isn’t being defined on the 5’ side

Question 22

How is alternative splicing regulated?

Answer 22

• By RNA-binding splicing factors
• They can bind to either splicing enhancers or silencers, which can be either intronic or exonic
• ISE, ESE, ISS and ESS
• Enhancers encourage the alternative exon to be included whereas silencers prevent this

Question 22

How are cryptic splice sites masked?

Answer 22

• The exon is typically marked with SR proteins and the intron is bound with hnRNPs
• This masks cryptic splice sites

Question 23

How is sex determined in drosophila?

Answer 23

• Determined by number of x chromosomes
• In the fly, ‘sex lethal’ is transcribed and translated functionally only in females
• This sets off a cascade of gene expression for either female or male development
• Sxl is an RNA-binding protein -> first target is ‘transformer’ (tra) which can be spliced to include or exclude the high affinity splice site (default)
• High affinity splice site is masked in females by sxl -> functional TRA
• This is an example of a variable 3’ splice site

Question 23

Give 4 examples of human disease caused by missplicing in lamin gene:

Answer 23

• Limb girdle muscular dystrophy 1B (intron retention)
• Familial partial lipodystrophy (intron retention)
• Hutchinson-Gilford progeria syndrome (exon depletion)
• Dilated cardiomyopathy (exon extension)

Question 24

+ How does alternative splicing occur? Name 6 processes:

Answer 24

• Exon skipping
• Intron retention
• Mutually exclusive exons
• Alternative first and last exons
• Alternative 5’ and 3’ splice sites
• Alternative ‘tandem’ 5’ and 3’ UTRs

Question 25

+ Prevalence of alternative splicing in human genome:

Answer 25

• 86-88% of human protein-coding genes are reported to undergo AS

Question 26

+ Relationship between alternative splicing and complexity:

Answer 26

• Higher number of alternatively spliced genes in broad groups with more cell types
• Vertebrates display a tissue-dependent regulation of AS splicing

Question 27

+ How do Alu sequences influence splicing?

Answer 27

• Intronic Alu sequences may regulate alternative splicing by shifting splicing patterns through secondary structure changes to pre-mRNAs
• They also contain cryptic splice sites

Question 28

+ Name the 2 anatomic sites where highest level of exon skipping events occur:

Answer 28

• Brain
• Testis

Question 29

+ How is alternative splicing involved in pathology of Shigella infection?

Answer 29

• Shigella infects epithelial cells and ultimately causes bacterial dysentry
• Delivers proteins to nucleus via type II secretion system; including IpaH9.8
• IpaH9.8 disrupts splicing activity upon binding to U2AF35 splicing factor
• Reduces expression of cytokines and chemokines -> dampening proinflammatory response

Question 30

+ TCGA data: how is transcriptomic diversity enhanced in many cancers? Overview of alternative splicing in cancers:

Answer 30

• An abundance of mRNAs in tumour cells may increase the transcriptional diversity of many cancers
• Alternative splicing is known to be involved in carcinogenesis, with well-characterised alternative splicing patterns in various key components of cancer hallmarks (PT53, hTERT, EGFR etc)