BIO 344 1st Edition Lecture 12Outline of Last Lecture I. Post transcriptional regulation of gene expressionII. Introns and exonsa. DNA hybridizationb. Conserved sequencesIII. Splicing stagesa. Assembly of spliceosomei. snRNPIV. intron vs. econ definitiona. mutations in exon vs intron definition Outline of Current Lecture I. Alternative splicing—4 patternsa. Optional exonb. Optional intronc. Mutually exclusive exclusiond. Internal splice siteII. Observing Alternative splicing in nerve growth and sex determination of Drosophiliaa. Nerve growthi. Dscamb. Sex determinationi. Sxl, tra, dsxii. Activators vs. repressorsCurrent Lecture4 Patterns of alternative splicing- Optional exonThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.o Skip or include exon- Optional introno Skip or include intron- Mutually exclusive excusion- Internal splice siteObserving Alternative splicing in nerve growth and sex determination of Drosophilia- Nerve growth-- Neural projections interact with others but not each othero as neurons grow, Dscam1 expression tells projections from the same neuron to grow away from each other (repulsion) and towards projections of other neurons(interaction)o Dscam gene is required for this wiring- How do they recognize themselves as different from other cells/neuronso Alternative splicing Dscam pre-mRNA can generate 38,016 splice variants- Different exons have several different variants each—many combinations- Increases complexity of the genome- Sex determinationo X chromosome number determines sex Need a greater X:A ratio to be a female- 2X:1A= female Sex specific transcription factors push female sex differentiation expression Sxl= sex lethal—starts generating female cells- Regulates itself and tra, and tra regulates dsx (double sex) In males, exon 3 is included in Sxl and excluded in females- Including exon 3 includes a stop codon, making the gene nonfunctional- Sxl nonfunctional= male In tra, exon 2 is included in males and excluded in females- Also includes a stop codon, making the gene nonfunctional In dsx, females have exon 4 and males have exon 5 and 6 at C terminuso Activators= enhance splice evento Repressors= inhibit splice event Repression is more efficient in absence of regulatoro Negative or positive regulation Is Sxl a repressor or activator?- If repressor:o and if loss of function (male)o blocks utilization of exon 2 (female)o will bind at 3’ splice site of exon 2o If exon 3 is mutated and there is no effect, sxl is a repressor- If activator:o And if loss of function (male)o Utilizes exon 3 (male)o Will bind at 5’ splice site of exon 3o Sxl is bound to the RNA target RNA sequence is GUUGUUUUUUUU (poly pyrimidine track)- Immediately downstream of 3’ splice site- Repressor—competes with U2AF binding- Association of U@AF with pyrimidine-rich region, assists U2 snRNP to associate with branch siteo Tra in combination with Tra2 regulates Dsx splicing Tra2 binds on Dsx exon 4 Activator—binding to exon enhancer elements, which bind SR proteins (tra2 is an SR- Tra-Tra2 bound enhances binding of U2AFo ESS (exon splicing suppressor) Also controls SR binding When bound to SR on an exon, exon is spliced- See slide for an overall summary of sex determination alternative
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