Module 6 ============ .. role:: underline :class: underline .. rubric:: Module 6: Alternative splicing :class: header1 :Author: Leocadia Paliulis (Bucknell University) :Last Update: |today| :Version: |version| Investigation 1: Construct the gene model for tra-RB ---------------------------------------------------------------------- .. admonition:: Learning Objectives :class: admonition-learning - Identify how alternative splicing of a gene can lead to different mRNAs. - Identify how alternative splicing can lead to the production of different polypeptides. In this investigation, we will focus on tra-RB, the second :term:`isoform` of the *tra* gene, and will explore how multiple different mRNAs and polypeptides can be encoded by the same gene. The story of tra-RB is an exciting story of sex, :term:`alternative splicing`, and poison :term:`exons `! 1. To begin, open a web browser 2. Go to the UCSC Genome Browser Mirror site at http://gander.wustl.edu/ and follow the instructions given in :ref:`module1/module1_exercise:Module 1` to open ``contig1`` of *Drosophila melanogaster*, using the ``July 2014 (Gene)`` assembly rather than the "Aug. 2014 (BDGP Release 6 + ISO1 MT/dm6)" assembly. Once you are on the Genome Browser page, set "Base Position" (under the "Mapping and Sequencing Tracks" bar) to ``full`` so that you will be able see the three possible reading :term:`frames ` (remember that you will not see individual :term:`bases ` or :term:`amino acids ` until you zoom in, though). Also set "FlyBase Genes" (under the "Genes and Gene Predictions" bar) to ``full``. Don't forget to click on one of the ``refresh`` buttons to see your changes. 3. Enter the following coordinates into the "enter position or search terms" text box: ``contig1:9,700-11,000`` and hit the ``go`` button to get a good view of the *tra* gene (:numref:`Figure %s `). .. figure:: /_static/images/module6/Figure1.png :alt: Genome Browser view of the *tra* gene :name: module6_figure_1 Center the browser on the *tra* gene. 4. Let's consider what we know about tra-RA and learn more about tra-RB. .. admonition:: Question 1 :class: admonition-question Given that exons are shown by the black boxes, and introns are shown by thin lines with arrowheads in the FlyBase Genes track, what does this tell us about the first intron of tra-RB compared to that of tra-RA? 5. Now let's look at the patterns of :term:`transcription`. Scroll down to "RNA Seq Tracks", click on the ``RNA-Seq Coverage`` link. Change the track display settings as we did in :ref:`module2/module2_exercise:Module 2`: - Set the "Display mode" to ``full`` - Set the "Data view scaling" field to ``use vertical viewing range setting`` - Set the "max" field under "Vertical viewing range" to ``37`` - Check both ``Adult Females`` and ``Adult Males`` under "List subtracks" - Hit the ``Submit`` button (:numref:`Figure %s `) .. figure:: /_static/images/module6/Figure2.png :alt: Configure the display settings for the RNA-Seq tracks :name: module6_figure_2 Enter settings for RNA Seq tracks. 6. Back on the browser main page - Under "RNA Seq Tracks", change the display mode for the "Exon Junctions" track to ``full``, then hit ``refresh``. .. tip:: To review the use of RNA Seq data, watch the `RNA Seq and TopHat video `_ Now we can see the RNA-Seq data for males (red) and females (blue). Recall that peaks in RNA-Seq Read Coverage tracks usually correspond to the regions of the genome that are being transcribed. These two samples generally show similar RNA-Seq read coverage along the entire span of the *tra* gene. However, the adult female sample shows substantially lower RNA-Seq read coverage at around 9,971-10,145 (red box in :numref:`Figure %s `). We can also see the RNA-Seq Exon Junctions track, which shows the location of splice sites supported by the RNA-Seq data (as you saw in :ref:`Module 4 `). Recall that the black boxes in the FlyBase Genes track are exons and the thin lines with arrowheads show the locations of the :term:`introns `. Notice that the diagrams for the first and second RNA-Seq Exon Junctions tracks have the same :term:`5'` splice site but different :term:`3'` splice sites. Let's see what we can find out about these splice sites. .. figure:: /_static/images/module6/Figure3.png :alt: Gene predictions and RNA-Seq data for *tra* :name: module6_figure_3 Browser showing gene predictors, RNA-Seq tracks, and RNA-Seq exon junctions for male and female *Drosophila melanogaster*. 7. First, we need to establish the reading frame for the first exon. Zoom in on the 5' end of the transcript around position ``contig1:9850-9860``. .. admonition:: Question 2 :class: admonition-question Given what you know about the initiation of translation, which of the 3 possible reading frames is used for both the tra-RA and tra-RB products? 8. Now zoom in on the location of the 5' splice site at the end of the first exon in both tra-RA and tra-RB (:numref:`Figure %s `). We will also be thinking about the concept of :term:`phase` here. To review :term:`splicing` and phase, watch the `Splicing and Phase video `_. .. figure:: /_static/images/module6/Figure4.png :alt: Splice donor site for intron 1 :name: module6_figure_4 Zoom in on the first 5' splice site. .. admonition:: Question 3 :class: admonition-question Give the coordinate for the last base of the first exon for tra-RA. .. admonition:: Question 4 :class: admonition-question Give the coordinate for the last base of the first exon for tra-RB. .. admonition:: Question 5 :class: admonition-question What is the consensus sequence for the 5' splice site (donor site)? .. admonition:: Question 6 :class: admonition-question What are the coordinates for the 5' splice site in tra-RA? .. admonition:: Question 7 :class: admonition-question What are the coordinates for the 5' splice site in tra-RB? .. admonition:: Question 8 :class: admonition-question What is the phase at this splice site? 9. Now zoom out and zoom in on the start of the second exon in tra-RB, just after the 3' splice site (:numref:`Figure %s `). We can identify the second exon by the RNA-Seq data, in particular using the RNA-Seq Exon Junctions data. .. figure:: /_static/images/module6/Figure5.png :alt: Splice acceptor site for intron 1 of tra-RB :name: module6_figure_5 Zoom in on the start of second exon in tra-RB. .. admonition:: Question 9 :class: admonition-question What are the coordinates for the first base of the second exon in tra-RB? .. admonition:: Question 10 :class: admonition-question What is the consensus sequence for the 3' splice site? .. admonition:: Question 11 :class: admonition-question What are the coordinates for the 3' splice site in intron 1 of tra-RB? .. admonition:: Question 12 :class: admonition-question What phase do we anticipate? .. admonition:: Question 13 :class: admonition-question Given this, what is the reading frame for tra-RB exon2? .. admonition:: Question 14 :class: admonition-question Does this make sense, given the location of stop codons? 10. Now zoom out and zoom in on the 3' splice site for tra-RA. (:numref:`Figure %s `). This can be identified from the RNA-Seq data, particularly the RNA-Seq Exon Junctions. .. figure:: /_static/images/module6/Figure6.png :alt: Splice acceptor site for intron 1 of tra-RA :name: module6_figure_6 Zoom in on start of second exon for tra-RA. .. admonition:: Question 15 :class: admonition-question What are the coordinates for the first base of the second exon in tra-RA? .. admonition:: Question 16 :class: admonition-question What is the consensus sequence for the 3' splice site? .. admonition:: Question 17 :class: admonition-question What are the coordinates for that sequence in intron 1 of tra-RA? .. admonition:: Question 18 :class: admonition-question Given the phase at the donor site, what phase are we looking for here? .. admonition:: Question 19 :class: admonition-question Given this, what is the reading frame for tra-RA exon 2? .. admonition:: Question 20 :class: admonition-question Does this make sense, given the location of stop codons? The 3' acceptor site for the second intron in tra-RA is found inside the second exon of tra-RB. This intron is :term:`alternatively spliced `. Alternative splicing is one way eukaryotes produce different proteins from the same coding regions of DNA. Here the alternative decision is made in a sex-specific manner; male fruit flies have targeted the spliceosome to use the first 3' acceptor site identified by the RNA-Seq Exon Junction data, while female fruit flies have targeted the spliceosome to use the second 3' acceptor site identified. This change in splicing has profound effects --- in fact, it drives the programming of male and female characteristics in the developing fly. To review alternative splicing, watch the `Genes and Isoforms video `_. 11. Reset your browser by entering ``contig1:9,700-11,000`` into the "enter position or search terms" text box and hit ``go``. Let's analyze the consequences of this alternative splicing on production of a protein product. .. admonition:: Question 21 :class: admonition-question From your analysis of the A isoform of *tra* in :ref:`Module 5 `, how many amino acids does the tra-RA protein product have? Now look at the tra-RB isoform: .. admonition:: Question 22 :class: admonition-question What are the coordinates for exon 1? .. admonition:: Question 23 :class: admonition-question Given the reading frame that you established for tra-RB, does translation continue past exon 2, or is it terminated by a stop codon in exon 2? .. admonition:: Question 24 :class: admonition-question What are the coordinates for the translated portion of exon 2? .. admonition:: Question 25 :class: admonition-question How many amino acids does the protein translated from the tra-RB isoform have? .. admonition:: Question 26 :class: admonition-question Is it likely that the protein translated from tra-RB could play the same functional role played by the protein translated from tra-RA? .. note:: The Tra protein has an important function in female *Drosophila*, and is itself a splicing factor that regulates splicing. Careful :term:`annotation` of genes, as we have done here, can provide many insights into biological control mechanisms. Investigation 2: Polypeptides produced from each isoform of *tra* ---------------------------------------------------------------------- .. admonition:: Learning Objectives :class: admonition-learning - Describe how alternative splicing producing different polypeptides might result in changes in phenotype. Now that we know that *tra* is alternatively spliced to make two isoforms, tra-RA and tra-RB, and that males express one isoform while females express the other, let's try to figure out how alternative splicing affects the polypeptides produced from translating these mRNAs. To do this, we need to produce a gene model for tra-RB and compare it to the gene model for tra-RA that you constructed in :ref:`module5/module5_exercise:Module 5`, showing where the :term:`start codons ` and :term:`stop codons ` appear in each isoform. Use what you learned in :ref:`Module 5 ` to construct a gene model for tra-RB. Locate the start codon, splice sites, and the stop codon. Construct the gene model below. .. admonition:: Question 27 :class: admonition-question :underline:`Gene model for tra-RB:` - Coordinate for start of translation: \_____________\_ - Coordinate for last base of exon 1: \_____________\_ - Coordinate for first base of exon 2: \_____________\_ - Coordinate for last base of exon 2: \_____________\_ - Coordinate for first base of exon 3: \_____________\_ - Stop codon coordinates: \__________________________\_ Summary ---------------------------------------------------------------------- .. admonition:: Summary Question 1 :class: admonition-question How does the polypeptide translated from the tra-RB isoform differ from the polypeptide translated from the tra-RA isoform? What are the consequences of these differences on protein function? .. admonition:: Summary Question 2 :class: admonition-question Discuss how the bigger mRNA leads to creation of a smaller polypeptide!! .. admonition:: Summary Question 3 :class: admonition-question Consider how alternative splicing could allow many different proteins to be encoded by the same gene. .. admonition:: Summary Question 4 :class: admonition-question Based on the gene structure of the two isoforms of *tra* shown in the "FlyBase Genes" track, provide a hypothesis that could explain this difference in RNA-Seq read coverage between the adult males sample and adult females sample.