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BIOL200 > RR9: RNA processing II > Flashcards

RR9: RNA processing II Flashcards

1
Q

Does only mRNAs undergo splicing and modifications?

A

No.
mRNA synthesized by RNA pol 1, 2 and 3 gets spliced, but not all in the same way.
rRNAs are changed also.
tRNAs are changed also.

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2
Q

What synthesized rRNAs?

A

RNA pol 1 in the nucleolus.

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3
Q

When does the modifications of rRNAs occur?

A

After transcription is done.

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4
Q

How is the transcription of rRNA happening?

A

RNA pol 1 will make a long pre-rRNA.
rRNAs are transcribed from repeated sections in the ribosomal DNA.
The transcription of rRNA is the same in all organisms.

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5
Q

How does the cleavage of rRNA happen?

A

Small nucleolar RNAs (snoRNAs) cleave the rRNA and it’ll give rise to 18S RNA, 5.8S RNA and 28S RNA, always in the same sequence in every organism. It’s conserved.

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6
Q

Are tRNAs spliced like the transcripts done by RNA pol 2?

A

No.

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7
Q

What synthesizes tRNAs?

A

RNA pol 3.

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8
Q

How are tRNAs modified?

A

tRNAs are not spliced, they’re modified.
1. 5’ end sequence is removed
2. a short segment is removed at the 3’ end
3. CCA is added on the 3’ end
4. More modifications in the tRNAs

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9
Q

Why is the addition of the CCA sequence on the 3’ end of tRNAs is so important?

A

Because it ensures that tRNA can participate in protein synthesis by linking with the aminoacyl tRNA.

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10
Q

What proteins help the modification process of all RNAs?

A

RNA binding proteins that have specific domains like:
- RRM domain (beta sheets positively charged that interact with negatively charged regions of RNAs)

Polypyrimidine tract binding protein (PTB) has the RRM domain and it interacts with that polypyrimidine tract in the introns

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11
Q

How do RNA binding protein help the modification of all RNAs?

A

RNA binding proteins help the splicing machinery recognize which sequences need to be taken out.

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12
Q

What does the protein U2AF do?

A

It’s an RNA-binding protein that helps identify which sections need to be spliced in mRNA.
Knows where the exons are.
The small subunit of U2AF interacts with the 3’ end of the intron (AG)
The bigger subunit of U2AF interacts with sequences around the polypyrimidine region.
Overall, U2AF splices the 3’ end of the intron (AG)

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13
Q

Which type of protein defines where the exons are?

A

Senior proteins (SR proteins)

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14
Q

In eukaryotes, are introns or exons smaller?

A

Exons are smaller compared to introns.

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15
Q

What do senior proteins do?

A

Interact with exonic splicing enhancers on exons.
Help U2AF identify where the AG is.
Help the polypyrimidine tract find the 3’ end of the intron.
Help U1 snRNP find the 5’ end of the intron (GU)

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16
Q

What is called the regrouping of senior proteins, U2AF, U1 snRNP and polypyrimidine tract called?

A

Cross-exon recognition complex.

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17
Q

What are exonic splicing enhancers doing?

A

Interact with senior proteins in the exon.
During splicing, they promote exon joining.

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18
Q

What is the overall role of the cross-exon recognition complex?

A

SR proteins + snRNPs (with their snRNAs) + polypyrimidine tract + U2AF
Adds efficiency to the splicing.
Make sure the right segments are spliced

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19
Q

What is alternative splicing?

A

It’s the splicing of a gene so that one promoter can give rise to different mRNAs, so different proteins.
Can be used when a gene is expressed in 2 different places in the body, but has 2 different roles.
It can splice exons to make sure that the proteins are different in the 2 different places.
It takes out sticky domains to make sure the cells in our blood don’t stick to the veins.

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20
Q

What’s an example we’ve seen about alternative splicing?

A

Sex-lethal (RNA binding protein)
Alternative splicing that controls sex determination in Drosophila

21
Q

What differentiates male and female flies Drosophila?

A

Males have 1 X chromosome
Females have 2 X chromosomes, but 1 is inactive. Sex lethal is activated in female flies

22
Q

Why is sex lethal only activated in female flies?

A

There’s a chromosome counting to make sure that male and female have the same X chromosome dosage. The activation of sex lethal in female is associated with that dosage.
In males, the sex lethal promoter is shut off because after the chromosome counting, the gene knows that the sex lethal protein doesn’t have to be formed.

23
Q

When does the sex lethal activation happen?

A

During zygotic transcription.

24
Q

What’s the reaction pathway of sex lethal?

A
  1. Sex lethal, the RNA binding protein, will bind at the 3’ end of the intron between exon 2 and 3.
  2. Binding of sex lethal to the 3’ end blocks U2AF from binding
  3. U2AF has to bind to the next intron at the 3’ end, which is the intron between exon 3 and 4
  4. Because U2AF couldn’t sit right before exon 3, it sat right before exon 4, which causes the splicing of exon 3
  5. Female flies don’t have an exon 3, while males have one
25
Q

If the mRNA in female flies encodes for sex lethal protein, what does the mRNA with the exon 3 will encode?

A

It doesn’t encode any protein, because there’s a stop signal in exon 3. So the ribosome drops the mRNA in males before it can make a protein.

26
Q

The sex lethal protein plays a role in alternative splicing in its own mRNA, but what else does sex lethal protein influence?

A

ONLY IN FEMALES because sex lethal protein is only found in female flies.
Sex lethal protein will bind to the pre-mRNA of that encode the TRA protein.
1. Sex lethal binds to the 5’ end of the intron between exon 1 and 2.
2. U2AF can’t bind to that 5’ end, so it has to bind to the next intron between exon 2 and 3.
3. In female flies, exon 2 will be spliced, but in male, exon 2 will be there.

27
Q

Why is the TRA protein only found in female flies?

A

There’s a stop signal in exon 2. Since the exon 2 is spliced in the TRA mRNA in females, it gives rise to the TRA protein.
Since male flies have the exon 2, the ribosome will stop translation and won’t encode the TRA protein.

28
Q

What is double sex?

A

It’s a transcription factor in drosophila flies that activate important genes for sexual characteristics.
There are 2 forms:
- one for females
- one for males

29
Q

What is the pathway to make the double sex protein in female flies?

A
  1. TRA protein, only found in female flies will form a complex with RBP1 and TRA2 on the 5’ end of the intron between exon 3 and 4 of double sex mRNA
  2. This complex that’s been formed defines exon 4 as an important exon for females.
  3. The splicing will only take out introns, they leave all the exons.
30
Q

What type of proteins are TRA2 and RBP1?

A

They’re senior proteins, so they will help with the splicing of introns.

31
Q

What’s the pathway to make double sex protein in male flies?

A

Males don’t have TRA protein. They don’t form a complex to define exon 4 as important.
The splicing will cut introns and exon 4.

32
Q

What are the differences in the functions of double sex protein in males and females?

A

In males, the double sex protein interacts with parts of the genome that activate male features.
In females, the double sex protein has an extra domain in exon 4 that interacts with genes that will block male features. It allows the expression of female features.

33
Q

What’s the cascade of events that needs to happen so that male and female flies are different?

A
  1. In early development, chromosome counting.
  2. Dosage compensation for X chromosomes (1 in males, 2 in females)
  3. Activation of sex lethal promoter in females
  4. Sex lethal protein binds to the 3’ end of the intron right before exon 3, splicing exon 3 in females
  5. The stop signal in exon 3 doesn’t allow males to produce sex lethal protein.
  6. Sex lethal protein in females binds to the 3’ end of the intron right before exon 2 of TRA pre-mRNA, splicing exon 2
  7. Stop signal in exon 2 doesn’t allow males to produce TRA protein
  8. TRA protein with RBP1 and TRA2 bind to the intron right before exon 4 on double sex pre-mRNA, defining exon 4 as important in females
  9. In males, exon 4 of double sex is spliced
  10. The extra domain in mRNA creates a different double sex protein that blocks male features in female flies
34
Q

What is another example of processing event that is not splicing?

A

RNA editing

35
Q

Where does RNA editing happen?

A

In mitochondria and plastids.
Rarely in nuclear genomes of eukaryotes

36
Q

What is RNA editing?

A

It’s when deaminases (enzymes) convert one nucleotide to another.
So the mature mRNA is different from the sequence of the coding region of the DNA.
(Transcription of ssDNA to make mRNA, so it’s supposed to be complementary)

37
Q

What does a deamination reaction do?

A

It removes amines to convert:
A to I
or
C to U

38
Q

In RNA editing, was the transcription of DNA to mRNA made correctly?

A

Yes. It’s deaminases that change the mRNA after it was correctly transcribed.
The DNA is not changed.

39
Q

How do we see that RNA editing happened?

A

Since the mRNA is modified, we’ll see the changes in the proteins that the modified mRNA will translate.

40
Q

Why can RNA editing make such big changes if only one nucleotide is changed?

A

Because during translation, the mRNA is read in codons, so 3 nucleotides at a time that correspond to 1 amino acid.
The protein will have 1 different amino acid than it was supposed to, which changes the functions of a protein.

41
Q

In which gene can we see regulated RNA editing happening? (an example)

A

the mammalian Apolipoprotein B made in the liver and the intestine.
In the intestine, the regulated RNA edition causes a conversion from C to U in the RNA.
It creates a UAA codon, which is a stop signal during protein synthesis.
So the protein in the intestin is half the size of the protein in the liver.

42
Q

How does the termination of transcription happen?

A

RNA polymerase recognizes the terminator and falls off.

43
Q

RNA polymerase 2 actually transcribes more nucleotide than necessary, so how is the mRNA transcript cleaved to make sure it doesn’t have too many sequences?

A
  1. AAUAAA xxxxx G/U sequence on the pre-mRNA is recognized by cleavage polyadenylation factors (CPSF).
  2. The AAUAAA sequence is in the 3’ end of pre-mRNAs and it’s the poly A signal.
  3. The poly A signal is recognized by a protein complex.
  4. The protein complex binds to the pre-mRNA, and catalyzes the cleavage of the pre-mRNA.
44
Q

What happens at the exact moment the cleavage of the pre-mRNA at the poly A signal happens?

A

Poly A Polymerase (PAP) adds adenosine nucleotides to the end of the pre-mRNA to create a poly A tail.

45
Q

What does the poly A tail do to the pre-mRNA?

A

It protects the 3’ end of the pre-mRNA from degradation by enzymes in the nucleus.

46
Q

Does Poly A Polymerase act alone in the polyadenylation of the 3’ end of the pre-mRNA?

A

No. The Poly A Binding Protein (nuclear) acts as a co-factor to help the addition of adenosine be more efficient.
So PABPN binds to the poly A tail to catalyze the reaction.

47
Q

What are the 2 phases in the polyadenylation of the pre-mRNA?

A
  1. Slow. PAP. Poly A Polymerase adds 12 A residues
  2. Fast. PABPN. Poly A Binding Protein adds 200 A residues and helps the reaction.
48
Q

Do all mRNAs require poly A tails?

A

No. Histone mRNAs don’t require a poly A tail. They are not polyadenylated.

49
Q

Why aren’t histone polyadenylated?

A

Because they have secondary structures (stem-loops) in their 3’ untranslated regions.
It protects histone mRNAs from degradation by the 3’ -5’ exoviralnucleases.

BIOL200 (16 decks)

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