Assignment 2

Mark Botirius11/16/16

Assignment 2

Describe the relationship between alternative splicing and sex determination in the fruit fly Drosophila melanogaster, including the characteristics and numbers of introns/exons, mechanism of splicing and proteins involved.

Sex determination in Drosophila melanogaster is primarily centered around the

presence or absence of the sex lethal (Sxl) protein, which then establishes the roles of two

other proteins (Transformer, or Tra, and Doublesex, or Dsx) in the molecular pathway that

determines the sex of the organism. In the presence of the Sxl protein, the organism

produces a functional Tra protein, which then causes the organism to produce a female

version of the Dsx protein that results in a female fly. On the other hand, in the absence of

the Sxl protein, a dysfunctional version of the Tra protein is produced, which then causes

the organism to produce a male version of the Dsx protein, resulting in a male fly. This is

related to alternative spicing in that the Sxl protein determines how the Tra and Dsx pre

mRNAs are spliced, which in turn determines which Tra and Dsx proteins are produced,

which finally establishes the sex of the fly. (Pierce, 2012, p. 471) Therefore, I will address

each protein as a step in the pathway and discuss how alternative splicing acts to ultimately

produce a female or male fly. Conveniently, the first step (whether or not the fly produces

the Sxl protein) is common to both the male and female pathways since it is, of course,

antecedent to where the two diverge.

Step 1. Sxl protein production.

Interestingly, the Sxl gene has two promoters, SxlPe (the “promoter for

Establishment”) and SxlPm (the “promoter for Maintenance”) and the products of these two

promoters is different. If transcription originates at the SxlPe promoter, then it produces a

fully functional Sxl protein. If transcription originates at the SxlPm promoter, then it produces

a transcript that contains an extra exon that has a stop codon. This stop codon results in a

truncated, defective Sxl protein. Therefore, in order to produce a functional Sxl protein,

transcription must begin at the SxlPe site. Obviously, in females, transcription begins at the

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SxlPe site, whereas in males it doesn’t. The reason why has to do with something called

“numerator” and “denominator” elements.

Transcription of the SxlPe gene requires a couple of activators called sisA and sisB.

The genes for these two proteins are located on the X chromosomes and they are the

numerator elements. The main denominator element is the repressor protein Dpn

(Deadpan) which is located on the autosomes. Since female’s have two X chromosomes and

two sets of autosomes, their numerator (X) is 2, and their denominator (A) is also 2, for an

X/A ratio of 2/2, or 1. For male’s, they have only one X chromosome and two sets of

autosomes for an X/A ratio of ½. Since the Sxl gene is located on the X chromosome,

females have double the Sxl, SisA, and SisB genes as males and the same number of Dpn

genes. As a result, females have enough SisA and SisB activators to overcome the Dpn

repressors and transcription proceeds from the SxlPe promoter, whereas males do not have a

sufficient number of activators for transcription to proceed from the SxlPe promoter due to

the Dpn repressor. At the end of the 14th embryonic cycle of the fly’s development,

(Ashburner, Golic, & Hawley, 2005) the SxlPe promoter becomes inactive, and all Sxl

transcription must thereafter proceed from the SxlPm promoter for both males and females.

(Salz & Erickson, 2010)

Step 2. Sxl maintenance. (Exon skipping)

At this stage, the transcription of the Sxl gene is proceeding from only the SxlPM

promoter in both males and females, yet females are still producing Sxl proteins while males

are not. The reason is because the splicing of the pre mRNA transcript of the Sxl gene is

controlled by non-other than the Sxl protein itself. Recall that the transcript produced from

the SxlPm promoter contains an extra exon that has a stop codon that produces a truncated,

defective Sxl protein. In the presence of pre-existing Sxl proteins, however, the pre-mRNA

spliceosome is directed to splice out the extra exon, producing an mRNA that produces

additional fully functioning Sxl proteins. The Sxl proteins do this by binding to poly U

sequences in introns above and below the bad exon (specifically, exon 3) causing the

spliceosome to “choose” splice sites on either side of the exon, thereby skipping it (cutting it

out). Since, at this stage, only females have Sxl proteins produced from SxlPe promoters,

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only females have the ability to splice out the offending exon and continue to produce

more, functional Sxl proteins from SxlPm promoters. As a result, only females have, and have

the ability to continue to have, Sxl proteins. This type of alternative splicing is well known. It

is called “exon skipping” (Ashburner, Golic, & Hawley, 2005, p. 901)

Step 3. Tra protein production (Alternative splicing)

Both female and male flies continually transcribe Tra genes, however only females

produce functional Tra proteins. The pre – mRNA transcript in males and females is

identical, and the first intron contains a nonsense codon in its open reading frame. Flanking

this nonsense codon upstream and downstream are two alternative splice sites. In female

flies, Sxl blocks the U2AF spliceosome subunit from binding to the upstream binding site.

Consequently, U2AF binds to an alternative, downstream binding site which excludes the

nonsense codon from the mRNA and a functional Tra protein is made. In male flies, the

absence of Sxl allows the spliceosome to bind to the higher affinity upstream site producing

an mRNA with a nonsense codon that makes a truncated, defective protein. The Tra protein

is important because it regulates the last protein under consideration, the Dsx (Doublesex)

protein.

Step 4. Two proteins from the same pre – mRNA transcript (DsxF

and DsxM).

Lastly, we arrive at the Dsx Protein. This is the only pre – mRNA that produces two

working versions of the protein from the same transcript. This time, the splicing responsible

for which version is made is not regulated by the Sxl protein. It is instead regulated by the

Tra protein which, as was already mentioned, is only produced in females. In males, the lack

of a working Tra protein causes the Dsx pre – mRNA to be spliced to produce the male Dsx

protein (which is, by the way, the default splicing pattern) (Penn, 2006) that acts as a

regulator controlling male specific genes and a repressor of female genes. (Watson, 2014, p.

495) In the presence of Tra, the opposite result obviously follows. This time, the Tra protein

resembles a splicing regulator (SR) that directs the spliceosome to female specific splice

sites.

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Conclusion

It should be noted that this paper is only intended to be a brief overview of the

relationship between sex determination and alternative splicing in D melanogaster. In order

to stay close to the three-page limit, many proteins and their splicing patterns have been

omitted that were related to, but not essential to, the topic. Examples are the Tra2 gene

(constitutively expressed, and therefore not regulated sexually), and the MSL-2 (Male –

specific lethal) gene which is actually related to dosage compensation (males have only one

X chromosome relative to two in females), and not sex determination.

Works CitedAshburner, M., Golic, K. G., & Hawley, R. S. (2005). Drosophila, A laboratory handbook (2nd ed.).

Cold Spring Harbor: Cold Spring Harbor Laboratory Press.

Penn, J. K. (2006). Regulation of, by, and for Sex-Lethal in Drosophila Melanogaster. Princeton University, Pro Quest Dissertations Publishing.

Pierce, B. A. (2012). Genetics, A Conceptual Approach. New York: W.H. Freeman and Company.

Salz, H. K., & Erickson, J. W. (2010). Sex Determination in Drosophila. The view from the top. PMC.

Watson, e. a. (2014). Molecular Biology of the Gene (7th ed.). Boston: Pearson.

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Assignment 2