Bentzen 7 - RNA molecules and mRNA processing part 2 Flashcards
What is a summary of transcription and translation in bacteria?
Transcription produces fully functional mRNA
The mRNA start and stop is determined by the Shine-Delgarno sequence (ribosome binding), start codon and stop codon.
How are genes organized in bacteria?
In operons
Is mRNA polycistronic or monocistronic in bacteria?
Polycistronic, meaning there are several open reading frames (ORF) in one mRNA (leading to multiple polypeptides)
What are two theories for how introns arose?
- Introns have been around forever, but were lost in prokaryotes
- Introns arose from group II introns, which themselves arose as parasitic DNA sequences (transposable elements). Subsequently invaded eukaryotes from endosymbiont ancestors of mitochondria and chloroplasts
What is alternative processing of pre-mRNA intorns? What are two ways to do this?
A single pre-mRNA can be processed in different ways to produce different mRNA molecules.
- Alternative splicing: pre-mRNA can be spliced in different ways
- Alternative PolyA site: PolyA tail can be added at different 3’ cleavage sites
What are four pre-mRNA splicing patterns?
- Exon skipped (an exon is spliced out with introns)
- Intron retention (an intron is left in the mRNA)
- Alternative 5’ or 3’ splice site (A splice site is moved, including some of a intron or excluding some of an exon)
- Mutually exclusive exons (Some exons are included only when another is excluded)
What is the DSCAM gene ans its protein?
A gene encoding the Down Syndrome Cell Adhesion Molecule. It has an essential role in establishments of neural circuits.
The protein is a trans-membrane protein presented on surface of neurons.
The gene is about 600 million years old and in Down Syndrome critical region of chromosome 21 in humans
How does the DSCAM RNA transcript demonstrate alternative splicing in Drosophilia?
There are 115 exons but only 20 are always expressed, and only 24 exons used in the final protein. There are 4 variable clusters of exons (total of 95 exons which can be alternatively spliced), one exon used from each cluster.
This leads to different isoforms of the protein. These isoforms are thought to be important in self recognition of neurons, and therefore important for proper neural wiring. In vertebrates it is different, the gene is duplicated, but much simpler in organization.
How do mRNAs produce similar proteins of differing sizes when translated by alternative Poly(A) 3’ cleavage site?
Pre mRNA contains multiple 3’ cleavage sites. The 3’ cleavage site used determines the length of the mRNA transcript.
How do alternative 3’ splice sites lead to production of a functional or nonfunctional protein in Tra pre-mRNA?
Transformer protein (Tra) in Drosophilia can be spliced at one site to be functional (and make a female phenotype) or nonfunctional (and cause male to stay male) depending on where the pre-mRNA is spliced
In males there is a premature stop codon when the upstream 3’ splice site is used (causing the Tra protein to be nonfunctional)
In female drosophilia, the presence of what protein causes the downstream 3’ splice site to be used instead of the upstream one (which would cause a male)?
The Sxl protein, this splicing causes the termination codon to be spliced out with the intron
Why do thyroid cells make Calcitonin and brain cells make calcitonin-gene-related peptide (CGRP) from the same DNA sequence?
In pre-mRNA processing, there is alternative processing based on the cell type.
What are two ways to edit the coding information of mRNA transcripts? (when a gene is found with a sequence of nucleotides that do not exactly match that of its RNA product)
- Substitution editing
2. Insertion/deletion editing
What is substitution editing of mRNA?
Chemical alteration of individual nucleotides by specific enzymes.
What is insertion editing of mRNA?
Addition of U nucleotides occurs by cleavage of the RNA, addition of the U, and ligation of the ends.
The reactions are catalyzed by a complex of enzymes under the direction of guide RNA
What does guide RNA do in insertion editing of mRNA?
Guide RNA adds nucleotides to the pre-mRNA that were not encoded by the DNA
What are RNA interferences (RNAi)?
Short regulatory RNAs that repress or ‘silence’ the expression of homologous genes
What do the short (21-25 nucleotides long) utilize RNA molecules, siRNA and miRNA do? What are the three mechanisms that allow them to do this?
siRNA (small interfering RNA) and miRNA (microRNA) inhibit the expressino of homologous genes via at least 3 mechanisms:
- mRNA degradation
- Inhibition of translation
- Inhibition of transcription
How was RNA interference (RNAi) discovered?
By observing RNA silencing, a gene incorporated into the genome (a transgene) could not only induce or stimulate gene activity but could also inhibit the expression of homologous sequences.
In a flower a extra copy of a pigment gene (transgenes) added by researchers made parts of the flower turn white, rather than have more colour.
What does double stranded RNA do in RNAi?
dsRNA triggers suppression of a gene in a homology dependent manner
What are siRNA and miRNA made from? How does this cause gene regulation?
- Cleavage of double stranded RNA by the Dicer enzyme.
- The siRNAs are then combined with protein complex RISC and pair with complementary sequences on mRNA
- The RNA-induced silencing complex (RISC) is made, which cleaves the mRNA and causes it to degrade
How is gene regulation by inhibition of translation done?
- Other double stranded regions of RNA molecules are cleaved by Dicer
- This produces microRNAs (miRNA)
- Some miRNAs combine with protein complex RISC and pair imperfectly with an mRNA
- Which leads to the inhibition of translation
How is gene regulation by inhibition of transcription done? What is RITS?
- Other miRNAs attach to complementary sequences in DNA and attract methylating enzyme (RITS is a miRNA that attaches to do this)
- Methylating the DNA or histones inhibits the transcription
RITS = RNA transcriptional silencing
What percent of genes are thought to be regulated by RNAi? When is RNAi most important?
30%. During development
Why might RNAi have evolved?
As a defence mechanism for silencing invading transposable elements
Identify four ways to vary the products of genes and how they are expressed:
Alternative splicing
Alternative 3’ cleavage & polyA sites
RNA editing
RNA interference
