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Alternative Splicing and Sex Determination Complete Summary
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Content covered during the alternative splicing module including the drosophila, medfly and bee case study for sex determination
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Alternative Splicing - Module 2Alternative Splicing – Gene Regulation
à Case study: sex determination in insects
RNA SPLICING OVERVIEW =
• Removal of introns from primary mRNA transcript + joining of exons
• Produces different mRNA variants that usually encode different proteins
o Differ in:
o Structure
o Stability
o Ability to interact with other proteins
• Disproves one gene one protein hypothesis in eukaryotes
• 2 trans-esterification reactions
o 1st nucleophilic attack at 5’ end of intron
o Formation of lariat intermediate
o 2nd nucleophilic attack at 3’ end of intron
o Results in removal of intron (lariat loop) + exons are joined together
SPLICEOSOME
• Massive molecular complex
• 5 small nuclear ribonucleoproteins (snRNPs) + up to 300 proteins
• snRNPs =
o U1
o U2
o U4
o U5
o U6
• Each snRNP = short RNA molecule (100-300 nucleotides)
o Role of RNAs in each snRNP
§ Recognition of intron exon boundaries
§ Definition of boundaries
• U1 snRNP à recognition of 5’ end of intron
• U2 defines 3’ boundary
§ Catalysis of trans esterification rxns
• Assembly of spliceosome = highly ordered
1) Splice site definition
a. U1 at 5’
b. U2 at 3’
2) Spliceosome assembly
a. Other snRNPs join
b. Catalytically inactive
3) Formation of catalytically active complex + splicing
a. Release of U1 and U4 activates complex
b. Now catalytically competent
RECOGNITION OF INTRON EXON BOUNDARIES
• Recognition of sequence motifs
• 5’ end has GU to be recognized by U1
• 3’ end has AG to be recognized by associated factor of U2 (U2AF35)
• Pyrimidine rich tract upstream of 3’ end is recognized by associated factor of U2
(U2AF65)
, o Cytosine and uracil rich
• Branch point site is recognized by U2 snRNP
*all four motifs are necessary in correct spacing!!!
• snRNPs = analogous to GTFs
o required for splicing but do not regulate the process
• splicing factors à analogous to RTFs
o determine how splicing occurs
ACTIVATION OF SPLICING
• SR proteins
o Serine and arginine rich
• Bind to nucleotide sequences in primary mRNA
o Inside an exon or intron
• Mechanism -- Help U1 and U2 to bind to 5’ and 3’ splice sites
REPRESSION OF SPLICING
• Mechanism -- Inhibit U1 and U2 binding by binding to splicing suppressors in exon
(ESS) or intron (ISS) sequences
• Recognize specific nucleotide sequences
• hnRNPs = heterologous nuclear RNP splicing factors
• every cell has same complement of snRNPs but NOT splicing factors
o different cells = different splicing factors
§ timing
§ development
§ different tissues
o different splicing of same pre-mRNA in different cells
eg. Alpha-tropomyosin gene à tissue specific splicing
• different tissue produces different final mRNA via alternative splicing
5 MODELS OF ALTERNATIVE SPLICING IN EUKARYOTES
• Alternative splicing à production of multiple final mRNA transcripts from a single
primary mRNA strand via different combinations of exons
1. exon skipping
a. most common
b. exon is present or absent in final mRNA
2. mutually exclusive exons
a. two exons but only one present in final mRNA
3. alternative 5’ donor sites
a. more than one site to be recognized by U1
4. alternative 3’ acceptor sites
a. more than one site to be recognized by U2
5. intron retention
a. most rare
b. sequence is removed or not removed
, c. contains an open reading frame that a ribosome can read
HOW TO STUDY SPLICING
• cDNA synthesis
o convert RNA to DNA and then perform PCR reactions
o addition of poly-T primer
o add reverse transcriptase to make DNA complementary to RNA
o treat with RNase and can now use as PCR template
• PCR primers outside of the region to be alternatively spliced
o Run an agarose gel
SEX DETERMIANTION IN INSECTS
à drosophila
• male = XY
• females = XX
• humans = active Y mechanism of sex determination
o presence of Y changes sex development from default female
• in drosophila- mechanism of sex determination is different albeit that genetic
complement that determines sex is the same
o number of X chromosomes determines sex
o XO flies = male
*sex determination depends on the number of X chromosomes and first occurs though
transcriptional regulation of the sex lethal gene
• Transcription of sex lethal leads to cascade of events that influence sex (3
independent pathways)
o Do flies look and act like females or males
o Do germ cells develop as eggs or sperms
o Dosage compensation by doubling rate of transcription of X linked genes in
males (transcribe X lined genes at double the rate)
• Male = default developmental pathway in flies
o Brought about by presence of 1 x chromosome
o 2 x chromosomes are required to activate transcription of sex lethal gene
X CHROMOSOME SIGNAL ELEMENTS
• Number of x chromosomes is determined by expression level of 4 X linked genes
o 3 of the genes à encode transcription factors that bind to a promotor in the
sex lethal gene
§ Scute
§ Sisterless
§ Runt
th
o 4 gene à encodes a ligand that activates a maternally supported TF
o à these activators = only on X chromosome
§ XX individuals have 2 x as much of the 4 genes
• Allows for sufficient concentration of activators to allow Sxl
transcription to occur
o All 4 TFs bind to the promotor of the SEX LETHAL gene (Sxl)
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