Exam 3: Lecture 6 Flashcards Preview

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Eukaryotic RNA Splicing (Removal of Introns)

-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


After RNA Splicing

-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


Intron-exon Junctions

-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


Intron Numbers in Single-celled Organisms

-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


Intron Numbers and Multicellularity

-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


Introns in Human Genome

-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


Gene Location of Introns

-some genes are located within introns of other genes

-Drosophila seven in absentia (sina) gene is located within intron of rhodopsin-4 (Rh4) gene


Richard Roberts and Philip Sharp

-awarded Nobel Prize for their demonstration that introns were spliced from primary mRNA transcript


Splicing Three Step Program-Sequences

-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


Splicing Three Step Program-Process

-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)


RNA Splicing and Transcription

-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


General Splicing Proteins

-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


Recursive Splicing

-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


Exon Splice Enhancers and Silencers and Intron Splice Enhancers and Silencers

-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


Alternate Splicing Method 2 (Graphic)

-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.


Drosophila Dscam and Axon Guidance

-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


Sex Regulation

-heavily dependent upon correct RNA splicing

-Sex-lethal (Sxl) gene encodes developmentally regulated splicing factor that is not part of the normal splicing machinery



-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



-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


End Result of Differential Transcription and Sxl Gene Splicing

-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


Males Lack of Sxl

-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