RR8: RNA Processing I

Question 1

what do the different polymerases do?

Answer 1

pol 1: ribosomal RNA
pol 3: tRNA
pol 2: all the mRNAs and other important RNAs like snRNA and micro RNA

Question 2

what is the distinguishing feature of RNA polymerase ii?

Answer 2

the fact that it has a CTD
it is very important, as RNA pol II does very different things than the other RNA polymerases

Question 3

what is the structure of the CTD and what happens if you change it?

Answer 3

has 52 heptapeptide repeats (in humans) and 26 in yeast
7 proteins repeated 52 times
if you remove sections of these repeats from the CTD, the yeast die and they can’t grow anymore
the CTD is essential

Question 4

what are the phosphorylations that happen on that heptapeptide sequence and what do they lead do?

Answer 4

one of the phosphorylations happens during transcriptional initiation by TFIIH (by the subunit called protein kinase module)
this protein kinase module will phosphorylate all serine 5’ in those heptapeptide repeats in the CTD at initiation
During elongation, serine 2 is also phosphorylated by another protein kinase
this protein plays a critical role in switching from post initiation to real true elongation

Question 5

what is promoter clearance?

Answer 5

once RNA pol II initiates transcription and starts to form an RNA molecule, it will leave the promoter and GTFs (with exception of TBP)
it will dissociate and reassociate later on

Question 6

how many nucleotides does pol 2 transcribe before it dissociates?

Answer 6

about 100 nucleotides, stops around the first nucleosome

Question 7

what happens to the heptapeptide repeat sequence during that pause of RNA pol 2?

Answer 7

serine 5 on the CTD is phosphorylated thanks to TFIIH
this gives rise to specific structures on that sequence that is recognised by a very important enzyme required for the processing of the emerging 5’ end of the pre-mRNA (recruits this enzyme)

Question 8

how are the exit channel and the CTD positioned relative to each other?

Answer 8

very close

Question 9

what is the enzyme that is recruited by the phosphorylation of serine 5 on the CTD? and what does it do?

Answer 9

the capping enzyme
interacts with the phosphorylated CTD and becomes very active
recognises the 5’ end of the pre-mRNA and adds a 7-methylguanosine cap to that end
that cap is added thanks to a triphosphate 5’-5’ linkage
this cap will protect the 5’ end of the emerging nascent pre-mRNA from any exoribonucleases that might attack that 5’ end

Question 10

what are other things that are done to protect the mRNA from ribonucleases?

Answer 10

in animal cells and higher plants the 2’ hydroxyl of ribose group of first nucleotide is methylated, and in vertebrates the second nucleotide is also methylated (on the 2’ hydroxyl)

Question 11

what other effect except protection from ribonucleases does the cap have?

Answer 11

plays an important step later in processing and expression through its ability to facilitate nuclear export
plays a critical role in recruiting factors important for efficient translation

Question 12

how is RNA pol II stalled?

Answer 12

• When RNA pol 2 leaves the pre-initiation complex it leaves an interface that was normally bound to those GTFs
• Once that region is freed, a very important protein interacts with it
• that protein is called negative elongation factor (NELF)
• That’s also associated with another protein called DSIF (Drb sensitive inhibitory factor)
• Drb is an inhibitor of elongation
• DSIF and NELF will bind RNA pol2 together, and NELF acts like a plug so that NTPs can’t get into the catalytic site —> elongation stops
• The presence of these two negative factors that slow RNA pol down and have it pause at or around the first nucleosome

Question 13

how does RNA pol ii continue with elongation after its pause is over?

Answer 13

• The pause complex is also recognized by the other protein kinase critical for elongation which Isn’t associated with a GTF
• Cyclin dependent kinase called cdk9, coupled with cyclin T (also called P-TEFb)
• This kinase will recognise this paused complex and will phosphorylated NELF, DSIF, serine 2 on the heptapeptide repeat and will therefore cause NELF to leave the complex
• Another protein replaces it so that it can’t rebind afterwards
• Phosphorylation of DSIF changes its conformation and becomes instead of being a negative elongation factor, it forces the clamp down, ensuring that RNA pol 2 becomes very processive
• Another elongation promoting factors also associate with the complex
• P-TEFb plays a critical role from changing from stalled to elongation complex

Question 14

what is the particular significance of the serine 2 phosphorylation?

Answer 14

it recruits factors required for RNA processing

Question 15

what are the different RNA processing factors and when do those things happen?

Answer 15

splicing factors, poly adenylation factors, export factors
recognise serine 2 phosphorylation and use it as platform to interact with the large domain of RNA pol 2
this happens as RNA is being synthesised, all at once

Question 16

what needs to happen for RNA to become mRNA?

Answer 16

introns need to be spliced out and exons have to be put together continuously so that they retain a reading frame so that translation works

Question 17

what do DNA segments that encode introns do?

Answer 17

they are not junk, they encode regulatory information (cis acting elements and enhancers)

Question 18

how was the discrepancy of the mRNA size and the DNA genes visualised in an experiment?

Answer 18

visualised with an EM micrograph
double stranded viral genes were separated and the mRNA that corresponds to that gene was allowed to bind and make a hybrid
mRNA-DNA hybrid
mRNA will bind to its complementary sequence and it will be seen that big loops in the DNA don’t correspond to anything
what mRNA interacts with is exons

Question 19

what are the regions in an intron that are recognised by the intron splicing machinery?

Answer 19

throughout the introns per se there is little conservation, but there are specific conserved sequences necessary in specific sequences
* At the 5’ end of the intron to be spliced out: GU dinucleotide sequence
* At the 3’ end of the intron to be spliced out: AG
* Some of the sequences of the other side of the boundary are also conserved, but GUAG stands, it is needed for it to work
* towards the 3’ end of the intron there are also sequences that seem to be present very commonly: the pyrimidine rich region
* There is always an adenosine a bit upstream of that pyrimidine rich region
* Those adenosine is very important to the biochemical understanding of the splicing reaction (branch point)

Question 20

what are the interactions between snRNPs and snRNAs that happen during intron splicing?

Answer 20

• splicing takes place by virtue of a number of RNA associated proteins
• Small nuclear RNAs (snRNA)
• snRNA are always associated with proteins
• The proteins that associated with snRNA are called small nuclear ribonucleoproteins (snRNPs)
• the flavor is determined by the snRNA with which it associates (U1, U2, U3, 4, 5, 6 snRNPs)
• RNA-protein complex that are responsible for splicing RNA
• There are 6 snRNAs that are associated with these RNPs and most of them are generated by RNA pol 2 with the exception of U6
• U1 snRNA plays a critical role in splicing out introns by interacting with the boundary at the 5’ end of the intron and 3’ end of upstream exon
• There is base pairing between snRNA (u1) and some of the residues in the 3’ exon end and 5’ end of intron sequence, particularly the GU
• U2 snRNA also interacts with regions a bit upstream of the poly pyrimidine region
• It base pairs with nucleotides around that adenosine residue, called the branch point
• The A never base pairs, the residues around it yes will base pair with the U2 snRNA leaving the A to bulge out from that double stranded RNA
• This changes the conformation and makes it critical for some of the downstream reactions
• U1 and U2 snRNA have to base pair with the RNA in the intron that is to be excised

Question 21

how was it tested that RNA:RNA pairing is critical for splicing?

Answer 21

• experiments were done to show how essential that is
• Experiments were done where that region that U1 snRNA binds to was modified, and that modification blocked splicing
• If you introduce oligonucleotides that interact with that region, you can also block splicing
• most convincing experiment:
• Transfected in a gene that had to be spliced but was mutated around those sequences, splicing didn’t happen
• But if they co transfected a cDNA that would give rise to a U1 snRNA that could interact to that mutated variant, splicing could be restored
• So, as long as U1 snRNA can interact with the sequence around that upstream boundary, then splicing will take place
• Compensatory mutation
• Same experiments were done with U2 to show that it to must base pair with nucleotides around the adenosine branch point

Question 22

what are the trans-esterification reactions that occur during intron splicing?

Answer 22

• the whole rection (intron splicing) takes place very rapidly and is dependent on two trans estérification reactions
• the bulged out A plays a critical role here
• This conformation change enhances its ability to interact in the first trans estérification reaction
• The hydroxyl group on 2’ end of the bulged out adenosine carries out a nucleophilic attack on the phosphorus present and attached to the upstream exon
• In doing so it creates a new phosphoester bond, lariat structure (2’-5’ linkage)
• Things are unstable after this
• The now free hydroxyl group on the 3’ end of the upstream exon can carry out nucleophilic attack again on the phosphorus present at the 5’ end of the downstream exon
• In doing so you readjust and you form a new phosphodiester bond and make the two RNA segments continuous (exons)
• Result is the removal of the lariat which contains the intron

Question 23

how can the intermediates in the intron-splicing reaction be visualised?

Answer 23

if you radiolabel the substrates, you can see them on a polyacrylamide gel

Question 24

how does the splicing reaction involve the snRNPs?

Answer 24

• the actual reaction also includes associated snRNPs
• In the first step of the reaction, the U1 snRNP will interact with the 5’ boundary
• The U2 snRNP will be recruited to the region around the branch point adenosine
• At that point the complex is recognized by U4 U5 and U6 snRNPs
• This collection of balloons readjusts
• through changes within the spliceosome, U1 and U4 will be ejected and the remaining snRNPs will be the active spliceoome (when the transesterification reaction happens)

Question 25

what is the result after the splicing reaction?

Answer 25

• is a continuous RNA molecule with two exons linked by that phosphodiester bond
• All the snRNPs leave the complex (U2, U5, U6 because 1 and 4 already left)
• And the lariat intron has to leave

Question 26

what happens to the lariat intron?

Answer 26

• The lariat intron is a problem because it’s got the weird 2’-5’ linkage and its really hard to get rid of it using conventional RNA degrading enzymes
• devoted enzyme responsible for the removal of the 2’ 5’ linkage and the linearization of the lariat, called the debranching enzyme
• Once the intron is linear, it can be degrade very fast by exo and endo ribonucleases within the nucleus

Question 27

what is an alternative mechanism for intron splicing without the need for any protein function? what does it need and what does it suggest?

Answer 27

• it was found that introns can be sometimes excised without the need of any protein function
• Removed by the RNA themselves
• RNA must have some sort of catalytic function that is independent of any protein
• The reaction only happens when you add magnesium to it
• Doesn’t work if you don’t have magnesium
• If you treat it with proteases and give it magnesium, it will still happen
• Nucleic acids themselves can do it, they dont need proteins
• RNAs can carry out some of the most essential functions critical for life
• Suggests that RNA molecules were formed first in the primordial world and them came proteins and DNA
  self splicing is the exception not the rule, proteins make everything more efficient