Topic 10 Rna Splicing Flashcards
What are the two types of splicing
Cis: have precursor mRNA , exon intron exon, the intron in the precursor mRNA gets slipped out and exon 1 exon 2 joined
Trans: have two diff mRNA, one has exon 1 intron the other has exon two
The intron is spliced out and the exon 1 from on mRNA a joins with exon 2 on the other mRNA
Trans happens in c elegans and not in humans
Explain the relationship in organism complexity and the average number of introns per gene
Proportional relationship
More complex organism (humans) have more introns per gene , which is why the mechanism of splicing is important in complex organisms
Explain the general process of cis splicing
When and where does it happen
Dark green is exon, coding
DNA has intron and exon sequences
After transcription Pre mRNA has a 5’ leader sequence and 3’ non coding region
In posttranscriptional processing the pre mRNA is spliced to remove introns and the exons are rejoined
Spliced mRNA has 5’ UTR and 3’ UTR before the first exon and after the last exon
Post transcriptional processing happens in the nucleus and can happen while transcription is happening
When are the RNA processing enzymes recruited
Splicing factors are Recruited to the CTD tail of rna pol II after the tail is phosphorylated and a bit of the pre mRNA is already made
So splicing can happen during transcription in the nucleus and
Explain the structure of an intron
The GU-AG rule: required that all introns start with GU and end with AG
Intron structure:
5’ splice site donor : GU, set the boundary between the 5’ end of the intron and the 3’ end of the exon (the 5’ exon)
Middle branch site: has loosely conserved 7 nucleotide sequence with adenine nucleotide in it (ends with RAY sequence) where r is purine and y is pyrimidines (C/U)
3’ splice site acceptor: AG, set the boundary between the 3’ end of the intron and the 5’ end of the exon (the 3’ exon) + poly pyrimidine tract: many pyrimidines
What are the minimum requirements need in the intron to splice it
The 5’ GU, 3’ AG, A in the branch site
All other sequences are loosely conserved
What is used to remove introns in humans
Transestericfication
What is transesterification
A reaction that breaks and makes chemical bonds (for splicing its phosphodiester bonds) so its energy independent
Explain step 1 in transesterification
The 2’ OH of the A in the branch site act as nucleophile to attack the phosphate backbone between the last nucleotide of the exon (3’ end) and the first nucleotide of the intron (5’ G)
The G from the intron now form phosphodiester bind with adenine in the branch site: makes the three way junction (since RAY with G on the A) making the lariat intermediate structure of the intron
Explain the structure of the 3-way junction in the first step of transesterification
5’ RAY 3’
these nucleotides are bound by 3’ to 5’ phosphodiester bonds in the chain
When the G comes in and forms the branch point, is a 2’ - 5’ phosphodiester bond since 2’ OH of adenine attacked
This means adenine has two bonds, one at the regular 3’ OH and one at the 2’ OH bound to G
This 2-5 makes the lariat intermediate structure during the first transesterification
Explain the second transesterification in intron removal
The 3’ OH from the freed exon acts as nuceleophile to attack the phosphodiester bond between the 3’ end of the intron and the 3’ exon (so after the AG 3’ splice site sequence)
The lariat structure is freed and the 5’ and 3’ exons are joined together
The intron lariat is rapidly degraded because no protection from the 5’ cap or 3’ poly A tail
What type of splicing is transesterification
Cis splicing
How is transesterification energetically neutral
Because the number of phosphate bonds is conserved (one broken and one made)
What is trans splicing
two exons in two separate prerna molecules are spliced together into a single exon
Similar to cis splicing, but instead of lariat forming it’s a y shaped branch
The A branch point in the second mRNA causes the first transesterification and attacks the GU in the first mRNA
The gu is flipped (now UGA) making the y shaped branched intron
The 3’ OH from the first exon in the first mRNA act as nucleophile for the second transesterification
Two exons joined, y shaped intermediate removed
What is transsplicing used in
Not in eukaryotes
In C elegans and trypanosomes
Why is trans splicing used
In c elegans there is a non coding microRNA called let-7 that is Matured through the trans splicing mechanism
It’s also found in human but doesn’t go through tea splicing in human
Clinical implications: This mechanism allows us to introduce foreign molecules to remove the mutated sequence that causes disorders through trans splicing
What are the spliceosomal components
They help with the splicing of intron
It’s small nuclear rna (snRNA) + protien which makes snRNPs (small nuclear ribonucleoprotien)
These snRNPs make the spliceosome (a catalytic protien and RNA complex)
Decribe the structure of snRNP
snRNA: small nuclear rna that is 100-300 BP long, called U1-U6
Each snRNP has one snRNA and many protien parts
The snRNA part can locate the target sequence elements at the intron exon borders (recognizing the splicing site)
The protien part of the spliceosome has the catalytic activty which lets it remove intron and catalyze the transesterification reaction
What are the major roles of snRNPs
Give an example
They recognize the 5’ and 3’ splice sites, and the branch site
Catalyze the 5’ splice site cleavage and joining with the branch site
- Mutiple snRNP can recognize one rna molecule (ex. U1 and U6 snRNP can both recognize one splicing sequence)
- The U2 snRNP recognizes the branch site to make the A a nucleophile, so even though they recognize the same sequences, snRNP have specific activities/roles
- Diff snRNP can form complexes with each other through their snRNA folding, so some region of snRNP recognize the splicing site and some recognize the other snRNP, work together with each other
- snRNP are sequentially recruited to the splice site when it is first recognized
Ex. U2AF recognizes the py tract/3’ splice site and helps the BBP (branch binding protien) bind to the branch sequence of the intron
Then U2 displaces BBP and binds to the branch site, U2 protrudes out the adenine in the branch site so it can act as a nucleophile
So overall snRNP are Sequentially recruited and stepwise displacement
Explain the steps in spliceosome assembly and splicing
Complexs E A B C are formed
E (early) complex:
5’ splice site recognized by U1 snRNP
U2AF has two subunits 35 and 65, U2AF65 binds to py tract and interacts with BBP
BBP is bound to the branch site
U2AF35 subunit binds to 3’ splice site
A complex:
U2 is recruited to the branch site, displaced BBP
Protruded unpaired A in branch site reacts with 5’ splice site
B complex:
U4,5,6 form a trimeric complex
The complex bridges between U1 and U2 to drag together and shorten the distance between them in the intron
This displaces U1, U6 replaces it at the 5’ splice site
This form the b complex
C (catalytic) complex:
U4 is released, U2 and U6 pair to form an active site at the 5’ splice site and the branch site to do the first transesterification and make lariat intermediate structure
U5 does the second transesterification to free the lariat intermediate and the mRNA exons
The snRNPs are released and recycled, the lariat rna is degraded
What was the first lab to discover ribozymes?
Cech lab
What are the three types of cis intron splicing
Pre-mRNA spliceosome: requires protiens, most common splicing we see
Group II self splicing: doesn’t require protiens, the rna can spliced themselves (ribozymes)
Group I self splicing
Explain the preMRNA spliceosome intron splicing
Very common, used for most eukaryotic genes
Two transesterification reactions, formation of lariat, joining of exons
The spliceosome complex mechanism
Explain the group II intron self splicing
Rare but Similar to preMRNA spliceosome
Have 2 transesterification reactions, adenine branch point but in vitro they don’t need protiens to catalyze the splicing
The 2’ OH in the A and the 3’ OH of the 5’ exon act as nucleophiles and catalyze this reaction themselves (as ribozymes)
Explain the group I intron self splicing
Rare
Also two transesterification reactions, don’t require protiens in vitro
Instead of adenine in the branch point there is Guanine 3’ OH not 2’ OH meaning no intermediate lariat structure forms
What is special about group I and II intron
They don’t require protiens in vitro to splice but in vivo they may have some protiens helping them
What are the properties between all three types of introns
All three:
use two transesterification reactions, meaning energetically neutral and no atp needed
RNA are all catalytic in all three types introns
Group I and II:
Found in some bacteria (prokaryotes), mitochondria, and chloroplasts, not in humans or eukaryotes
Self splicing (the rna catalyze splicing without protiens)
Group two introns and nuclear preMRNA (soliceosomal introns):
Use the same splicing pathway with a lariat intermediate
Because of the parallels between the two , group II introns are through to be the ancestors/primitive precursors of the spliceosomal introns
Explain the structural similarities between spliceosomal complex snRNA and the group II introns
What does this tell us
The premrna:
The U2 and U6 snRNPs pair together with the 5’ exon and branch site, this paring is catalytic
The group II intron:
Has the catalytic domain 5 which is equivalent to the U2:U6 pairing
Has domain 6 which is equivalent to the U2 binding to the branch site
Tell us that group II introns could be the primitive ancestors of the spliceosomal introns, but not sure because there are differences
What types of errors in splicing are there
Caused by mistaken splice site selection
Exon skipping:
3 exons two intron, the exon two is skipped and missing in the final product so just exon 1 and 3
Pseudo splice site selection:
Have exon 1 intron exon 2
Spliced a part of exon 2 out with the intron so only a part of exon 2 is joined to exon 1 and the rest is spliced out
This pseudo splice site is chosen because of the loose consensus sequence of the splice site
Explain the example of why there is a problem with the appropriate splice site being chosen
The drosophila Dscam gene has 24 exons which can make 38,000 diff mRNA transcripts though splicing
So if so many diff splice products, how does the cell know to make all these diff splice product
What methods are used by the spliceosome to ensure accurate splice site regocnition
- Formation of an active site by the sequential recognition/recruitment of snRNP (makes it so that one has to come in first to displace the other, a step can’t be skipped)
- Co transcriptional loading of spliceosomal protiens via RNA pol II c-term tail to allow recognition of the 3’ splice site. This make it so splicing happens during transcription to make it less chaotic when splicing all at once (help accuracy)
- SR (ser-arg rich protiens) that bind to ESE (exonic splicing enhancers) sequences to recruit splicing machinery to the correct sites. They set the boundary of where splicing occurs and helps binding of snRNP to the correct location
Explain how SR protiens binding to ESE helps proper assembly of the spliceosome
The exons has the ESE sequences which the SR bind to
These SR mark the correct splice site and recruit U1 snRNP to the 5’ splice site and U2AF to the 3’ splice site
This is done by:
- the SR protien interacting directly with the snRNP to recruit it to mRNA
- The SR protien stabilizing the RNA:RNA interaction between the snRNP and the preMRNA (since snRNP bound to the mRNA for during assembly of the spliceosome)
What is the minor spliceosome
In rare cases the normal spliceosome isn’t formed and the minor one is formed instead to recognize the unique AU-AC introns
Its called the AT-AC spliceosome, uses the same chemical pathway as the regular spliceosome, but different snRNP in the spliceosome (but some same like U5)
In minor spliceosome:
U4 and U6 in the trimeric complex have a different recognition sequence (AT-AC) but U5 stays the same
5’ AU and the 3’ AC splice site recognized by U11 and U12 snRNP respectively)
But in major:
the 5’ GU and 3’ AG is recognized by U1 and U2 snRNP respectively
What is alternative splicing
Different splicing of the same preMRNA to make diff isoforms of mature mRNA (with diff exons)
Explain alternative splicing in the human Troponin T gene
How can the gene function be turned off
Diff spicing can lead to alpha or beta Troponin T mRNA
5 exons in the preMRNA
Alpha Troponin has exon 4 skipped, Beta has exon 3 skipped
To turn off the gene, There is usually a stop codon built into the alternatively spliced exon to make a truncated non functional product
What is beneficial about alternative splicing
Shows how don’t need diff primary transcripts to make diff proteins
Only need one primary transcript to make many diff protien transcripts/isoforms, less work
Explain the 5 ways mRNA can be alternatively spliced
Have 3 exons and 2 introns
Normal: the 2 introns are removed
Exon skipped: exon 2 is removed with the introns
Exon extended: intron 1 is only partially removed, so final product has exon 1 extended
Intron retained: intron 1 is not spliced out, retained with exons
Alternative exons:
Can make two products
- Intron 1 2 and exon 3 are removed, giving exon 1 and 2 as the product
- Intron 1 2 and exon 2 are removed giving exon 1 and 3 as the product
But we always see exon 1 retained
How are diff splicing products formed during alternative splicing
By the competion between different splicing factors/ snRNPs which can lead to diff splicing
What is the SV40 T-antigen
Causes cells to become immortal
The preMRNA has 2 exons and one intron
Also 2 different 5’ splice sites, the 5’ SST and 5’ sst
The intron has a stop codon inside it
The ratio of the 2 forms of the mRNA (t or T) produced depends on the levels of the splicing regulator (SR): SF2/ASF
Explain the splicing of the SV40 T-antigen
Upon infection either the small t antigen or the large T antigen is made to make cells immortal
The t antigen blocks apoptosis, T induces cell cycle re entry (more cell division)
mRNA of t is longer and made when there is exon extension (so part of the intron is retained)
T is shorter and only has exon 1 and 2
How is alternative splicing regulated
By activators and repressors
The repressor compete with the splicing machinery to suppress splicing and retain the intron
The activators can cooperatively bind to the splicing molecules and enhance the splicing
These splicing regulator protiens have an RNA recognition motif (RRM) that lets them bind to rna and interact with other splicing protiens like the RS (arg serine) rich protiens
Explain the sex determination gene in drosophila
The females make double amount of chromsomes than males
Female ratio: 2 X chromsome : 2 autosomes
Male ratio: 1 X chromsome : 2 autosomes
The Sxl gene (sex lethal gene) determine the female sex and is activated in females and repressed in males
The Sxl gene is regulated by the Sis-a and Sis-b genes which activate transcription of Sxl , they are on the x chromsome
the dpn inhibitors inhibits transcription of Sxl and is found on autosome.
it gets outcompeted by the activators in the female because there are more x chromsomes (meaning more activators)
In males: the dpn repesses the expression of Sxl preMrna
In females: the sis a and b bind to the Pe to make Sxl premRNA
What is the Pm and Pe in the drosophila sex determination
Pm is the promoter for maintence which maintains the Sxl expression in embryos
Pe is the promoter for establishment which is controlled be sis a and b bind
Explain the alternative splicing of the Sxl gene in drosophila
After being transcribed in the female, the Sxl premrna needs to be spliced
The Sxl is a splicing factor that induces its own alternative splicing (auto regulation)
Sxl is also a alternative splicing factor for the tra (transformer gene)
Tra is a splicing regulator for Dsx (double sex)
So Sxl allows formation of tra and tra allows formation of the female dsx gene , this dsx gene represses male genes and promotes female development
For male the Sxl isn’t expressed so dsx is repressed, to male genes not repressed and male formed
Explain how terminally differentiated cells can be reverted into induced pluripotent stem cells
Grow the differntated cells in culture with transcription factors :
Oct4
Sox2
cMYC
NANOG
This allows de differentation of the cells to revert them back to stem cells
Explain the process of how terminally differentiated cells can be reverted into induced pluripotent stem cells
A transcription factor FOXP1 regulates/activates other transcription factors like OCT4 and NANOG
Depending on the alternative splicing of FOXP1 and which exons are retained the FOXP1 is expressed and the stem cells are made
If the 18a exon present, differentiated cells
If 18b exon present, pluripotent stem cells
What is RNA shuffling
The creation of new genes through evolution
Expalin the origin of intron models
Intron Early model:
Says that introns were initially present in all organsims early in evolution
Counter: why do we see most Bacteria have less introns than eukaryotes
Response: simpler primitive organisms need fast cell division and replicate chromsomes quickly to survive, to do this they need less introns to replicate (smaller genome)
Intron late model:
Says that introns were inserted into genes that previously had no introns
Why are many introns found in multicellular organisms
- To make mutiple protien products from a single gene though alternative splicing
- To make new genes by reshuffling exons:
When exons are translated they are protien domains with independent folding and function
If we reshuffle diff exons through recombination in dna, diff gene can get diff domain and finctions
Give an example of the exon reshuffling
The LDL receptor gene i made up of exons that are from other genes such as the C9 compliment gene and the EGF precursor gene
Shows how reshuffling of individual exons gives a new product
Also the histone modifier protien has similar exon domains across diff organsims, showing how exons can be reshuffled to make families of protiens in diff species
Overall why do scientist use rna to study evolution
Because they can observe what exons are reshuffling and what organisms rna have similar domain that are conserved between each other
Showing that they have similar origins
Other than rna splicing what post transcriptional process is there for rna
RNA editing:
Changes the rna sequence post transcriptionally
the translated protien is diff than what the gene sequences predicted it should be (because edited)
Explain rna editing by deamination
The enzyme cytidine deaminase specifically recognizes the correct location on cytosine to deaminate and turn into uracil
Explain the deamination of apolipoprotien B
Apolipoprotien B is a lipid and transports cholesterol in our bodies and is found in liver and intestines (in diff tissues)
The preMRNA has a CAA codon in the exon
In the liver The the C is not deaminated, stays CAA, protien with glutamine
In the intestine the C is demainated, UAA, gln changes to stop codon and make shorter protien
So this deamination rna editing happens in a tissue specific manner to make diff apolipoprotien B products
What is adenine deamination
The ADAR (adenine deaminase act on RNA) demanites adenine to inosine
Inosine is translated as guanine
This deamination important in the nervous system and neuronal development
Explain insertional editing : uridine insertion
Found in trypanosomes coxII genes
After the preMRNA is made the uridines (U) are inserted into specific region of the pre mRNA
This changes the codons/amino acid sequence to make the correct reading frame following insertion
What are the types of rna editing
Cytosine deamination
Adenine demaination
Insertional editing: Uridine insertion
After the rna transcript it made how it it sent to the cytoplasm
Through nuclear export
in the nucleus The mRNA is coated with protiens during splicing and rna processing
After export through the nuclear pore, some of these protiens are recycled back to the nucleus but some stay
The export is regulated so that rna isn’t exported prematurely
But there are also RNA processing granules next to the nuclear envelope on the surface of the cytoplasm, This includes the stress granule, which have rna that are undergoing further processing
40:48 may be an extra Slide
