Sex Determination

Question 1

What is splicing?

Answer 1

= The removal of introns from primary RNA & joining together of exons

• Involves 2 trans-esterification reactions
  o Nucleophilic attack at 3’ end of intron
  o Nucleophilic attack at 5’ end of intron forms Lariat loop
  o Lariat released

Question 2

What is the spliceosome?

Answer 2

• Massive macromolecular complex containing 5 small nuclear ribonucleoproteins (snRNPs) and up to 300 other proteins
• The snRNPs are called U1, U2, U4, U5 & U6
• Each contains a single short RNA molecule (100-300 nucleotides in length)
• RNA mols in snRNPs play 2 major roles:
  1. Play a role in recognising intron-exon boundaries
  
  2. Play a role in the catalysis of trans-esterification reactions

Question 3

Steps in the assembly of the spliceosome

Answer 3

1. Splice site definition
   a. U1 is snRNP responsible for defining 5’ splice site and U2 defines 3’ splice site
2. Spliceosome assembly
   a. Catalytically inactive at this point
3. Formation of catalytically active complex (Only becomes active when U1 and U4 are released) and splicing occurs.

Question 4

How do U1 and U2 know where the 3’ and 5’ ends of the introns are?

Answer 4

• U1 recognizes dinucleotide sequence G-U at 5’ end of every intron
• Associated factor of U2 (U2AF35) recognizes dinucleotide sequence AG at 3’ end of every intron.
• Associated factor of U2 (U2AF65) recognizes pyrimidine rich tract upstream of U2AF35.
• Branch-Point site upstream of U2AF65 is recognition site for snRNP U2 itself.
• ALL 4 of these motifs have to be PRESENT and SPACED CORRECTLY for splice sites to be defined.
  o i.e. only introns with all 4 of these motifs in the right place will be spliced out

Question 5

What is alternative splicing?

Answer 5

• Splicing of a primary RNA molecule into different mRNA variants that (usually) encode different proteins.
• Different proteins formed might differ in function, stability, ability to interact with other proteins in the cell etc.
• snRNPs are present in all cells and are required for splicing but do not regulate this process (think general transcription factors). Splicing is instead regulated by splicing factors (think regulatory transcription factors).
• Different cells have different complements of splicing factors.
• Complement of splicing factors present in cell determines how an mRNA is spliced
  o i.e. the same mRNA can be spliced in different ways in different cells.

Question 6

What are the 5 modes of alternative splicing in eukaryotes?

Answer 6

1. Exon skipping
2. Mutually exclusive exons
3. Alternative 5’ donor sites
4. Alternative 3’ acceptor sites
5. Intron retention

Question 7

What are the 2 types of splicing factors?

Answer 7

• SR proteins (Promote splicing):
  o Serine & Argenine – rich splicing factors that bind to splicing enhancers in exon (ESE) or intron sequences (ISE) and promote binding of U1 & U2 snRNPs.
  o i.e. they are promoters
• hnRNPs (repress splicing):
  o Heterologous nuclear RNP splicing factors that bind to splicing suppressors in exon (ESE) or intron sequences (ISS) and inhibit binding of U1 & U2 snRNPs.

Question 8

How do we study splicing?

Answer 8

1) cDNA synthesis
a. Extract RNA from cell of interest
b. Add poly-T primer that binds to poly-A tail of mRNA
c. Add Reverse transcriptase which makes a DNA mol complementary to RNA strand.
d. Treat with RNase to get rid of RNA and use DNA as template for PCR reaction.

2) Perform standard PCR

Question 9

X-chromosome signal elements (XSEs) & Threshold Response Model

Answer 9

• Number of X chromosomes is reflected by the protein levels of 4 X-linked genes:
  o 3 of these encode transcription factors: scute, sisterless & runt
  o The fourth (unpaired) encodes a ligand that activates a maternally supplied TF
  o All 4 TF’s bind to the promoter of a single gene – sex lethal (Sxl)
• These 4 X-linked genes are only expressed for a very small window of time during development (about first 3 hours after fertilization, when blastoderm forms (after 14 divisions))
• In those 3 hours, XX individuals produce enough proteins to reach the threshold and activate the Sxl gene, while XY individuals don’t, and the gene is not activated.
• In the rare haploid individuals (single X chromosome and autosomes, X:A) also form females (i.e. activate the Sxl gene). This is because the nuclear division cycle at which the blastoderm forms is later (at 15 divisions rather than the normal 14)

Question 10

Sex lethal gene

Answer 10

• Sxl lethal is a splicing repressor
• The presence of the Sxl activates an alternative splicing cascade that results in female development
• The first target of Sxl is its own mRNA
• In XX Sxl protein is produced which drives female development and blocks male dosage compensation
• In XY, no Sxl protein is produced so the embryo proceeds down the default pathway which is to become male and activates male dosage compensation.

Question 11

Sex lethal gene Structure

Answer 11

Has 2 promoters:
- Establishment promoter (Pe):
o X-signaling element (XSE) transcription factors bind to this element to activate transcription prior to cellular blastoderm formation (in XX flies only).
o This produces the early Sxl transcript – exon 2&3 is spliced out.
o Early Sex lethal protein is produced.

• Maintenance promoter (Pm):
  o Not regulated by XSEs.
  o Activated in both XX and XY flies after cellular blastoderm formation.
  o This produces the late Sxl transcript.
  o Splicing of this is regulated by Early Sxl.
  o Early Sxl acts as a splicing repressor causing skipping of exon 3 in late Sxl pre-mRNA in females.
• Exon 3 is only present in males (XY), only XX (females) produce early Sxl which causes skipping of exon 3.

Question 12

Splicing of late Sxl pre-mRNA in XX flies

Answer 12

• Exon 3 contains a premature (poison) stop codon, so unless exon 3 is removed, when the ribosome translates this mRNA into protein it will reach this premature stop codon and cease translation. If that occurs you will get the production of a truncated protein that has no biological activity
• In order to get the full length protein being produced, you need to remove that exon.
• So functional late Sxl protein is only produced in XX individuals. Functional late Sxl is NOT produced in XY, because exon 3 isnt removed so premature stop codon is there.

Question 13

Late Sxl protein

Answer 13

• Also a splicing regulater (like early Sxl) but doesn’t regulate itself, it regulates splicing of transformer (tra) pre-mRNA
• tra pre-mRNA has a premature stop codon on exon 2
• both XX and XY flies produce exon 2, however sex specific splicing does occur
  o in XY flies, the full exon 2 is there so translation is prematurely halted
  o in XX flies, the 5’ end of exon 2 has been removed, the premature stop codon is gone, functional full length protein is translated.
  o i.e. functional transformer proteins only produced in XX

Question 14

How does late Sxl direct alternative splicing of tra?

Answer 14

• First clue: there are potential 3’ splice sites (AG dinucleotide motif and pyrimidine rich tract upstream) on either side of the premature stop codon.
  o Proximal 3’ SS = at end of intron 1
  o Distal 3’ SS = (downstream of proximal 3’ SS) end of exon 2
• Concluded that mode of splicing = use of alternative 3’ acceptor sites by U2 snRNP; and presence of Late Sxl protein might force U2 snRNP to bind to Distal 3’ SS rather than the proximal one.

Question 15

Mechanism of gel shift assay

Answer 15

1. Label nucleic acid
2. Add labelled nucleic acid to protein A and B (separately)
3. Load mixtures onto acrylamide gel, perform gel electrophoresis.
4. If labeled nucleic acid + protein mixture has the same banding pattern as the labeled nucleic acid alone.. then there was no interaction between the nucleic acid and the protein
5. If there is an interaction, there should be a higher molecular weight band as well as the ‘free probe’ band.

Question 16

How was Sxl directed alternative splicing of tra investigated?

Answer 16

• Knew that there were splice sites at either end of premature stop codon in exon 2 on tra (Proximal 3’SS and distil 3’ SS)
• Mixed RNA sequences associated with each SS with the 2 proteins (Sxl and U2AF) - did Gel shift
• Findings:
  o Both proteins can bind to the Proximal 3’SS but ONLY U2AF65 can bind to the Distal 3’ SS
  o Different doses of the protein were used to test different affinities of the proteins for this RNA sequence
  o In proximal, at very low concentrations of the proteins (10x10-8) U2AF has virtualy no interaction, while Sxl still has strong interaction.
  - This tells us that Sxl has a stronger affinity for this sequence than U2AF65
  o Sxl has NO affinity for distal SS; binding is basically abolished between U2AF and Distal at 10x10-6 which is much lower than the proximal site.
  - This tells us that although U2AF CAN bind to the Distal SS, it would prefer to bind to the Proximal SS (has a higher affinity for it)
• Conclusion:
  o Sxl competes with U2AF to bind to the proximal 3’ SS of tra; so if it is present it will force the U2AF protein to bind to the distal SS
• Implication for sex determination:
  o In XY flies the Sxl protein is not present, so U2AF binds to the proximal 3’ SS at the end of intron 1, exon 2 is not spliced out so a truncated, inactive protein is produced.
  o In XX flies, the Sxl protein is present so it forces U2AF to use the distal 3’ SS in tra pre-mRNA so the 5’ end of exon 2 is spliced out (including the premature stop codon) and a functional Tra protein is produced.

Question 17

Transformer Protein

Answer 17

• Tra is a SR protein that promotes splicing of a transcript
• It prevents the skipping of exon 4 in dsx pre-mRNA
• Tra will bind to exon 4 in dsx pre-mRNA
• 3’ SS at end of intron 3 is a ‘weak SS’ – sequence there doesn’t match perfectly the sequence UAF are looking for
  o Meaning that they bind to the next available splice site, which is at the end of exon 4
• This means in XY fly (no tra gene because no late Sxl because no early Sxl) that exon 4 will be skipped from dsx pre-mRNA during splicing
• In XX fly, tra will bind to exon 4 and will recruit U2AF to the weak splice site on exon 3, so exon 4 will be in the final mRNA product
  o Important because exon 4 has a polyadenylation sequence, so if its present then a poly-A tail is added to the protein.
• This means that different versions of dsx protein are produced in XX and XY flies
  o XX: have exons 1,2,3,4
  o XY: have exons 1,2,3,5,6

Question 18

Double sex transcription factor

Answer 18

• This is the final point of the alternative splicing cascade
• XX & XY forms of the dsx TF bind to the same enhancers but have opposite effects on gene expression
• N-terminus of 2 forms of the protein are identical (coded from exons 1-3)
  o This encodes DNA-binding region of TF; so both regulate the same set of genes
• C-terminus of 2 forms are different (Female contains protein coding info from exon 4; male contains protein coding info from exons 5&6)
  o Regulatory region; influences how gene expression is controlled
  o Therefore they have opposite effects
• Female version of dsx TF activates female specific genes and represses expression of male specific genes
• Male version of dsx (default) TF activates male specific genes and represses expression of female specific genes.

Question 19

Summary of Sex Determination in Drosophila

Answer 19

XX individual:

• Early Sxl produced -> M dosage comp blocked
• Late Sxl produced
• Tra produced
• Dsx-F produced
• FEMALE

XY Individual:

• No early Sxl produced -> M dosage comp activated
• No Late Sxl produced
• No Tra produced
• Dsx-M produced
• MALE

Question 20

Does Sxl act as a binary switch to determine sex in the Medfly?

Answer 20

No. The transcript is spliced the same way in males and females, and the protein is produced in both males and females.

Question 21

What is the difference between tra in males and females in MedFly?

Answer 21

o Final mRNA in males is larger than that in females becomes males transcript contains exon 2 and 3’ end of exon 1; both of which are absent in female version of final mRNA

o 2 premature stop codons in exon 2, and 1 at 3’ end of exon 1 which are therefore present in males but not in females.

o Therefore; females have functional transformer protein, but males have a truncated, nonfunctional version.

Question 22

How did they test if the transformer protein played a role in sex determination in MedFly?

Answer 22

o Knocked the tra gene out of the mRNA by using RNAi (dicer)
o Injected embryo (anterior pole) with dsRNA; dicer complex that’s present in all eukaryotic cells binds to dsRNA and degrades it into small fragments
o Small fragments loaded onto Argonaute complex which forms the RISC complex.
o 1 or other of those strands on the complex will be degraded
o RISC searches cell for RNA mols complementary to RNA strand it carries. When it finds them it binds to them and degrades them.
o Therefore RISC loaded with ds transformer RNA will seek out native transformer RNA in the cells and degrade that mRNA.
o Therefore by injecting cells with ds traRNA it will lead to the degradation of native transformer mRNA that the embryos are producing

• Findings:
  o Of 272 flies tested, 231 were male
  o This could have been because tra is essential for female development, so by knocking it out only males developed.
  o OR it could be because not have transformer is lethal in XX flies, so by knocking it out, all the females died.
  o The intersex individuals (37 of the 272) were XX, but had become male at the head/apex (where the RNA was injected) but remained female at the tail
• Chromosomal evidence:
  o If first possibility is true, then half of the flies should be XX and half sould be XY (the XX just developed as male, but still have the XX chromosomal arrangement)
  o If the second is true, then there should only be XY individuals because all the XX individuals died.
  o Tested by doing a PCR analysis with primers to a specific region of Y chromosome to distinguish between these 2 hypothesis.
  o RESULT: 4 out of 10 flies were XY individuals, the remaining 6 were XX
  = Means that transformer is essential for female development and by knocking it out we were changing females to males

Question 23

How is differential splicing of the Medfly tra pre-mRNA achieved?

Answer 23

o 8 splice sites in Medfly tra RNA that have a very high similarity to the site on tra RNA in Drosophila that Sxl binds to. All of them are clustered around region of transcript that is removed in females and not males.
o Meaning that transformer regulates the splicing of its own mRNA like Sxl does in drosophila.
o Tra is still responsible for female-specific splicing of dsx like in Drosophila.

This is achieved by:
o Embryo is provided with correctly spliced transformer mRNA from the mother
o Both XX and XY embryos contain maternal derived, female-spliced transformer mRNA
o In females that is translated into TRA protein and acts to splice transformer mRNA produced by embryo itself into the female specific version which leads to production of female version of dsx
o In males, active Y mechanism, that prevents the establishment of autoregulatory loop.
- Could be protein synthesis of that TF is blocked
- Or the maternal protein is destroyed
- Or there is another protein factor that inhibits that

Question 24

Sex Determination in Honeybees

Answer 24

• No sex chromosomes
• Csd (complementary sex determination) haploid or homozygous diploid (rare) = male
• Csd heterozygous diploid = female
• Dsx is also present in bees & is spliced in a sex-specific manner
• Females can produce 2 different transcripts, males can produce 1
• Like in drosophila, 5’ end of these transcripts is the same – so the N-terminus of the proteins produced is the same which encode for DNA-binding region of the TF
• Exons at 3’ end of transcripts are different, so C-terminal of protein is different
• ‘fem’ protein is required for female-specific splicing of dsx
  o only produced in individuals that are heterozygous at csd locus.
• Loss of fem results in male development