1
Q
- Understand what RNA processing includes
A
- Splicing
- 5’ cap addition
- polyadenylation.
2
Q
definition of an exon
A
- Sequences that are translated into protein
3
Q
definition of an intron
A
- intervening sequences that interrupt exons.
4
Q
- Know what R looping experiments are used for
A
- RNA hybridized to its DNA template and the product is examined by electron microscopy.
5
Q
how R looping experiments led to the identification of introns.
A
- These experiments revealed introns in adenovirus
- RNA-DNA hybrid was interrupted by 3 ss DNA loops that represented introns
6
Q
- Know what hnRNA is and where it is found in the cell.
A
- hnRNA: heterogeneous nuclear RNA, a class of large nuclear RNAs
- located in the nucleus.
- It is a precursor to mRNA
- hnRNA turns over very rapidly
- Converted into smaller RNAs very quickly.
7
Q
- In splicing of eukaryotic mRNA, know the significance of the GU…..AG splice site signals and the A at the branch point. Be able to recognize at 5’ splice site, 3’ splice site and branch site within an intron.
A
- 5’ splice site - GU
- 3’ splice site - AG
- A - located 13-22 nucleotides upstream from 3’ splice site
8
Q
- Know the two step splicing mechanism.
A
- Formation of the lariat-shaped intermediate:
forms when 2’ hydroxyl group of an adenosine nucleotide in the middle of the intron attacks phosphodiester bond between first exon and G at beginning of intron - forms lariat and simultaneously separates first exon from the intron
- 3’ hydroxyl group left at end of first exon attacks the phosphodiester bond linking intron to second exon
- This forms the exon-exon phosphodiester bond and releases the intron, in lariat form, at the same time
9
Q
- Be able to identify the two phosphodiester linkages that are lost and the two that are formed in the splicing of eukaryotic mRNA.
A
- Phosphodiester bonds lost between the two exons binding to the one intron
- Phosphodiester bond formed between the two exons and a bond in intron forming a lariat structure.
10
Q
- Understand what happens to the splicing pattern of a transcript when a branch site is mutated or deleted.
A
- Mutant 1: remove a region 35-70 bp upstream of intron 3’ splice site
- Mutant 2: insert extra DNA segment between this special sequence and exon 2
- Mutant 3: place special sequence within exon 2
11
Q
Result of Mutant 1: remove a region 35-70 bp upstream of intron 3’ splice site
A
- Result: splicing is blocked, suggesting that it is important for splicing
12
Q
Result of Mutant 2: insert extra DNA segment between this special sequence and exon 2
A
- Result: different AG used
- 1st AG downstream of special sequence (with inserted DNA)
13
Q
Result of Mutant 3: place special sequence within exon 2
A
- Result: 1st AG downstream of new special sequence used as a 3’ splice site
14
Q
- Understand what the spliceosome is
A
the spliceosome is a ribonucleoprotein complex consisting of a number of subunits and requires ATP for assembly.
15
Q
- Understand the involvement of 5’-2’ phosphodiester linkages in splicing of eukaryotic mRNA.
A
- an intron is removed as a lariat structure in which the 5’ G of the intron is joined in a unusual 2’-5’-phosphodiester bond to an adenosine near the 3’ end of the intron.
- The adenosine is called the branch point because it forms an RNA branch in the lariat structure
16
Q
- Understand what the spliceosome consists of and what each contain
A
- the spliceosome consists of 5 ribonucleoprotein complexes called snRNPs.
- These contain snRNAs that help catalyze the splicing reactions.
17
Q
- Know what the U1 snRNA base pairs with.
A
- U1 snRNA base pairs with the 5’ splice site.
- It is not sufficient but it is necessary
18
Q
- Understand how compensatory mutations in a snRNA can be used to show its interaction with sequences within a pre-mRNA.
A
- Disruption of base pairs and introduction of new potential base pairs will weaken the overall base pairing
- The number of contiguous base pairs is lower.
- Compensatory mutations restore base pairing and may or may not restore splicing
19
Q
- Know how an RNase protection assay works and can be used to identify splicing patterns of mRNAs.
A
- Synthesize in vitro transcript that is radioactively labeled and allow it to hybridize to RNA.
- RNase digests single-stranded RNA and dsRNA is detected
20
Q
- Understand what the U6 snRNA interacts with.
A
- the U6 snRNA interacts with the 5’ splice site of eukaryotic mRNAs.
- In complexes with both the substrate and the lariat, binds both before and after the initial step
- Also base pairs with U4
21
Q
- Know what U2 base pairs with.
A
- base pairing between U2 and U6 help to form a structure that forms the active site of the spliceosome.
- U2 also base pairs with the branch site.
- U6-substrate complex and U6-lariat complex can be detected experimentally
22
Q
- Understand what U4 snRNA interacts with
A
- U4 snRNA interacts with the consensus branch sequence in pre-mRNAs.
23
Q
- Know what U2 base pairs with prior to splicing
A
- U2 also base pairs with U6 prior to splicing.
24
Q
- Understand what U5 binds to during the second step of splicing
A
- U5 binds to the 3’ end of the first exon and the 5’ end of the second exon
- to bring the splice sites together during the second step of splicing.
25
10. Know what happens between U4 and U6
- U4 dissociates from U6 after splicing is underway
- must be removed before U6 can interact with U2 to form an active spliceosome.
- Some U6 bases that participate in base pairing with U4 to form stem I are also involved in the essential base pairing to U2
26
11. What was the surprising observation made by Tom Cech and his group that led to a Nobel Prize?
- RNA itself catalyzed its own splicing (self-splicing introns)
27
12. Understand the similarities between Group II self-splicing introns and the spliceosome.
- Group II use a lariat intermediate - similar to nuclear mRNA splicing
- U5 loop = domain ID - positions exons 1 and 2 for splicing
- U6 region that base pairs with the 5’-splice site substitutes = domain IC
- U2-U6 helix I = domain V
- U2 branchpoint helix - domain VI
- In both cases, base pairing around the branchpoint A causes it to bulge out to form the branch
28
13. Know how snRNAs function as ribozymes
- snRNAs likely catalyze the splicing reactions in the spliceosome and, in this way, function as ribozymes.
29
14. Understand the concept of the "ribozyme" as an RNA molecule with catalytic activity.
Ribozymes are catalytic RNA that are capable of carrying out enzymatic reactions
30
15. Know the RNA world hypothesis.
- Evolution of self-replicating systems.
- RNAs catalyzed biological reactions, cleavage, and self replication
- In a world before proteins with 20 amino acids added versatility
31
1. Understand how splice site selection is regulated
- splice site selection is regulated by controlling where a spliceosome forms.
- A cell can regulate the quality and quantity of splicing and thereby regulate gene expression.
32
2. Understand the role of the protein U2AF in splicing.
U2AF is the splicing factor involved in 3’-splice site recognition
33
3. Understand the roles of the branch point binding protein in splicing.
- Branch point binding protein (BBP) helps define the intron and helps bring 2 ends of the intron together for splicing
34
Understand the roles of SC35 in splicing.
- SC35 is a member of a group of RNA-binding proteins called SR proteins
- contains domains that are rich in serine and arginine
- it is a commitment factor that helps select splicing sites for the spliceosome
35
4. Understand how different mRNAs can result from the transcription of a single gene by alternative splicing.
- Alternative splicing generates 2 or more alternative RNAs that encode different proteins
36
5. What percentage of transcripts are subject to alternative splicing in humans?
- at least 40% of transcripts have the potential for alternative splicing.
37
6. Know what alternative splicing can do.
- alternative splicing can result in the production of more than one protein from a single gene.
38
7. Understand the variable splice sites in transcripts.
- can have variable 5’ splice sites that are spliced to a common 3’ splice site
- can have variable 3’ splice sites that are spliced to a common 5’ splice site;
- have exons that are sometimes skipped
- can have mutually exclusive exons
- can have introns that are used only under certain circumstances.
39
8. Understand the difference between splice sites that match the consensus sequence versus splice sites that diverge from the consenus.
- splice sites that match the consensus sequence are less likely to undergo alternative splicing compared to splice sites that diverge from the consensus.
40
9. Know the role of cis-acting enhancers
- cis-acting enhancers can help regulate the utilization of certain splice sites.
41
10. Understand the function of cells expressing specific splicing factors.
- cells express specific splicing factors that regulate the splicing pattern of certain pre-mRNAs in specific ways.
42
11. Understand the sex determination pathway
- the sex determination pathway consists of several alternative splicing steps.
- Loss of function mutations are lethal in females and do not affect males
- Gain of function mutations are lethal in males and do not affect females.
- All due to the role of Sxl in dosage compensation
43
12. Know the default sex determination pathway
- the default sex determination pathway is the male pathway in fruit flies.
44
13. Know what the X:A ratio does
- initiates the female sex determination pathway in fruit flies through a female-specific early promoter that is ON is females and OFF in males.
45
14. Understand the importance of the sex-lethal protein in flies.
- the Sex-lethal protein in flies is an RNA binding protein that binds to, and regulates its own splicing pattern by preventing splicing to the male-specific exon.
46
15. Know the level of regulation of initiation and maintenance of the sex determination pathway.
- the initiation of the sex determination pathway in flies is regulated at the transcriptional level
- maintenance of the pathway is regulated by alternative splicing.
47
16. Understand the roles of the Doublesex zinc finger transcription factor in the sex determination pathway of flies.
- Specify different aspects of male differentiation
48
16. Understand the roles of the Fruitless zinc finger transcription factor in the sex determination pathway of flies.
Controls most steps of male courtship behavior.
| Specifies aspects of sexual differentiation responsible for male sexual behavior
49
1. Know what capping does
Capping adds a special blocking nucleotide (a cap) added to the 5’-end of a pre-mRNA
50
Know what polyadenylation is
polyadenylation is a string of AMPs (poly[A]) added to the 3’-end of a pre-mRNA.
51
2. Understand that the 5’ cap has 4 functions:
- To protect mRNAs from degradation
- To enhance the translatability of mRNAs - most important
- To enhance the transport of mRNAs from the nucleus to the cytoplasm
- To enhance the splicing efficiency of mRNAs.
52
3. Understand when the 5’ cap is added.
- the 5’ cap is added early, before the pre-mRNA chain is about 30 base pairs long.
- The 5’ cap is added by the condensation of a molecule of GTP w/ triphosphate at the 5’ end of the transcript
53
4. Know the average size of a poly A tail
- the average size of a poly A tail is about 250 nucleotides.
54
5. Understand when the poly A tail is added.
- the poly A tail is added post-transcriptionally and not encoded in the DNA.
55
Know the evidence that poly A is added post transcriptionally:
- poly(A) digested quickly with an enzyme that degrades RNAs from 3’
- Located at the 3’ end of messages
- Genome do not have run of T’s long enough to encode polyA tail
- No T’s at ends of thousands of sequenced genes
- Actinomycin D inhibits DNA-directed transcription, does not inhibit polyadenylation
56
6. Understand what poly(A) polymerase does
- poly(A) polymerase (PAP) is the enzyme in the nucleus that adds AMP residues to mRNA precursors.
- It is found on hnRNA
57
7. Know the function of the poly A tail
- the poly A tail protects mRNAs from degradation and stimulates translation.
58
8. Know what poly(A)-binding protein I does
- poly(A)-binding protein I binds to the poly A tail and enhances the efficiency of translation.
59
9. Understand how poly A+ mRNA forms polysomes more successfully than poly A- mRNA and is, therefore, translated more efficiently.
- mRNA with >1 ribosome translating it = polysome
- Poly(A)+ showed the greatest increase in polysome formation - as poly(A) grows from 15 to 30 nt and a more gradual increase as more A’s added
- Enhancement of translatability by poly(A) seems to be more important than mRNA stabilization
60
10. Understand the relationship between the transcript and poly(A) polymerase
- The transcript does not have to be completely synthesized before poly(A) polymerase adds A’s to its 3’-end
- polyadenylation and transcription termination are not necessarily linked.
GN 421 Final
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