Module 3 ============ .. |br| raw:: html
.. rubric:: Module 3: Transcription Part II: What happens to the initial (pre-mRNA) transcript made by RNA pol II? :class: header1 :Authors: S. Catherine Silver Key (North Carolina Central University) and |br| Chiyedza Small (Medgar Evers College CUNY) :Last Update: |today| :Version: |version| Introduction ---------------------------------------------------------------------- In :ref:`module2/module2_exercise:Module 2`, you identified the transcription start site (TSS) for the A :term:`isoform` of the *tra* gene (tra-RA). In this module, we will explore each of the three steps of :term:`pre-mRNA` processing. Setting up our Browser page (review): ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ 1. Open a new web browser window and go to the UCSC Genome Browser Mirror site at http://gander.wustl.edu/. Follow the instructions given in :ref:`module1/module1_exercise:Module 1` to navigate to the ``contig1`` project in the *D. melanogaster* ``July 2014 (Gene)`` assembly. 2. As you may remember from :ref:`module1/module1_exercise:Module 1`, contig1 is derived from chr3L in the *D. melanogaster* genome. This contig contains three different genes (*CG32165*, *spd-2*, and *tra*). Enter ``contig1:9,500-11,000`` into the "enter position or search terms" textbox and then click on the ``go`` button to navigate to the genomic region surrounding the *tra* gene. 3. Because the Genome Browser remembers your previous display settings, you should click on the ``default tracks`` button to reset the display to the default settings. Change the display mode for the "Base Position" track to ``full`` and verify that the "FlyBase Genes" track is set to ``pack``. Click on the ``refresh`` button. 4. Scroll down to the "RNA Seq Tracks" section and then click on the ``RNA-Seq Coverage`` link. Change the track display settings to the following, as we did in :ref:`Module 2 `: - Change the "Display mode" field to ``full`` - Select the "Data view scaling" field to ``use vertical viewing range setting`` - Change the "max" field under "Vertical viewing range" to ``37`` - Under the "List subtracks" section, select BOTH the ``Adult Females`` and the ``Adult Males`` subtracks (Select the check box next to subtrack to turn the subtrack on.) - Click on the ``Submit`` button. Verify that the RNA-Seq Coverage track on the browser page is set to ``full``. Investigation: mRNA processing ---------------------------------------------------------------------- .. admonition:: Learning Objectives :class: admonition-learning - Use the genome browser to identify the relationships among; pre-mRNA, 5' capping, 3' polyadenylation, splicing, mRNA. - Describe how the transcript generated by RNA polymerase II (the pre-mRNA) is processed to become mature mRNA, using the sequence signals identified in :ref:`module2/module2_exercise:Module 2`. The processing of pre-mRNA into mRNA involves three key steps (:numref:`Figure %s `) - The addition of a **5' cap** - The addition of a **3' poly(A) tail** - The removal of introns through **splicing** Removal of the :term:`introns ` during this process results in adjacent :term:`exons ` being brought together in the final mRNA message. .. figure:: /_static/images/module3/Figure1.png :alt: Diagram of mRNA processing :name: module3_figure_1 Diagram of mRNA processing that converts a pre-mRNA to a processed mRNA. Addition of a 5' cap ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ The **first step in pre-mRNA** processing occurs at the :term:`5'` end of a messenger RNA. Recall that mRNA is synthesized in a 5' to 3' direction, so the 5' end of the mRNA was synthesized first. Let's examine the beginning of the *tra* gene. Type ``contig1:9,825-9,870`` into the "enter position or search terms" textbox and then click on the ``go`` button. In :ref:`Module 2 `, we identified the **transcription start site** (TSS) of the A isoform of *tra* at position **9,851**. To show the TSS's that have been annotated by the modENCODE project, scroll down to the "Genes and Gene Prediction Tracks" and change the display mode for the "TSS Annotations" track to ``pack``, and click ``refresh``. The modENCODE project looked for TSSs by using a chemical method to tag the special structure that occurs at 5' ends of transcript, fishing out the RNA molecules that carried these tags, and mapping the sequence back to the genome, a method called "CAGE" (cap analysis of gene expression). In addition, we will also display the "D. mel. cDNAs" track (also under the "Genes and Gene Prediction Tracks" section); change this to ``pack``. This track shows the alignment of *D. melanogaster* :term:`cDNAs ` (complementary DNAs, made by copying the mRNA) that have been sequenced by the Berkeley Drosophila Genome Project (BDGP). Click on the ``refresh`` button (:numref:`Figure %s `). These two tracks both provide an analysis based on the RNA population, and mapping the positions of these sequences indicates where the transcript started. .. figure:: /_static/images/module3/Figure2.png :alt: Change the display modes of two tracks to "pack" :name: module3_figure_2 Change the display modes of the "D. mel. cDNAs" and "TSS Annotations" tracks to "pack". Remember from :ref:`Module 2 ` that we also found a match to TCAKTY, a common initiation signal just upstream, at 9,834 (display this using the "Short Match" track). All of these pieces of evidence argue for a TSS in this region. The new Genome Browser image (:numref:`Figure %s `) shows the 5' end of the pre-mRNA transcript (i.e. the start of :term:`transcription`) based on the CAGE experiment (modENCODE track) with the additional lines of support. On this end of the pre-mRNA, a modified guanine nucleotide (7mG) is added to the nucleotide at position 9,851, forming the **5' cap**. Note that this additional nucleotide is **NOT** visible in the DNA track. It is added AFTER the transcript is made. This is the **first step** in **pre-mRNA processing**: capping. .. figure:: /_static/images/module3/Figure3.png :alt: Addition of a 5' cap to the transcript :name: module3_figure_3 Addition of a 5' cap to the 5' end of the transcript. .. admonition:: Question 1 :class: admonition-question What is the coordinate of the first nucleotide that is transcribed? In the DNA sequence, is it an A, C, T or G? .. admonition:: Question 2 :class: admonition-question What are the coordinates for the start codon that codes for the first amino acid of the A isoform of the *tra* gene? (Assume reading frame +3.) .. admonition:: Question 3 :class: admonition-question The region of the transcript from the 5' cap to the nucleotide just upstream of the start codon is called the 5' untranslated region (5'UTR) because it is part of the transcript that is not translated. How long (in ribonucleotides) is the 5'UTR? Addition of a 3' poly(A) tail ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ The **second step** in pre-mRNA processing is **polyadenylation**. 5. To view the :term:`3'` end of the tra-RA gene, change the "enter position or search terms" field to ``contig1:10,633-11,000`` and then click on the ``go`` button. Polyadenylation means that **many** (poly) **adenine nucleotides** (ribonucleotides) are added to the 3' end of the pre-mRNA **AFTER** transcription termination. This generates a :term:`poly-A tail ` (typically ~20 to ~250 As) that will be retained in the final mRNA but it is not present in the "Base Position" track of the Genome Browser. This is because the poly-A tail does not exist in the DNA template but is simply added to the RNA by a special polymerase as a long run of adenine nucleotides. Our previous analysis in :ref:`Module 1 ` has shown that the last :term:`coding exon` of tra-RA is in frame +2 and the :term:`stop codon ` is located at 10,754-10,756. We can use the Genome Browser to determine the end of the tra-RA transcript indicated by the cDNA track (in blue). (Note that this aligns with the cDNA although there is some discrepancy between the two as to the exact end of the transcript.) .. admonition:: Question 4 :class: admonition-question How long (in base pairs) is this 3' untranslated region (3'UTR) as indicated by the cDNA track (in blue)? .. admonition:: Question 5 :class: admonition-question Zoom into the 3' end of the FlyBase Gene, near the termination site. What is the longest stretch of A nucleotides that you observe? .. admonition:: Question 6 :class: admonition-question Do your findings support the conclusion that the poly(A) sequence observed in the mature mRNA transcript is not in the template DNA? 6. Perform a "Short Match" search for the poly-A signal using the protocol you learned in :ref:`Module 2 `. This search should place the poly-A signal at 10,818-10,823 (:numref:`Figure %s `). As mentioned in :ref:`Module 2 `, the transcript is cleaved 11 to 30 nucleotides downstream of the poly(A) signal sequence, and then 150-200 adenines are added to the pre-mRNA. The nucleotides between the stop codon and the end of the poly-A tail comprise the 3' :term:`UTR`. .. figure:: /_static/images/module3/Figure4.png :alt: Short Match search results for AATAAA :name: module3_figure_4 Previous analysis placed the stop codon for the A isoform of *tra* in frame +2 (blue arrow) and the poly-A signal at 10,818-10,823 (red arrow). We can see the polyadenylation sequence that is associated with the processed mRNA by examining the cDNA (BT028774) that has been aligned to this region. 7. Click on the ``BT028774`` feature under the "cDNAs from *D. melanogaster*" track and then click on the ``View details of parts of alignment within browser window`` link (:numref:`Figure %s `). .. figure:: /_static/images/module3/Figure5.png :alt: Configuring the genome browser display modes :name: module3_figure_5 Examine the alignment of *D. melanogaster* cDNA BT028774 against contig1. The next figure shows the actual alignment between the *D. melanogaster* cDNA BT028774 and the genomic sequence in contig1 (:numref:`Figure %s `). Nucleotides that are identical between the two sequences are shown in blue capital letters while nucleotides that differ are shown as black lowercase letters. The light blue :term:`bases ` denote the start and the end of the gap in the alignment. The side-by-side alignment shows the pairwise alignment between the cDNA (top) and the contig1 sequence (bottom) within the viewing region (i.e. contig1:10,633-11,000). .. figure:: /_static/images/module3/Figure6.png :alt: BLAT alignment of D. melanogaster cDNA BT028774 against contig1 :name: module3_figure_6 Alignment of the *D. melanogaster* cDNA BT028774 with the end of contig1. .. admonition:: Question 7 :class: admonition-question Scroll up to the ``cDNA BT028774`` area. After which coordinate (number in the cDNA) do you see the polyadenylation track (in lower case black letters)? .. admonition:: Question 8 :class: admonition-question How many "A" ribonucleotides have been added to the *tra* mRNA (represented in the cDNA)? .. admonition:: Question 9 :class: admonition-question Locate the ``AATAAA`` termination signal in the cDNA sequence. How many nucleotides 3' of the final "A" in the signal sequence does the poly(A) run start? (This number should be between 11--30 nucleotides.) Removal of introns through splicing ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ The final step in pre-mRNA processing is :term:`splicing` out of introns and merging adjacent exons into one continuous open reading frame so that the mRNA is ready for :term:`translation` into a protein. 8. Change the "enter position or search terms" field to ``contig1:9,870-10,170`` and then click on the ``go`` button to navigate to the first intron of the tra-RA transcription (:numref:`Figure %s `). .. figure:: /_static/images/module3/Figure7.png :alt: First intron of tra-RA :name: module3_figure_7 The genomic region surrounding the first intron (red arrows) of tra-RA. 9. Zoom in to the region near the end of the first exon of tra-RA. .. admonition:: Question 10 :class: admonition-question Which two nucleotides are found just after the end of the first exon of tra-RA? Repeat this determination, identifying the two nucleotides at the start of intron 2 of tra-RA. These two nucleotides are a signal for **splicing** to occur at the 5' end of an intron; these represent the first two bases of the intron, often called the donor site (or 5' splice site). .. admonition:: Question 11 :class: admonition-question At which base does exon 1 end? 10. Zoom out and then zoom in to the region near the beginning of the second exon of tra-RA. .. admonition:: Question 12 :class: admonition-question Which two nucleotides are found right before the start of tra-RA exon 2? 11. Zoom out and then zoom in to the region near the beginning of the third exon of tra-RA. .. admonition:: Question 13 :class: admonition-question Which two nucleotides are found right before the start of tra-RA exon 3? These two nucleotides are the signal for **splicing** out of the 3' end of the intron, often called the acceptor site (or 3' splice site). These represent the last two bases of the intron. .. admonition:: Question 14 :class: admonition-question At which base does exon 2 of tra-RA begin? What is its coordinate? Summary ---------------------------------------------------------------------- In this module, we learned about the three key steps that are involved in converting the pre-mRNA into a :term:`mature mRNA`. .. cssclass:: compact-list 1. The addition of a **5' cap** 2. The addition of a **3' poly(A) tail** 3. The removal of introns through **splicing** .. note:: Introns are removed during this process and adjacent exons are brought together in the mRNA message. After mRNA processing, the mature mRNA (tra-RA) can now exit the nucleus so that it can be translated into a protein (tra-PA) by the cytoplasmic ribosomes. .. admonition:: Summary Question 1 :class: admonition-question Describe how the transcript generated by RNA polymerase II (the pre-mRNA) is processed to become mature mRNA, using the sequence signals identified in :ref:`module2/module2_exercise:Module 2`.