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Define hnRNA and hnRNP
- 1. hnRNA - heterogeneous nuclear RNA is the unprocessed RNA
- 2. hnRNP - consists of hnRNA with various RNA binding packaging/transport proteins
Describe the structure of and mechanism behind the 5' cap
- 1. Initial 5' end is 5'pppA/GpNpN
- 2. Guanylyl-transferase adds cap
- 3. 5'Gppp + 5'pppApNpN --> Gppp5'-5'ApNpNp+pp+p (cap structure)
- 4. The 7 position on the terminal G is methylated by G-7 methyl transferase
What is the importance of adding the cap in the nucleous?
- 1. RNA pol II pauses ~30 nucleotides downstram of initiation site
- 2. Capping enzymes recruited
- 3. Capping protects 5' end from 5'-3' endonucleases avoiding dissociation of RNA pol II form DNA template strand
- 4. 20/80 heterodimer binds cap to stimulate splicing of first intron and facilitates mRNA transport
- 5. In cytoplasm, eIF4F binds cap to initiate translation
What are splice sites?
Short consensus sequences immediately surrounding the exon-intron boundaries
What are the two types of introns
- 1. Major - U2
- 2. Minor - U12
How are the correct intron junctions recognized?
- 1. Recognition of specific Gu-AG sequence pairs at the ends of introns
- 2. All 5' splice sites are equivalent and all 3' sites are equivalent
- 3. Splice sites/apparatus are generic
- 4. Splicing is coupled w/transcription - sequences in both exons/introns may regulate splice site selection
What are the steps in a splicing reaction?
- 1. Nucleophilic attack by 2'OH (A - branch site) on 5' splice site breaks bond
- 2. Lariat is formed via transesterification of 5'G and 2'A linkage
- 3. Exon 3'OH attack on 3' splice site breaks bond
- 4. Exons are joined 3' to 5'
- 5. The branch site is important in determining the nearest 3' splice site
What are spliceosomes?
- 1. Consists of the snRNPs and other proteins (splicing factors) involved in splicing.
- 2. 5 snRNP's - U1, U2, U5, U4, U6
- Small nuclear RNPs - consists of 1 snRNA and several proteins
- 3. Each snRNP contain 1 snRNA and several (<20) proteins
- 4. anti-Sm - autoimmune antiserum that recognizes a conserved sequence in all of the snRNPs excepts U6
- 5. Transesterification - rxn that breaks and makes bonds in a coordinated transfer so that no energy is required
How does the structure of the U1 snRNA promote splicing?
1. The 5' end is ss stretch of 4-5 bases that binds to 5' end of intron.
What is the mechanism for E (early - pre spliceosome) complex formation?
- 1. Intron definition - SR proteins and U1 complex recognize (define) 5' splice site
- 2. U2Af65/35 bind Pyrimidic tract (Py tract) between branch point and 3' end
- 3. BBP binds to branch point
- 4. SR proteins loop out intron in completion of complex by binding U2AF35/65
- 5. This mechanism can be applied to exon definition where introns are too long
Describe the spliceosome assembly pathway
- 1. E complex formation
- 2. A complex - U2 binds at branch point in the presence of ATP
- 3. B1 complex - U4, U6, and U5 join - mature spliceosome with catalytic center
- 4. B2 complex - U1 and U4 release; U6 binds 5' splice site and U2; U5 interacts w/exons
- 5. C1 complex - first step of transeterification where 5' site is cleaved
- 6. C2 complex - second step of transesterification where 3' site is cleaved and exons ligated
Describe spliceosome function for minor introns.
- 1. Same as for major introns
- 2. U11 binds 5' splice site
- 3. U12 binds branch point
- 4. Alternate forms of U4 and U6 perform analogous functions
Compare and contrast Group I, Group II, and Nuclear intron splicing
- 1. Nuclear and group 2 introns - 1st transesterification between 2' OH on A in branch site and 5'P on G at splice site
- 2. Nuclear introns require proteins
- 3. Group II and Group I are autosplicing
- 3. Group I - 1st transesterification is between 3'OH on G and 5'P on G
T or F Splicing can occur during or after transcription
- 1. 5' introns are removed cotranscriptionally
- 2. 3' introns may be removed cotranscriptionally or post-transcription
How is splicing physically and functionally coupled with transcription?
- 1. CDT serves as a loading pad for mRNA processing proteins
- 2. Keeps splicing events moving in a 5' to 3' direction
- 3. 5' capping enzymes (gualylyl transferase and 7 methyltransferase) - help RNA pol II overcome initial transcriptional pausing near promoter
- 4. splicing factors - facilitate elongation
- 5. poly-A - instrumental in transcription termination
How is splicing connected to mRNA export and stability?
- 1. mRNA that has undergone splicing process is exported more efficiently than cDNA derived RNA
- 2. Exon junction complex (EJC) - inserted onto exon-exon junction during splicing
- 3. REF protein - part of EJC to which TAP/Mex binds to transport mRNA through nuclear pore
How does splicing in the nucleus effect mRNA stability and translation in the cytoplasm
Nonsense-mediated mRNA decay (NMD) - EJC bound to aberrant mRNA (such as premature stop codon) will recruit 5' decapping proteins during translation to induce rapid degradation of mRNA
What is alternative splicing
- 1. Splicing that produces more than one mRNA sequence from one gene
- 2. Results in the functional diversity of gene products from a single gene locus
Describe the alternative splicing events that determine sex in Drosophila.
- 1. Exon 3 of sxl has a termination codon that prevents synthesis of Sxl functional protein
- 2. Alternative 3' splicing of Exon 3 included exon 3 in males and not in females
- 3. Males don't produce functional protein, females do
- 4. Functional Sxl protein changes the splicing of transformer (tra) gene
- 5. Females produce a Tra and Tra2 proteins that cause the dsx gene to splice out intron 3 which causes termination at the end of exon 4
- 6. Males produce Tra and tra2 proteins that cause the dsx gene to splice out exon 4 and continue translation throught exon 6.
- 7. The dsx products produced by each sex repress the expression of the others sex-specific genes
What is the correlation between alternative splicing and weak splice sites
The sequences at splice sites deviate from the consensus sequence