L5, Post Transcriptional Modifications of Gene Expression Flashcards
+ At what points does the spiceosome recognise exon-intro boundaries and points within introns?
- cis elements present at exon-intron boundaries
- branch point sequence and polypyrimidine tracts within introns
+ Types of splicing regulator:
- heterogenous nuclear ribonucleoproteins (hnRNPs)
- serine-arginine-rich proteins (SR proteins)
- Auxiliary proteins
These groups comprise exonic and intronic splicing silencers and enhancers
Key benefits of PTMs:
- 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)
What key processes occur during PTMs?
Coupled processes:
- Cleavage
- Splicing
- 5’ capping
- Polyadenylation
Why is the CTD of RNA PII useful in PTM?
- 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
Phosphorylation status of CTD of RNAPII at different stages:
- 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
What is used to cap the 5’ end?
- 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
Outline the 3 step capping process:
- 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)
Contrast: enzymes for the 3 steps of 5’ capping in yeast vs mammals
- Yeast: all 3 steps are carried out by separate enzymes
- Mammals/c.elegans: First two reactions are carried out by the same enzyme
How is 3’ polyadenylation carried out?
- Cleavage after consensus sequence (by XRN2)
- Polyadenylation and specificity factors associate, then poly A polymerase (PAP) adds the A tail
Sequences associated with polyA sites
- 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
How can polyadenylation regulate gene expression?
- 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
What common mechanism is used in all splicing events?
- Transesterification reactions
What 3 ways can an intron be removed?
- Removed by proteins
- By RNPs
- By themselves
What two ways can splicing confer specificity?
- Alternative splicing
- Exon shuffling
What is the spliceosome made up of?
- 5x snRNPs: U1, 2, 5, 4, 6
- Each has a snRNA of about 100-300 nts (sn = small nuclear)
- Additional proteins also involved
How is the spliceosome assembled?
- 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
What feature typically defines the ends of introns
- 5’-GU
- 3’-AG
Why might some spliceosome components remain on transcript after splicing?
- If they have roles in subsequent processes
- e.g. EJC
How does the spliceosome recognised the correct splice sites? What is to be avoided?
- 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
Exon definition process:
- 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
How is alternative splicing regulated?
- 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
How are cryptic splice sites masked?
- The exon is typically marked with SR proteins and the intron is bound with hnRNPs
- This masks cryptic splice sites
How is sex determined in drosophila?
- 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
Give 4 examples of human disease caused by missplicing in lamin gene:
- Limb girdle muscular dystrophy 1B (intron retention)
- Familial partial lipodystrophy (intron retention)
- Hutchinson-Gilford progeria syndrome (exon depletion)
- Dilated cardiomyopathy (exon extension)
+ How does alternative splicing occur? Name 6 processes:
- Exon skipping
- Intron retention
- Mutually exclusive exons
- Alternative first and last exons
- Alternative 5’ and 3’ splice sites
- Alternative ‘tandem’ 5’ and 3’ UTRs
+ Prevalence of alternative splicing in human genome:
- 86-88% of human protein-coding genes are reported to undergo AS
+ Relationship between alternative splicing and complexity:
- Higher number of alternatively spliced genes in broad groups with more cell types
- Vertebrates display a tissue-dependent regulation of AS splicing
+ How do Alu sequences influence splicing?
- Intronic Alu sequences may regulate alternative splicing by shifting splicing patterns through secondary structure changes to pre-mRNAs
- They also contain cryptic splice sites
+ Name the 2 anatomic sites where highest level of exon skipping events occur:
- Brain
- Testis
+ How is alternative splicing involved in pathology of Shigella infection?
- 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
+ TCGA data: how is transcriptomic diversity enhanced in many cancers? Overview of alternative splicing in cancers:
- 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)
