Topic 10 Rna Splicing

Question 1

What are the two types of splicing

Answer 1

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

Question 2

Explain the relationship in organism complexity and the average number of introns per gene

Answer 2

Proportional relationship

More complex organism (humans) have more introns per gene , which is why the mechanism of splicing is important in complex organisms

Question 3

Explain the general process of cis splicing

When and where does it happen

Answer 3

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

Question 4

When are the RNA processing enzymes recruited

Answer 4

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

Question 5

Explain the structure of an intron

Answer 5

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

Question 6

What are the minimum requirements need in the intron to splice it

Answer 6

The 5’ GU, 3’ AG, A in the branch site

All other sequences are loosely conserved

Question 7

What is used to remove introns in humans

Answer 7

Transestericfication

Question 8

What is transesterification

Answer 8

A reaction that breaks and makes chemical bonds (for splicing its phosphodiester bonds) so its energy independent

Question 9

Explain step 1 in transesterification

Answer 9

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

Question 10

Explain the structure of the 3-way junction in the first step of transesterification

Answer 10

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

Question 11

Explain the second transesterification in intron removal

Answer 11

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

Question 12

What type of splicing is transesterification

Answer 12

Cis splicing

Question 13

How is transesterification energetically neutral

Answer 13

Because the number of phosphate bonds is conserved (one broken and one made)

Question 14

What is trans splicing

Answer 14

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

Question 15

What is transsplicing used in

Answer 15

Not in eukaryotes

In C elegans and trypanosomes

Question 16

Why is trans splicing used

Answer 16

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

Question 17

What are the spliceosomal components

Answer 17

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)

Question 18

Decribe the structure of snRNP

Answer 18

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

Question 19

What are the major roles of snRNPs

Give an example

Answer 19

They recognize the 5’ and 3’ splice sites, and the branch site

Catalyze the 5’ splice site cleavage and joining with the branch site

1. Mutiple snRNP can recognize one rna molecule (ex. U1 and U6 snRNP can both recognize one splicing sequence)
2. 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
3. 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
4. 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

Question 20

Explain the steps in spliceosome assembly and splicing

Answer 20

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

Question 21

What was the first lab to discover ribozymes?

Answer 21

Cech lab

Question 22

What are the three types of cis intron splicing

Answer 22

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

Question 23

Explain the preMRNA spliceosome intron splicing

Answer 23

Very common, used for most eukaryotic genes

Two transesterification reactions, formation of lariat, joining of exons

The spliceosome complex mechanism

Question 24

Explain the group II intron self splicing

Answer 24

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)

Question 25

Explain the group I intron self splicing

Answer 25

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

Question 26

What is special about group I and II intron

Answer 26

They don’t require protiens in vitro to splice but in vivo they may have some protiens helping them

Question 27

What are the properties between all three types of introns

Answer 27

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

Question 28

Explain the structural similarities between spliceosomal complex snRNA and the group II introns What does this tell us

Answer 28

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

Question 29

What types of errors in splicing are there

Answer 29

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

Question 30

Explain the example of why there is a problem with the appropriate splice site being chosen

Answer 30

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

Question 31

What methods are used by the spliceosome to ensure accurate splice site regocnition

Answer 31

1. 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) 2. 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) 3. 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

Question 32

Explain how SR protiens binding to ESE helps proper assembly of the spliceosome

Answer 32

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: 1. the SR protien interacting directly with the snRNP to recruit it to mRNA 2. 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)

Question 33

What is the minor spliceosome

Answer 33

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

Question 34

What is alternative splicing

Answer 34

Different splicing of the same preMRNA to make diff isoforms of mature mRNA (with diff exons)

Question 35

Explain alternative splicing in the human Troponin T gene How can the gene function be turned off

Answer 35

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

Question 36

What is beneficial about alternative splicing

Answer 36

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

Question 37

Explain the 5 ways mRNA can be alternatively spliced

Answer 37

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 1. Intron 1 2 and exon 3 are removed, giving exon 1 and 2 as the product 2. Intron 1 2 and exon 2 are removed giving exon 1 and 3 as the product But we always see exon 1 retained

Question 38

How are diff splicing products formed during alternative splicing

Answer 38

By the competion between different splicing factors/ snRNPs which can lead to diff splicing

Question 39

What is the SV40 T-antigen

Answer 39

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

Question 40

Explain the splicing of the SV40 T-antigen

Answer 40

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

Question 41

How is alternative splicing regulated

Answer 41

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

Question 42

Explain the sex determination gene in drosophila

Answer 42

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

Question 43

What is the Pm and Pe in the drosophila sex determination

Answer 43

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

Question 44

Explain the alternative splicing of the Sxl gene in drosophila

Answer 44

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

Question 45

Explain how terminally differentiated cells can be reverted into induced pluripotent stem cells

Answer 45

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

Question 46

Explain the process of how terminally differentiated cells can be reverted into induced pluripotent stem cells

Answer 46

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

Question 47

What is RNA shuffling

Answer 47

The creation of new genes through evolution

Question 48

Expalin the origin of intron models

Answer 48

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

Question 49

Why are many introns found in multicellular organisms

Answer 49

1. To make mutiple protien products from a single gene though alternative splicing 2. 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

Question 50

Give an example of the exon reshuffling

Answer 50

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

Question 51

Overall why do scientist use rna to study evolution

Answer 51

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

Question 52

Other than rna splicing what post transcriptional process is there for rna

Answer 52

RNA editing: Changes the rna sequence post transcriptionally the translated protien is diff than what the gene sequences predicted it should be (because edited)

Question 53

Explain rna editing by deamination

Answer 53

The enzyme cytidine deaminase specifically recognizes the correct location on cytosine to deaminate and turn into uracil

Question 54

Explain the deamination of apolipoprotien B

Answer 54

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

Question 55

What is adenine deamination

Answer 55

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

Question 56

Explain insertional editing : uridine insertion

Answer 56

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

Question 57

What are the types of rna editing

Answer 57

Cytosine deamination Adenine demaination Insertional editing: Uridine insertion

Question 58

After the rna transcript it made how it it sent to the cytoplasm

Answer 58

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