lf206 lecture 6 - eukaryotic genomes, post transcriptional control

name 3 eukaryotic polymerase II post-transcriptional events

why do these events not always occur?

1. 5’ capping

2. splicing

3. 3’ end processing

they require factors that bind to the phosphorylated C-terminal domain of RNA Pol II

what does CTD stand for?

it is found on __karyotic RNA Pol __

it is comprised of a 7 amino acid sequence → what is it?

phosphorylation of ____ 2 and 5 marks the transition from transcriptional initiation to _______

C-terminal domain

found on Pol II

YSPTSPS

seryls

elongation

what is transcriptional initiation and elongation?

initiation → first steps of transcription where RNA polymerase unwinds DNA and begins to read to synthesise RNA

elongation → RNA polymerase building RNA in 5’ to 3’ direction

addition of 5’ cap is an early event (nascent mRNA ~ __bp)

it distinguishes Pol II ______ from other RNA molecules

stabilises RNA, there is no 5’ _____, so it is resistant to 5’ _________

aids in further processing and export to the ________

is required for efficient ________ of mRNAs

25

transcripts

phosphate

exonucleases

cytosol

translation

the 5’ cap is a modified ________ nucleotide

name the 3 capping enzymes - these enzymes are recruited by _____

guanine

1. RNA triphosphatase

2. guanylyl transferase

3. methyltransferases

the phosphorylated C-terminal domain (CTD) of Pol II

a multi-subunit ________ ATP-________ decapping enzyme ______ removes the 5’ cap

it restores the 5’ ______ on the mRNA

now the mRNA can no longer be bound by ______ so cannot be translated

the mRNA is ______ by a 5’-3’ RNAse

cytosolic

dependent

complex

phosphate

ribosomes

degraded

what is rDNA?

ribosomal DNA that encodes for rRNA (small subunits that make up a ribosome)

Tetrahymena thermophilia rDNA encodes for two large ribosomal subunits (___ and ____) and a small ribosomal rRNA (___)

28S 17S

5.8S

what is splicing? how does this relate to group I introns?

removing introns from pre-mRNA transcript to only have exons making mature mRNA

group I introns can self-splice

Cech discovered that an ______ in Tetrahymena thermophilia rRNA could remove itself - this is called?

the steps:

1. the intron ____

2. a _______ co-factor (GMP, GDP, GTP) is held in a pocket

3. the 3’ -__ of the co-factor is a nucleophile that attacks the ______ at the 5’ splice site

4. the 3’-OH of the upstream ____ attacks the phosphate of the 3’ _____ site

5. the ______ are fused and the intron in degraded.

introns

self-splicing

folds

guanosine

OH

phosphate

exon

splice

exons

group I introns are ___-splicing

group II introns utilise 2’-__ group of the branch site ______ as an in-built co-______

both group I and II introns encode protein maturases - what are these?

nuclear spliceosomal introns (what are these?) use the same catalytic mechanism as group __ introns.

self

OH

adenine

factors

proteins that increase efficiency of excision by stabilising folded 3D structure

introns found in pre-mRNA of eukaryotic cells to be removed by the spliceosome

II

how are eukaryotic introns spliced?

1. introns ___ and the 2’-OH of the _____ ___ adenosine attacks the phosphate at the _’ splice site

2. adenosine now has 3 _____________ bonds. one includes a strange bonds which is?

3. the 3’ -OH group of the _____ exon attack the _______ at the 3’ splice site

4. the exons are ____ and the intron is released as a ______

fold

branch site

5

phosphodiester

2’,5’ phosphodiester bond

upstream

phosphate

fused

lariat

the spliceosome is an RNA and _____ complex

it is composed of 5 subunits called ______. name all 5

what 3 specific sequences does the spliceosome recognise?

protein

snRNPs (small nuclear ribonucleoproteins)

U1, U2, U4, U5, U6

5’ splice site, branch site, polypyrimidine tract (poly Y)

where are the following located?

5’ splice site, 3’ splice site & branch site

5’ splice site: beginning of intron

3’ splice site: ending of intron

branch site: located upstream from ending (3’ splice site) of intron

spliceosome mechanism

1. U1 snRNP binds to the _’ splice ___

2. _____ _____ _______ (BBP) and the protein U2 auxiliary _____ (U2AF) bind the branch site and associated pyrimidine tract.

3. a trimer of U4, U5 and ___ snRNPs associates with the complex

4. U2 snRNP is recruited and displaces ___

5. base pairing of U2 snRNP with the ______ causes the branch site _____ to bulge towards the _’ splice site

6. loss of _ and _ snRNPs activates the complex

7. the 2’ -__ of the branch site adenine attacks the 5’ ____ site phosphate

8. the ____ is formed

9. U6 snRNP guides the 3’-OH of the upstream ____ that attacks the 3’ _____ site phosphate

1. 5, site

2. branch binding protein, factor

3. U6

4. BBP

5. intron, adenine, 5’

6. OH, splice

7. lariat

8. exon

9. splice

how does U1 snRNP find the 5’ splice site?

1. U1 is already close to the RNA Poll II tail - what is it called?

2. capping of the pre-mRNA transcript by the _______ enzyme (CE) is an early event

3. U1 snRNP is recruited by the _________ CTD

4. U1 snRNP _____ the growing mRNA for the 5’ ____ site and binds it

1. C-terminal domain (CTD)

2. capping

3. phosphorylated

4. scans, splice

what does U2AF do.

• it is bound by the ___ of RNA Pol __

• whilst the pre-mRNA chain is growing, BBP/U2AF scans for __________ and _________

• once recognised, the ________ is assembled

true or false - it is apart of the spliceosome structure?

CTD, II

pyrimidine tract and branch site

spliceosome

false

splicing by spliceosome is regulated by _______ sequences.

individual motifs (what are these?) have different affinities for spliceosome components.

consensus

motifs - recurring patterns (can be nucleotides, can be amino acids)

biogenesis of U1, U2, U4 and U5 snRNPs

1. transcription and _______ in the nucleus

2. _______ loading of a ring of ____ Sm proteins onto the snRNA by ___ proteins

3. the cap is __________

4. transport to the _____ and maturation by association with other proteins

5. U6 lacks __ proteins so remains nuclear.

1. capping

2. cytosolic

3. 7, SMN (Survival Motor Neuron)

4. hypermethylated

5. nucleus

6. Sm

function of the protein components of snRNPs

stability of the snRNP

difference between snRNA and snRNP

snRNA: just a small sequence of RNA (e.g. U1, U2 etc.)

snRNP: snRNA + associated proteins that can then catalyse intron removal (e.g. U1 snRNP, U2 snRNP etc.)

exons are defined by the binding of proteins during ________

this allows U1 to find __________

1. SR protein (rich is Ser and ___) bind to _____ ______ ______ (ESEs)

2. splicing ______ flank the 5’ and 3’ ____ sites ensuring precise intron removal

3. ________ hnRNP proteins can bind to exon sequences and act as _______ repressors as they compete with __ proteins.

transcription

5’ splice site

1. Arg, exonic splicing enhancers

2. factors, splice

3. heterogenous nuclear ribonucleoproteins, splicing

4. SR

what is the poly Y tract involved in?

alternative splicing

what is alternative splicing & what is it also called?

a single gene can produce multiple different mRNA transcripts

exon skipping

a strong consensus poly Y tract leads to?

1. strong binding of BBP and ____

2. BBP is displaced by __

3. results in a _’ end of an intron strongly defined → normal splicing

1. U2AF

2. U2

3. 3

a weak consensus poly Y tract leads to?

1. BBP and ____ binds

2. U2 is _____

3. 3’ end (which part of the intron?) of intron defined and you get normal _____

OR

1. U2AF ______

2. BBP and __ are not bound

3. _’ end of intron is not defined

4. _________ scans for the next U2AF and exon is _____ (alternative splicing)

1. U2AF

2. recruited

3. the beginning of the intron, splicing

1. disengages

2. U2

3. 3

4. spliceosome

alternative splicing is regulated by the sequence of _______

poly Y tract (polypyrimidine tract)

give an example where splicing goes wrong

• Alexei (great grandson of Queen Victoria) had a G residue in blood clotting factor IX

• no masking the mutation due to having only 1 X chromosome

• this caused splicing to occur 2 nucleotides early and produced an ineffective blood clotting factor

• Anastasia had a heterozygous A/G genotype in blood clotting factor IX

• therefor has a normal phenotype as the disease is recessive

how is the poly(A) tail added?

1. GU rich region is cleaved leaving 3’ -OH

2. the GU rich region is degraded in the nucleus

3. roughly 250 adenine residues are added at the 3’ end

how is the GU rich region cleaved?

1. RNA is folded to bring _ key sites together

2. the poly(A) tail is bound by __ ____ __ ___ (CPSF)

3. the cleavage site is bound by _______ _______ (CF)

4. the GU rich region is bound by ____ ____ ____ __ (CStF)

5. these provide a recruitment site for the polymerase called ___

6. cleavage provides a 3’-__ for PAP which adds ______ residues

7. this is a very ____ process

1. 3

2. cleavage and polyadenylation specificity factor

3. cleavage factor

4. cleavage stimulation factor F

5. PAP

6. -OH, adenine

7. slow

how to go from slow adenylation to fast adenylation? [3]

1. PAP slowly adds nucleotides

2. poly(A) binding protein then associates and extends tail faster

3. ~200 nucleotides are added and PAP disengages

functions of polyadenylation

1. nuclear transport → nuclear export receptor is loaded onto mRNA by Pol II CTD (a ‘ticket’ to leave the nucleus)

2. translation → pseudo circularisation (making loop) of transcript promotes translation (poly(A) tail & PABP interact w/ methylated 5’ cap)

3. stability → mRNA often degraded from 3’ end first, tail extends lifespan by reducing degradation rate