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Genes and Genomics > Alternative Splicing & Introns > Flashcards

Alternative Splicing & Introns Flashcards

1
Q

Splicing Components

A

Within introns, a donor site (5’ end of the intron), a branch site (near the 3’ end of the intron) and an acceptor site (3’ end of the intron) are required for splicing. The splice donor site includes an almost invariant sequence GU at the 5’ end of the intron. The splice acceptor site at the 3’ end of the intron terminates the intron with an almost invariant AG sequence. Further upstream is the branchpoint, which includes an adenine nucleotide involved in lariat formation

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2
Q

Self-Splicing

A
  1. The 2’OH of a specific adenosine in the intron attacks the 5’ splice site, thereby forming the lariat
  2. The 3’OH of the 5’ exon triggers the second transesterification at the 3’ splice site, thereby joining the exons together.
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3
Q

Spliceosome

A
  • formed of 5 small nuclear RNAs associating with proteins to form small nuclear ribonucleic particles
  • snRNAs: U1,2,4,5,6
  • snRNPs + accessory proteins = spliceosomes
  • attach to transcript in sequential manner
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4
Q

Assembly of Spliceosome

A
  1. U1/2 assemble onto pre-mRNA in cotranscriptional manner
    - U1 binds 5’ end of intron (donor site)
    - U2 binds 3’ intron end (acceptor site)
  2. U1/2 snRNPs interact with each other to form prespliceosome (complex A)
    - 2 exons brought together
  3. preassembled tri snRNP U4,6,5 recruited to form complex B
  4. complex B forms catalytically active complex
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5
Q

Activity of Spliceosome

A
  1. complex B carries out the first catalytic step of splicing generating complex C containing free exon 1 and intron-exon 2 lariat intermediate
  2. complex c undergoes additional rearrangements and carries out second catalytic step
    - ligates 5’ exon to 3’ exon
    - post splice complex with spliced exons and lariat intron
  3. release of lariat and spliced mRNA
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6
Q

Alternative Splicing

A
  • one transcript can have 2 or more splicing pathways
  • related but different mRNAs
  • exons retained or skipped
  • introns excised or retained
  • 5’ & 3’ splice site positions moved to change length
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7
Q

DSCAM Splicing

A
  • membrane anchored cell surface protein with noles in neuronal development
  • 24 exons permit 38,016 protein variants
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8
Q

Splicing Regulation

A
  • can play a role in regulation supplementing transcriptional control
  • insertion/deletion of specific domains controlling gene expression
  • regulated antibody and neuropeptide production
  • act of splicing coupled to nuclear export (intron presence reduces export)
  • regulates balance of function to nonfunctional RNAs
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9
Q

Effect of mRNA and Protein

A
  • rate of translation
  • mRNA degradation
  • insertion/deletion of residues or domains
  • truncation with stop codons
  • changes localisation of protein
  • change affinity of protein
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10
Q

5’ transcript splice types

A
  • 5’ transcript ends differ
  • differential start sites causes different processing and different splicing complexes
  • strength of donor sites differs
    eg. mouse a-amylase has 2 start sites forming a salivary amylase or liver amylase
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11
Q

3’ transcript splice types

A
  • 3’ ends differ
  • different polyA sites used for termination
    eg. immunoglobin chains of antibodies have different stop sites either coding for an additional TM anchor or making free antibodies
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12
Q

Middle portions splice types

A
  • not explained by differential promoter use or cleavage
  • internal exon combinations give permutations
    eg. troponin T gene in skeletal muscle
  • spliced in 64 different ways due to tissue specific splicing factors acting on pre-mRNA
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13
Q

Tissue Specific Splicing Factor

A
  • recognise cis-acting sequences in transcript

- promote or inhibit splice sites in different cases

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14
Q

SXL in Drosophila

A
  • autoregulates own splicing
  • in absence exon 2 splices with exon 1 to form a stop codon and a truncated male protein
  • sxl binds intron and inhibits factors at splice acceptor site
  • exon 3 included for a functional sxl protein
  • initial sxl expression caused by imbalance of X via autosomes
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15
Q

Tra in Drosophila

A
  • females have sxl protein
  • sxl binds at proximal splice sites in intron 1 preventing U2AF binding
  • binds cryptic site in exon 2: splicing out of stop codon
  • males have splicing from 1 exon to the next
  • no SxL so short protein formed (non functional)
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16
Q

Dsx in Drosophila

A
  • encodes transcriptional repressor determining development
  • tra recruited to exon splicing enchancer by tra 2, promoting exon 2 inclusion
  • skips exon 4 (terminates)
  • transcriptional termination
  • males have dsx mRNA ending with exon 4 so a shorter protein isoform
17
Q

Fru in Drosophila

A
  • fru splicing regulated by Tra
  • in males tra promotes splicing from end of exon 2 causing stop codon inclusion (no female isoform made)
  • splicing of exon 1 to exon 3
  • male fru regulates male behaviour/phenotypes and courtship
  • knockouts court other males
  • in females a stop codon is included
18
Q

Splicing and Signal Resposne

A

eg. SLO
- potassium channel gene
- affected by intracellular calcium levels
- high levels of calcium phosphorylates factor binding protein CaRRE site in premRNA
- causes splicing of STREX domain out of protein
- homeostatic mechanism
- STREX causes increased sensitivity to calcium levels

19
Q

Regulating Factors

A

serine arginine rich proteins (SR proteins)
- influences splice site recognition by binding teh 5’ splice site and promoting U1 binding (promotes splice)
- bind ESE in downstream exon (use of splice acceptor site)
heterogenous nuclear ribonucleoproteins
- inhibit splicing

20
Q

Splicing and Transcription

A
  • splicing occurs as transcription is occuring
  • rate of elongation affects splicing
  • slower: more likely to include weak exon acceptor sites
21
Q

Targeted DamID

A

For DamID, Escherichia coli DNA adenine methyltransferase (Dam) protein is fused to a DNA- or chromatin-binding protein of interest. When the fusion protein is expressed in vivo, its binding sites are tagged by adenine methylation of the sequence GATC, enabling DNA fragments containing the sites to be isolated after enzymatic digestion.

  • used to profile interaction of splicing factors with genome in cell type specific manner??
  • results looked promising but not repeatable
22
Q

Introns Origins

A

Intron-Early:
- originated in prokaryotes but were lost by genome compression
- exon theory of gene evolution: shuffling permitted by exons allowed more genes/complex genes/more proteins
- no indications of prokaryotes having any introns/snRNAs
Intron-Late:
- only evolved in eukaryotic branch of life
- random placement into genes
Current Theory:
- last common eukaryotic ancestry had intron rich genes thanks to invasion during eukaryotic cell formation after endosymbiosis

23
Q

Intron Life Phases

A
  • genomic intron
  • transcribed intron
  • intron being spliced
  • excised intron
  • EJC-haboring transcript (sites where intron was excised)
24
Q

Genomic Intron

A
  • transcription initiation with TF binding sites, silencers, enhancers
  • location of genes cis-regulatory elements
  • can have alternative promoters for different tissues (a-fetoprotein
  • regulate transcription termination, polyadenylation, cleavage
  • nested genes found in introns
25
Q

Flt-1 gene

A
  • soluble version inhibits angiogenesis by binding to vascular endothelial growth factor
  • gene has 3 polyA signals for termination sites
  • soluble form: no exon 14
  • membrane form: includes exon 14
26
Q

Transcribed Introns

A
  • regulates timing and expression of protein

- feedback loops

27
Q

HES7 gene

A
  • oscillation of protein levels important for directing mesoderm cells to form somites during development
  • oscillation dependent on delay in transcription, mRNA, and protein time
  • HES7 represses its own transcription
  • removing introns mutates somite development
28
Q

Spliced Introns

A

splicing linked to transcription via RNAPII C terminal domain
- affects all stages of transcription
Initiation:
- intron inclusion site close to 5’ end
- recruits splicing factors that then recruit transcriptional machinery
Elongation:
- interact with elongation factors for increased efficiency
Termination:
- endonucleolytic cleavage and polyA tail addition

29
Q

Excised Introns

A
  • excised nitrons undergo debranching and degradation
  • embedded RNA genes expressed on removal
  • non protein coding RNAs
30
Q

Mirtrons

A
  • non coding RNAs
  • spliced as hairpin intron
  • cleaved by dicer
  • forms RISC complex targeting mRNA degradation
31
Q

snoRNAs

A
  • RNA modifications
  • released after spliing
  • forms snoRNA assembly where they enter the nucleus and cause methylation and pseudouridytion
32
Q

EJC-Haboring Transcripts

A
  • exon junction complex
  • binds upstream of exon-exon junction on mRNA transcript
  • 4 core proteins
  • present from splicing to translation
  • involved in nuclear transport, translation activation, mRNA localisation, nonsense mediated decay
33
Q

Nuclear Transport (EJC)

A
  • mature mRNA binds to mRNA specific transport factors
  • shuttled through nuclear pore
  • EJC increases rate of export
34
Q

Nonsense Mediated Decay (EJC)

A
  • degrade mRNA with premature stop codon to prevent dominant negative/gain of function proteins
  • mRNA marking model: splicing dependent, EJC downstream of termination codon = premature = degradation
  • ribosome reaches EJC and displaces
  • if premature stop codon is before the EJC the decay process is triggered (position is a regulator)
  • ribosome pauses and UPF1,2,3 bind to EJC
  • causes RNA degradation via UPF phosphorylation

Genes and Genomics (13 decks)

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