Week 9 - Regulation of RNA Splicing

Question 1

Mechanisms of RNA splicing

Answer 1

1. Self splicing
2. Splicing that required additional factorsS

Question 2

Self-splicing

Answer 2

where intron removes itself without other factors (Discovery of ribozyme)

Question 3

splicing that required additional factors

Answer 3

assembly of the spliceosome, RNA components important for catalysis of splicing
- also the ribozyme

Question 4

5’ SS

Answer 4

pGU
- first 2 nucleotides of intron

Question 5

3’ SS

Answer 5

AGp
- last 2 nuc

Question 6

Branch point (BP)

Answer 6

A: ribo-base

Question 7

Mechanism of splicing

Answer 7

1. cleaved at 5’ SS, joins to A (BP) –> produces lariat
   
   • lariat and intro still attached to exon 2
2. exon 1 and 2 are joined by removal of intron which is in lariat structure
3. U1 and U2 (ribonucleoprotein particles) bind to 5’ and 3’ SS respectively
4. Then U4, 5 and 6 enter complex (pre-catalytic spliceosome)
5. U1 and U4 dissociate from complex
   
   • leaving the activated spliceosome
6. 5’ SS cleaved, transesterfication hooked up to A ribonuc. of the BP
   
   • creates lariat, producing the catalytic spliceosome where 2nd reaction occurs
7. exon 1 and 2 are fused together to create postspliceosomal complex
8. mRNA will dissociate from intron and associated proteins
9. associated proteins to the intron will dissociate and degrade

Question 8

transcription hubs are also ____ and composed of ___

Answer 8

splicing hubs, intrinsically disordered proteins

Question 9

is splicing co-translational?

Answer 9

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

Question 10

Alternative Splicing mechanisms

Answer 10

1. Alternative PolyA Sites
2. Alternative 5’ SS
3. Alternative 3’ SS
4. Alternative promoters
5. Mutually exclusive inclusion
6. Exon included for excluded (differentially spliced in mRNA)
7. Retained intron

Question 11

Positive Regulatory elements

Answer 11

RNA Cis elements:
- ISE intron splicing enhancer
- ESE exon splicing enhancer

Trans elements:
SR serine arginine repeat proteins

Question 12

Negative Regulatory elements

Answer 12

RNA Cis elements:
- ISS intron splicing silencer
- ESS exon splicing silencer

Trans elements:
hnRNP heterogenous nuclear ribonucleaoprotein particle

Question 13

Sex determination ratios in drosophila

Answer 13

• difference in X chromosome to autosome ratio
  2X:2A = 1 –> Female
  1X:2A = 0.5 –> Male

Question 14

Sex determination

2X:2A = 1 –> Female

Answer 14

• 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

Question 15

Sex determination

1X:2A = 0.5 –> Male

Answer 15

• 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

Question 16

What is the level of regulation for TRA and DSX?

Answer 16

Alternative Splicing

Question 17

DSX splicing using _______

Answer 17

Alternative polyA sites

Question 18

Dosage compensation of SXL in drosophila

Answer 18

when SXL removed from females = die
when SXL expressed in males = die

Question 19

TRA and TRA2; between sexes

Answer 19

• 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

Question 20

Loss of dsxM and dsxF

Answer 20

flies with both M and F genitals

Question 21

TRA is regulated by _______

Answer 21

Alternative 3’ splice sites

Question 22

SXL protein recognizes _____ interrupted by _____

Answer 22

polyU tracts, Gs

ex: GUUGUUUU

Question 23

Male specific lethals (MSL)

Answer 23

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

Question 24

MSL2 expression

Answer 24

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

Question 25

Retained introns and SXL

Answer 25

• SXL (male): intron removed = EXPRESSION OF MSL2
  + SXL (female): retained intron

Question 26

SXL expression and transcription of lambda repressor are examples of a _________________

Answer 26

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

Question 27

Promoter maintenance (Pm)

Answer 27

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

Question 28

Differential splicing - Exon included or excluded: Pm

Answer 28

+ 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

Question 29

Drosophila male courtship behaviours

Answer 29

1. Orienting
2. Tapping
3. Singing
4. Licking
5. Attempting copulation

Question 30

fru

Answer 30

important for M courtship behaviours
- activated in M but not F
- fru activates from which relates to male behaviour

Question 31

Splicing form the P1 fru promoter

ALTERNATIVE 5’ SS

Answer 31

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

Question 32

Riley day syndrome

Answer 32

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

Question 33

Familial dysautonomia FD gene

Answer 33

IKBKAP gene –> very long
- exon 20 is skipped because U1 cannot bind to 5’ SS
- causes a 6 bp change

Question 34

CLIP Seq

Answer 34

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

Question 35

Intragenic trans-splicing

Answer 35

DNA has 1 exon ‘2’ - > mature mRNA exon 2 has been duplicated

Question 36

Intergenic trans-splicing

Answer 36

pre-mRNA from gene 1 and 2 are joined through trans-splicing