The Drosophila Transcriptome
Example 1: Transcription Start Sites
Transcription start sites in the genome correspond to the first transcribed base of RNAs.RNAs are chemically modified after they are transcribed (posttranscriptionally) in a number of ways to ensure stability, facilitate recognition by the ribosome, and generate transcript and protein diversity. In the example below, the first nucleotide at the 5' end of each mRNA is protected by the enzymatic addition of a small molecule called a 7-methylguanylate cap, This "5' cap" is an identifier that declares to the cell that this is the beginning of a mRNA, that should be translated into a protein.
In the modENCODE project, RNAs containing a 5' cap were profiled using cap analysis gene expression (CAGE) to determine transcription start sites of most of the mRNAs expressed during development of the fruit fly embryo (R. A. Hoskins et al., Genome-wide analysis of promoter architecture in Drosophila melanogaster. Genome Research 21, 182 (Feb, 2011).). Another way to map 5' ends is to remove the cap and attach a linker (short string of known nucleotides) through the biochemical process known as ligation. This technique is known as RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE). We have used RLM-RACE to validate the transcription start sites identified by CAGE (Read more on transcription). Examples of transcription start sites illustrate the range of promoter types.
Example 2: Alternative RNA Splicing
Like C. elegans and human genes, Drosophila genes are often alternatively spliced to create a diverse set of transcripts and protein products from a single gene. In Drosophila there are about 4,000 protein-coding genes that produce multiple transcripts.When alternative splicing occurs in the 5' untranslated region of a gene, it leads to different mRNAs that can encode the same protein product, often in different tissues. Each alternatively spliced gene on average produces about 3.2 transcripts. When alternative splicing occurs within the coding region of a gene, it leads to mRNAs that encode different protein products. This process allows a single gene to generate more than one protein and is an additional source of the complexity found in multicellular animals.
Example 3: RNA Editing
The term RNA editing describes the molecular processes in which the primary sequence of an RNA molecule is altered through a chemical change in one of the RNA bases. It is thought that RNA editing is restricted to double-strand templates. In the modENCODE project we studied A> I editing in which adenosine is deaminated to form inosine. This is the only observed editing mechanism in flies. The inosine is read as a G in both translation and in secondary structures. A-to-I editing can be specific in which a single adenosine is edited (in flies 447 genes have only one site edited) or promiscuous, multiple adenosines are edited (150 genes have multiple edited sites). An example of one of the modENCODE discoveries is the multiply edited transcripts for the gene quiver. The base edits result in up to four amino acid changes from the genomic encoded predicted protein. Note that not all transcripts show every edit so multiple proteins can be produced as a consequence of the editing process. quiver is involved in circadian rhythm or the daily waking/sleep cycle and is thus a very important gene to understand and know all it's protein variants.
- When DNA is transcribed, successive ribonucleotides are assembled to make a direct copy of the DNA template, producing a single strand of RNA starting with a 5’ end and ending with a 3’ end. That new RNA is rapidly processed. What functions does the 5' mRNA cap serve?
- To determine how expression of a gene is regulated, it is important to know where transcription starts. Why is the 5' mRNA cap used to map the start site of transcription?
- Explain a scenario in which alternative splicing produces two different mRNA transcripts, but both mRNAs encode identical proteins.
- Why might alternative mRNAs that encode identical proteins be useful for an organism?
- Alternative splicing can produce different mRNAs that in turn produce different protein products. Why is this useful for an organism?
- Explain how RNA editing can allow one gene to encode different proteins.
- RNA editing must be carefully controlled within cells to ensure that only a subset of mRNAs are modified at specific bases. Why?