Exam 3: Lecture 6 Flashcards
1
Q
Eukaryotic RNA Splicing (Removal of Introns)
A
- in eukaryotic transcription RNA Pol II transcribes all introns and exons of gene
- exons contain important info for protein sequence and must be retained
- introns posses no info to be transferred to encoded protein so must be eliminated from primary transcript
- process of RNA splicing is tasked with recognizing and removing introns and with splicing together exonic sequences
2
Q
After RNA Splicing
A
-mature mRNA transcript (w/out introns) can then be edited by RNA editing enzymes within nucleus and exported to cytoplasm where it will serve as a template for translation machinery
3
Q
Intron-exon Junctions
A
- contain sequence information that is recognized by a set of proteins that are collectively referred to as Spliceosomes
- many proteins that comprise the spliceosome contain non-coding RNA species that helps mediate interactions with mRNA transcript
- these proteins recognize 5’ and 3’ end of each exon and internal sequence called branch site
4
Q
Intron Numbers in Single-celled Organisms
A
- nearly all bacterial genomes completely lack introns
- first organisms to contain introns are yeast (single-celled Eukaryote) -most genes within single-celled organisms still lack introns
- genes that contain introns usually have no more than one
5
Q
Intron Numbers and Multicellularity
A
- first increase in intron numbers appears to correlate with onset of multicellularity
- numbers continue to rise along with increasing complexity of organism
- ex: average number of introns in typical gene within fruit fly genome is 3 while number increases to 7 for typical gene in human genome
6
Q
Introns in Human Genome
A
- will be genes that lack introns
- along these lines, genes can have significantly higher than average number of introns
- human titin gene holds most number of introns with 178
- interspersed within 2.4 million bases that make up primary transcript
7
Q
Gene Location of Introns
A
- some genes are located within introns of other genes
- Drosophila seven in absentia (sina) gene is located within intron of rhodopsin-4 (Rh4) gene
8
Q
Richard Roberts and Philip Sharp
A
-awarded Nobel Prize for their demonstration that introns were spliced from primary mRNA transcript
9
Q
Splicing Three Step Program-Sequences
A
- correct splicing requires spliceosome proteins recognize three sequences within each intron
- includes 5’ intron-exon junction, 3’ intron-exon unction and internal branch site
- each site conserved within all introns of a given species
- sequences can vary from species to species
- spliceosome machinery in one species may not recognize and bind the sequence found in another species
10
Q
Splicing Three Step Program-Process
A
- first step is recognition and cleavage of 5’ intron-exon junction (splice donor)
- second step 5’ end of intron is attached to branch site which lies within intron creating a bound lariat
- last step involves cleavage of 3’ intron-exon junction (splice acceptor) and joining of two flanking exons -3’ end of upstream exon is joined to 5’ end of downstream exon
- intron lariat is targeted for destruction while spliced exons are exported to cytoplasm for translation *specific proteins do all these things but don’t need to know will be referenced in question (check slide for general information)
11
Q
RNA Splicing and Transcription
A
- occur simultaneously
- means entire primary transcript does not have to be synthesized before splicing can occur
- each intron can be spliced out of primary transcript nearly immediately after it is transcribed
- allows for rapid exportation of mature transcript after transcription is completed
12
Q
General Splicing Proteins
A
- U1-6, U2AF and BBP
- recognize sequences that are located at 5’ splice site, 3’ splice site and branch site within mRNA transcript
- each splicing protein contains short non-coding RNA (ncRNA) that has nucleotide sequence complementary to one of three splice recognition sites within mRNA transcript
- therefore splicing factors recruited to mRNA through RNA:RNA interactions
- types of interactions are very different than that seen with DNA binding protein
13
Q
Recursive Splicing
A
- allows for removal of large introns in piecemeal fashion
- in instances in which recursive splicing has been observed at least one hybrid splicing site is found within large intron
- this hybrid site contains sequences that correspond to both 3’ and 5’ splice sites
- this site is used tor remove a portion of the intron while recreating a 5’ splice site within intron
- this site can then be used in future splicing event:
- some large introns will have multiple such ratchet proteins (RP) thereby allowing for intron to be removed nearly simultaneously with transcription
14
Q
Exon Splice Enhancers and Silencers and Intron Splice Enhancers and Silencers
A
- ESEs and ESSs located within exon
- similar sequences are located within in introns, ISEs and ISSs
- these sites are bound by proteins that either promote or inhibit splicing
- ex: ESE sites bound by SR proteins whc physically interact with and stabilize UA2F35 at branch site
- TIA1 protein binds to ISE sites within introns and physically interacts with U1 at 5’ splice junction
- Mutations in these sites lead to inaccurate splicing of mRNA primary transcript
15
Q
Alternate Splicing Method 2 (Graphic)
A
- cases in which alternate splicing is employed all exons and introns are transcribed but all exons are not retained during the splicing process
- most famous example of a gene that is alternately spliced is the Drosophila Down syndrome cell adhesion molecule (Dscam) gene.
- contains several variable regions which can be alternately spliced to yield 38,016 different mature mRNA transcripts
- each of these unique transcripts will encode a unique protein that is used in axon guidance.
16
Q
Drosophila Dscam and Axon Guidance
A
- wiring of the nervous system depends on the correct contacts being made between neurons and muscle cells.
- often these cells types can be located at significant distances from each other.
- axons of a neuron will grow and extend towards its target
- neurons make very specific connection and thus require “directions” to their targets
- proteins encoded by the Drosophila Dscam locus are cell adhesion molecules and these play a role in attracting and repelling advancing axons
- Larry Zipursky (at UCLA) discovered Dscam locus and demonstrated that it is alternately spliced
- has the most extensive splicing of any known gene
- different cells in Drosophila will splice the Dscam primary transcript differently thus there is a combinatorial code to Dscam protein isoform throughout the animal
- some isoforms will repel each other thus an axon will not make a contact with a cell containing a repulsive cue
- other combinations of isoforms are attractive and these can serve to bring an axon of one neuron into contact with another neuron
- Dscam gene and the need for it to be alternatively spliced is conserved across evolution and is found in humans
- number of alternative exons varies from species to species however
17
Q
Sex Regulation
A
- heavily dependent upon correct RNA splicing
- Sex-lethal (Sxl) gene encodes developmentally regulated splicing factor that is not part of the normal splicing machinery
18
Q
Males
A
- in flies destined to become male, transcriptional repressors shut off early embryonic promoter preventing Sxl from being transcribed
- later in embryogenesis a second promoter is activated and gene is transcribed by RNA Pol II and spliced by splicing machinery
- during translation, stop codon in exon 3 forces ribosome to abort process -smaller Sxl protein is non-functional
19
Q
Females
A
- in flies destined to become females, transcription is activated from early embryonic promoter which leads to production of Sxl isoform
- this protein then binds to sequences within exon 3 of large primary transcript that is derived from late embryonic promoter
- as a consequence exon 3 is skipped -functional Sxl protein is produced
20
Q
End Result of Differential Transcription and Sxl Gene Splicing
A
- production of functional Sxl protein that is destined to be female while those that will become male will lack Sxl protein
- consequence Sxl is able to suppress splicing at 3’ splice site that is located within first intron of transformer (tra) mRNA
- normal splicing machinery is able to then correctly splice tra mRNA thereby producing mature transcript that can be translated into fully functional protein
- Tra goes on to aid in splicing of doublesex (dsx) mRNA transcript -
short female specific transcript is produced
-Dsx protein is a transcription factor that in females can repress male genes and activate female genes
21
Q
Males Lack of Sxl
A
- results in mis-splicing of tra mRNA which in turn prevents production of Tra prtoein
- without Tra protein, dsx mRNA transcripts are spliced differently
- resulting mature mRNA and proteins are larger
- this Dsx protein isoform represses female specific genes
