Bio 3000 - Splicing
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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.
What is the role of Serine in SR proteins?
Phosphorylation of Serine allows for activation of the SR protein.
How do SR proteins regulate splicing?
By binding to ESE's ESS's or snRNP's.
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.
What does hnRNP stand for?
Heterogeneous Nuclear RNP.
What do hnRNP's do?
- Alter splicing patterns
- Mark mature-mRNA for nuclear export.
Where would you find PRP8?
Associated with U5 snRNP.
What are some characteristics of PRP8?
- Largest splicosomal protein.
- Lies at Catalytic Core.
- Mutations can cause blindness.
What molecules are responsible for cross-exon interactions?
Where on the mRNA do SR proteins bind?
What are Cross-Intron Interactions?
Bridging of U1 and U2 by SR proteins.
What is the purpose of Cross-intron interactions?
What is the role of the CTD (RNA Pol.) in splicing?
To deposit snRNA's and splicing factors onto the transcript.
What is the exon definition model?
The addition of splicing factors from the CTD to exons that result in splicing.
Which CTD is responsible for exon definition?
What effect does the GST have on the CTD?
It makes it more soluble.
Can the GST stimulate splicing alone?
Yes but not as effectively as CTD+GST.
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.
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.
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).
What protein was responsible for fluorescence in Burge's study?
Green Fluorescent Protein.
What is a Ribozyme?
- An RNA with catalytic activity.
- (Not a true Enzyme, not multiple turnover).
Breifly describe RNA World Hypothesis.
- RNA predates DNA and Enzymes.
- DNA and Enzymes evolved because they are better at that they do.
What was the first discovered Ribozyme?
The Tetrahymena Group I intron.
What are four examples of Catalytic RNA's?
- Hammerhead Ribozymes.
- Hairpin Ribozymes.
- RNAase P.
What does RNAase P do?
Cleaves precursor tRNA (complexes with protein).
What are the two types of Hammerhead Ribozyme?
Cis and Trans.
Which form of Hammerhead Ribozyme can form from two RNA's
How does the Hammerhead Ribozyme work?
- Cis - Folds on itself and cleaves.
- Trans - Folds on target and cleaves.
What are the three types of Introns?
Group I, Group II and Splicosomal.
Which group of introns are Autocatalytic?
Why are Group I introns not considered Autocatalytic?
Because they require Guanosine residues to splice.
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.
What is the hypothesis on Splicosome Evolution?
That Splicosomes formed from Group II introns found in Mitochondria and Chloroplasts.
What are the two self-splicing introns?
Group I and Group II
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.
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.
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.
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.
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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