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Adv Cell Bio Lecture 12
Transcription: Regulation IV (Splicing)
Terms in this set (31)
What will happen to RNA once it's been made? (3 modifications that will occur)
How does splicing occur?
What is splicing?
Cutting out introns and pasting different exons together to get different proteins during translation
humans use it a lot and how we are so complex
similar to regulatory sequences...
splicing increases with organismal complexity, almost 8 introns per human gene
How many genes do Drosophila have?
How many genes do C. elegans have?
How many genes do humans have?
~20,000 protein coding genes
~34,000 when you include ncRNA genes
What is the average different spice form for every human gene?
likely average 3 different splice forms for every human gene
Increasing complexity largely due to....
alternative protein production and proliferation of regulatory sequences (complexity in regulation of expression)
What are the biggest portion of transcribed material?
Introns are the biggest portion of transcribed material
How long are human exons?
How long are human introns?
Why is most of conservation in the Introns
because you dont want the branch site in the coding region
Conserved sequence guide recognize...
of the 5' and 3' splice site
Branch site A
located near 3' end of intron
has 2'-OH group that can mediate attack on the end of one of the exons
Invariable information needed to guide splicing is found in..
Introns because you dont want it part of coding region
Splicing occurs through?
- 2'-OH group on Branch site A attacks the 5' end of the intron
- this frees the 3' end of exon and frees 3'-OH group of exon
- 3'-OH attacks 3'end of intron and frees 5' end of another exon
- Results in Intron lariat and spliced exon
Can mRNA splice on its own?
Most cant. Ones that can are considered Ribozymes (ex. hammerhead)
Group 1 and Group 2 Introns
Can catalyze their own cleavage. Can splice themselves.
What guides splicing?
known as small nuclear ribonuclear proteins (snRNPs)
~150 proteins similar to ribosome
- U1, U2, U4, U5, and U6- known as small nuclear RNAs (snRNAs)
What are spiceosomes composed of?
Proteins and RNA
Why would RNA be good for splicing?
Because it uses complementary nature of nucleic acid to identify particular sequences
What is the function of snRNAs in Spiceosomes?
Splice Junction Interactions
use complementary to detect the 5' end of the intron
Alternative splicing can lead to...
differences in protein activity
bind to a specific sequence to prevent snRNAs in Spliceosome from binding
inhibit binding of U1, U2 and/or U2AFs
Splicing Receptor Protein
Splicing repressor proteins (hnRNPs) bind to:
exonic splicing silencers
intronic spicing silencers
bind to RNA
Promote the binding of snRNAs U1, U2, and/or U2AFs to help to activate splicing to that particular site
Splicing activator proteins (SR) bind to:
exonic splicing enhancers
intronic splicing enhancers
RNAse Protection Assay
in this technique, a labeled probe is hybridized to mRNA creating dsRNA, and an RNase is used to chew up any remaining ssRNA
- Which particular sequence of a nucleic acid is present within a gel or a block?
- What portion of RNA is present?
- Have an mRNA that is getting bigger, where is it getting bigger? What's being differentially splicing?
- Shows that you not only have a certain gene present but a certain portion present
1. Purify RNA from Northern Blot
2. Hybridize with RNA antisense probe
3. Digest with RNAse (cut single stranded RNA)
4. Purify digest dsRNA fragments
5. Run on polyacrymide gel to see what regions are present and commentary to know how many bp your probe and mRNA have in common
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