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What is the structure of an SR protein?
An RNA binding domain at the N-Terminus and an argenine/serine rich region at the C-Terminus.
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What is the role of Serine in SR proteins?
Phosphorylation of Serine allows for activation of the SR protein.
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How do SR proteins regulate splicing?
By binding to ESE's ESS's or snRNP's.
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What are the twp main subunits of U2AF?
- - One subunit recognizes the 3' AG on the intron.
- - The other subunit binds to the polypyrimidine tract.
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What does hnRNP stand for?
Heterogeneous Nuclear RNP.
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What do hnRNP's do?
- Alter splicing patterns
- Mark mature-mRNA for nuclear export.
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Where would you find PRP8?
Associated with U5 snRNP.
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What are some characteristics of PRP8?
- Largest splicosomal protein.
- Lies at Catalytic Core.
- Mutations can cause blindness.
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What molecules are responsible for cross-exon interactions?
SR Proteins.
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Where on the mRNA do SR proteins bind?
on ESE's
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What are Cross-Intron Interactions?
Bridging of U1 and U2 by SR proteins.
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What is the purpose of Cross-intron interactions?
Splicing efficiency.
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What is the role of the CTD (RNA Pol.) in splicing?
To deposit snRNA's and splicing factors onto the transcript.
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What is the exon definition model?
The addition of splicing factors from the CTD to exons that result in splicing.
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Which CTD is responsible for exon definition?
Rpb 1.
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What effect does the GST have on the CTD?
It makes it more soluble.
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Can the GST stimulate splicing alone?
Yes but not as effectively as CTD+GST.
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How did Burge est for ESS's?
- He used a reporter plasmid.
- A 10-mer exon was flanked by two introns and two parts of the gene encoding the Green Fluorescent Protein.
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How do Report Plasmids work?
- The two Introns flanking the exon will always be spliced.
- If the 10-mer exon contains ESE's (Enhancers) it will not be spliced and will interrupt the GFP gene.
- If the 10-mer exon contains ESS's the exon will be splied and the GFP gene will be complete.
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How can you determine if ESS's are present using Burge's reporter plasmid.
If the Cell fluoresces, ESS's were present (the 10-mer exon was spliced).
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What protein was responsible for fluorescence in Burge's study?
Green Fluorescent Protein.
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What is a Ribozyme?
- An RNA with catalytic activity.
- (Not a true Enzyme, not multiple turnover).
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Breifly describe RNA World Hypothesis.
- RNA predates DNA and Enzymes.
- DNA and Enzymes evolved because they are better at that they do.
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What was the first discovered Ribozyme?
The Tetrahymena Group I intron.
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What are four examples of Catalytic RNA's?
- Hammerhead Ribozymes.
- Hairpin Ribozymes.
- RNAase P.
- Ribosomes.
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What does RNAase P do?
Cleaves precursor tRNA (complexes with protein).
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What are the two types of Hammerhead Ribozyme?
Cis and Trans.
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Which form of Hammerhead Ribozyme can form from two RNA's
Trans.
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How does the Hammerhead Ribozyme work?
- Cis - Folds on itself and cleaves.
- Trans - Folds on target and cleaves.
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What are the three types of Introns?
Group I, Group II and Splicosomal.
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Which group of introns are Autocatalytic?
Group II.
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Why are Group I introns not considered Autocatalytic?
Because they require Guanosine residues to splice.
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Is there a difference between Autocatalytic and Self Splicing?
Yes. Autocatalytic means splicing without any help at all whereas self splicing means some help is needed, but a splicosome is not.
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What is the hypothesis on Splicosome Evolution?
That Splicosomes formed from Group II introns found in Mitochondria and Chloroplasts.
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What are the two self-splicing introns?
Group I and Group II
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All Self Splicing introns are Autocatalytic, but not all Autocatalytic introns are Self Splicing. True or False?
- False, the statement should read,
- All Autocatalytic Introns are Self Splicing, but not all Self Splicing Introns are Autocatalytic.
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Describe Group I intron splicing.
- 1. Guanosine uses it's -OH as a nucleophile to attack the p in the 5' UpA site.
- 2. The Exposed -OH on the 5' U attacks the p on the 3' GpU site.
- 3. The Linear Intron is now spliced with the intitial Guanosine at the 5' end and a G-OH at the 3' end.
- 3a. The exon contains the sequence UpU at the splice site.
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What is the most significant difference between splicing of Group II Introns and Splicosomal Introns?
Splicosomal Introns use the Splicosome, Group II Introns don't.
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How does a Group II intron splice without snRNP's?
The Group II Intron folds into a Continuous Secondary Structure that loosely resembles snRNP's.
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What is the Hypothesis on the similarities between snRNP's and Group II Introns?
- That snRNP's Evolved from Group II introns due to their structural similarity.
- An alternative hypothesis says that their similarity is not a result of Evolution but of Structure and Function (Convergant Evolution)
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