Week 9 - Regulation of RNA Splicing Flashcards
Mechanisms of RNA splicing
- Self splicing
- Splicing that required additional factorsS
Self-splicing
where intron removes itself without other factors (Discovery of ribozyme)
splicing that required additional factors
assembly of the spliceosome, RNA components important for catalysis of splicing
- also the ribozyme
5’ SS
pGU
- first 2 nucleotides of intron
3’ SS
AGp
- last 2 nuc
Branch point (BP)
A: ribo-base
Mechanism of splicing
- cleaved at 5’ SS, joins to A (BP) –> produces lariat
- lariat and intro still attached to exon 2
- exon 1 and 2 are joined by removal of intron which is in lariat structure
- U1 and U2 (ribonucleoprotein particles) bind to 5’ and 3’ SS respectively
- Then U4, 5 and 6 enter complex (pre-catalytic spliceosome)
- U1 and U4 dissociate from complex
- leaving the activated spliceosome
- 5’ SS cleaved, transesterfication hooked up to A ribonuc. of the BP
- creates lariat, producing the catalytic spliceosome where 2nd reaction occurs
- exon 1 and 2 are fused together to create postspliceosomal complex
- mRNA will dissociate from intron and associated proteins
- associated proteins to the intron will dissociate and degrade
transcription hubs are also ____ and composed of ___
splicing hubs, intrinsically disordered proteins
is splicing co-translational?
can be detected using RT imaging of splicing because transcript in being synthesized with RNAP2
- can use RNA b-box fused to lambda N fused GFP
- compare, if one intron low, while other is high / vice versa. can be indicator of cotranslational splicing
Alternative Splicing mechanisms
- Alternative PolyA Sites
- Alternative 5’ SS
- Alternative 3’ SS
- Alternative promoters
- Mutually exclusive inclusion
- Exon included for excluded (differentially spliced in mRNA)
- Retained intron
Positive Regulatory elements
RNA Cis elements:
- ISE intron splicing enhancer
- ESE exon splicing enhancer
Trans elements:
SR serine arginine repeat proteins
Negative Regulatory elements
RNA Cis elements:
- ISS intron splicing silencer
- ESS exon splicing silencer
Trans elements:
hnRNP heterogenous nuclear ribonucleaoprotein particle
Sex determination ratios in drosophila
- difference in X chromosome to autosome ratio
2X:2A = 1 –> Female
1X:2A = 0.5 –> Male
Sex determination
2X:2A = 1 –> Female
- SXL bind to intron of TRA pre-mRNA and pushes U2 to use 3’ SS further down pre-mRNA (SXL BS = ISS)
- SXL blocks male 3’ SS from being used THEREFORE IS A NEGATIVE REGULATOR OF SPLCING
- transcriptional event leads to active. of Pe on exon 2 (exon 3 excluded through splicing)
- downstream 3’ SS on exon 2 used
- SXL protein expressed
- TRA protein expressed
- TRA2 binds
- REGULATES SPLICING OF DSX
- exons 1-4 expressed
- dsxF expressed
Sex determination
1X:2A = 0.5 –> Male
- U1 binds to 5’ SS and U2AF binds to the male 3’ SS.
- different 3’ SS used on exon 2 compared to F transcript – > premature stop codon = truncated protein with missing information
- No SXL expressed
- no TRA expressed
- TRA 2 expressed but cannot bind
- no splicing, exon 4 excluded
- exons 1,2,3,5,
- dsxM expressed
What is the level of regulation for TRA and DSX?
Alternative Splicing
DSX splicing using _______
Alternative polyA sites
Dosage compensation of SXL in drosophila
when SXL removed from females = die
when SXL expressed in males = die
TRA and TRA2; between sexes
- TRA2 BS are in Exon 4 and are ESE
- TRA2 binds to exon 4 in females because needs TRA present to bind
- TRA2 present in males but cannot bind due due to -SXL resulting in DSXm expression
- removal transforms F to M
- expression of TRA in M transforms them to F
Loss of dsxM and dsxF
flies with both M and F genitals
TRA is regulated by _______
Alternative 3’ splice sites
SXL protein recognizes _____ interrupted by _____
polyU tracts, Gs
ex: GUUGUUUU
Male specific lethals (MSL)
5 proteins + ROX RNA form a complex (localized on X chromosome in males)
- x chromosome in M drosophila are hyper activated and ensures genes here are transcribed 2x
MSL2 expression
activates hyper-transcription in males:
+ SXL (female): SXL binds to intron of exon 2 and suppresses excision of intron = MSL2 NOT EXPRESSED
- SXL (male): no inhibition of intron excision
- MSL2 expressed and X hyper transcribed
Retained introns and SXL
- SXL (male): intron removed = EXPRESSION OF MSL2
+ SXL (female): retained intron
SXL expression and transcription of lambda repressor are examples of a _________________
stable binary switch because SXL expression initially established during early embryogenesis from a transiently expressed Pe promoter, later SXL expression maintained by + auto regulation of splicing transcript expressed from Pm
Promoter maintenance (Pm)
SXL transcribed in M and F by Pm in embryogenesis
- on exon 1, results in pre-mRNA on this structure where SXL binds to either side of exon 3
Differential splicing - Exon included or excluded: Pm
+ SXL(female) = transcript with NO exon 3
- active SXL that binds to SXL pre-mRNA auto regulating its expression (AT LEVEL OF SPLICING)
- SXL (male) = all 5 exons and premature stop codon
- express a truncated INACTIVE SXL
Drosophila male courtship behaviours
- Orienting
- Tapping
- Singing
- Licking
- Attempting copulation
fru
important for M courtship behaviours
- activated in M but not F
- fru activates from which relates to male behaviour
Splicing form the P1 fru promoter
ALTERNATIVE 5’ SS
female: TRA + TRA2 = spliced so that there is an extension of exon 2
- no protein has been detected from this transcript
male: TRA2 = spliced with shorter exon 2
- transcript is translated to give the from protein
Riley day syndrome
congenital sensory neuropathies found in the Ashkenazi Jewish population
- small population have genetic conditions due to flounder effect –> 1 individual in a population creates a disproportionate freq. of the allele in the small pop
Familial dysautonomia FD gene
IKBKAP gene –> very long
- exon 20 is skipped because U1 cannot bind to 5’ SS
- causes a 6 bp change
CLIP Seq
maps what RNA is recognized by RNA binding factor
1. crosslink with UV light (protein w RNA)
2. digest RNA with nuclease (removes most RNA except RNA surrounding protein)
3. Immunoprecipitate (need antibody )
4. Convert RNA to DNA and sequence (sequences are mapped to seq of pre-mRNA transcribed from genome)
5. Align sequences to pre-mRNA sequence –> CAN DETERMINE WHERE PROTEINS HAVE BOUND
Intragenic trans-splicing
DNA has 1 exon ‘2’ - > mature mRNA exon 2 has been duplicated
Intergenic trans-splicing
pre-mRNA from gene 1 and 2 are joined through trans-splicing
