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BIOL 200 > RNA processing > Flashcards

RNA processing Flashcards

1
Q

What type of organism possess 3 RNA polymerases ?
What are the common features between all 3?

A

Eukaryotes

Common features:
- Are multimeric protein complexes
- Some subunits show significant homology with bacterial RNA polymerase

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2
Q

What is specific to RNA pol II?

A

Has CTD (C-terminal domain) tail on large subunit

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3
Q

What is the structure of the CTD tail?

A

Humans: YSPTSPS (52)
52 repeats of heptead-peptide sequence
*serine 5 phosphorylated during initiation (TFIIH/CDK7)
serine 2 phosphorylated after pausing (p-TEFb/CDK9)

Yeast: YSPTSPS (~26)

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4
Q

What must occur for nascent transcriptsas soon as transcription start to help them reach functional mRNAs?

A

*one of most important post-translational modification

  • Precursor mRNAs (pre-mRNA) are modified at their 5’-end
  • 7’ methylguanulate CAP added to 5’-terminal nucleotide through unusual 5’-5’ triphosphate linkage by Capping enzyme
  • 2’ OH (hydroxyl of ribose group) of 1st base for animal cells and higher plants
  • In vertebrate, second nucleotide base also methylated (2’ OH)
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5
Q

When does the addition of the 7’ methylaguanylate CAP occur? What is its importance?

A

Occurs during transcriptional pause (post-initiation)

CAP = common to all mRNAs:
- protects pre-mRNA
- facilitates nuclear export
- recognition by translation factors (eIF4)

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6
Q

Why is RNA pol II pausing after initiation an important regulated step?
How does it occur?

A
  1. NELF (negative elongation factor) associated with DSIF (DRB sensitive inhibitor factor) bind RNA pol II → NELF plugs so that NTPs can’t get into catalytic site → pausing of RNA pol II around 1st nucleosome
  2. Paused complex recognized by p-TEFb cyclin T coupled with CDK9 → will phosphorylate NELF, DSIF, Serine 2 on CTD (change dependent on this step)
  3. NELF leaves complex and other proteins takes its place so it can’t rebind
  4. Phosphorylation of DSIF changes conformation and forces clamp down ensuring RNA pol II becomes very processive
  5. Elongation factors associate with the complex: DSIF (stays), SPT6, PAF
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7
Q

What is the impact of phosphorylation of Serine 2 on the CTD?

A
  • Recruits splicing factors
  • Recruits polyadenylation factors
  • Recruits Export factors
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8
Q

How is the presence of introns in bacteria? yeasts? humans?
What is the importance of introns?

A

Bacteria = no introns
Yeast = small introns
Human = long introns
(eukaryotic genes have introns unlike bacterials genes)

Introns contain regulatory sequences (enhancers for ex)

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9
Q

How were introns discovered?

A
  • They were discovered because of a discrepancy between mRNA size and gene size
  • Visualized by hybridization experiments

mRNA-DNA hybrid of the adenovirus gene → intron sequences in the DNA loop out bc no complementary regions in mRNA

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10
Q

Which are the highly conserved sequences of introns?

A
  1. GU just downstream of 5’ splice site (5’ on intron)
  2. A of the branch point (followed by 20-50 pyrimidine-rich(C and U))
  3. AG just upstream of the 3’ splice-site (3’ on intron/5’ on exon)
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11
Q

What is the spliceosome?

A

Large ribonucleocomplex which catalyses splicing of pre-mRNA

Consists of 5 snRNPs (small nuclear ribonucleoprotien particles)

snRNPs = snRNA (U1, U2, U3,U4, U5 or U6) + 6-10 proteins subunits

*RNA:RNA interactions required for splicing (perfect Watson-Crick base pairing)

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12
Q

What is the role of U1 snRNP?

A

Recognizes 5’ splice site of intron (GU included)
Needs perfect Watson-Crick base pairing

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13
Q

What is the role or U2 snRNP?

A

Recognizes the branch point sequence within the intron
Key role in formation of spliceosome complex
Needs perfect base pairing except for A which never base-pairs to U2 snRNA creating conformational change (bulges out)

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14
Q

How did researchers find out RNA:RNA interaction was absolutely required for splicing?

A

By introducing snRNAs that possess a compensatory mutation corresponding to the one un the pre-mRNA, splicing is restored

*Especially for U1 function

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15
Q

What is the role of U4/U6 and U5 snRNAs?

A

They for an tri-snRNP complex
Stabilize the spliceosomal complex, facilitating the catalytic steps of splicing

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16
Q

Which reactions are involved in intron splicing?

A

2 trans-esterification reactions: (lariat formation + 2 exons connecting)

  1. OH of A at the branch point attacks 5’phosphate (the phosphate group between the intron and the exon) of 1st introns residue (G) → formation of a “lariat” → 5’-exon is detached
  2. Free 3’-end (OH) of detached exon attacks 5’-PO4 of the first residue of the 3’-exon
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17
Q

VOIR SCREENSHOT

A
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18
Q

How can the splicing reaction be visualized in vitro?

A

Using radiolabelled RNA substrate (probes)
Each intermediate splcing product can be separated and quantified in polyacrylamide gel
- 3’-Exon + lariat = most heavy
- lariat by itself (bc longggg introns)
- 2 exons not rlly linked
- final mRNA (2 exons linked by phosphodiester bond)
- 5’-exon by itself detached between the 2 steps

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19
Q

What are the important steps of the spliceosome cycle?

A
  1. Recognition of intro-exon boundaries: U1 snRNP, U2 snRNP and U2AF bind
  2. Assembly of rest of spliceosome: U6/4 U5-tri snRNP added → conformational change
  3. Activation of spliceosome: U1 and U4 leave + interaction between U2 and U6
  4. 1st trans-esterification reaction
  5. 2nd trans-esterification reaction
  6. Spliceosome disassembly
  7. Debranching enzyme → 2’-5’ linkage is a problem as lariat can’t be degraded in this form
  8. Exo- and endo-nucleases digest the linear intron RNA
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20
Q

Is the splicing reaction ATP-dependent?

A

NO, no energy expenditure
But snRNP binding/assembly of the spliceosome is ATP-dependent

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21
Q

How can self-splicing be possible?

A

Normal splicing = snRNA + proteins = Spliceosome
Self-splicing = only snRNAs meaning RNA must have some catalytic function (ex: as long as have Mg in the mix)
Seen in polyacrylamide gel electrophoresis, columns = pronase, proteinase K (strong proteasome chewing up all proteins)
Can still see a circular lariat trait and a linear exons trait

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22
Q

What is special with self-splicing group II introns? Where are they present?

A

They form structure resembling the spliceosome
Now only present in mitochondria and chloroplast genes, but may be evolutionary predecessors of other introns
*Still have snRNAs

23
Q

What is the importance of the phosphorylation of Serines 2 on the CTD?

A

It is recognized by proteins and enzymes involved in subsequent RNA processing → capping, splicing, polyadenylation
pre-mRNA → mRNA

24
Q

When does processing of pre-mRNA to mRNA starts?

A

It starts as pre-mRNA gets out of the transcriptional complex
Splicing is often co-transcriptional

25
Q

are rRNA, tRNAs and mRNA all processed in the same way?

A

No, because not transcribed by same RNA pol
But they are all processed in eukaryotes in different ways

26
Q

Explain rRNA processing.

A
  • rRNA cleaved AFTER transcription is terminated
  • Synthesized by RNA pol I in the *nucleolus (section of the nucleus devoted to rRNA and othe ri

Always same regions are conserved in rRNA:
18S + 5.8S + 28S in all eukaryotes!!

Doesn’t require snRNA, but snoRNA (small nucleOlar RNA)

27
Q

Where are the sequence coding for rRNA found in the DNA?

A

In repeat sequences (RNA pol I goes through them)
In tandem repeats of the genome

28
Q

Which enzyme transcribes rRNA and tRNA?

A

RNA pol I → rRNA
RNA pol III → tRNA

29
Q

What modifications are brought to pre-tRNA to have it mature into tRNA?

A

1) 5’ end sequence is removed
2) short segment is removed (not same in all tRNA, not the case in all tRNA)
3) CCA tri-nucleotide added onto 3’-end
4) Extensive modification of internal bases → substitution of nucleotides for other bases + modification of ribose

30
Q

What is the role of the 3’-CCA trinucleotide added during processing of tRNA?

A
  • recognition by Aminoacyl-tRNA synthetases to bring correct tRNA into the ribosome during translation
  • Flexibility and structure of tRNA
  • Serves as binding site for amino acid corresponding to specific tRNA
31
Q

What is required for proteins to be RNA binding?

A

They have to have characteristic RNA binding domain:

  1. RNA recognition motif (RRM):
    Beta sheets enriched in +vely charged residues interact with -ively charged backbone of RNA
  2. Polypyrimidine tract binding protein (PTB):
    Interacts with polypyrimidine tract (just 3’ from branch point A in introns)
  3. KH domains
  4. RCG domains
  5. PUF domains
    *Domains that confer to proteins its ability to bind to a specific region
32
Q

What is the role of U2AF protein?

A

Recognizes the 3’ splice site of introns

2 subunits: U2AF65 (binds to polypyrimidine tract) and U2AF35 (binds to AG)

Helps recruit U2 snRNP

33
Q

Which of exons and introns are usually longer in higher eukaryotes?

A

Exon are usually much smaller than introns in higher eukaryotes
Exons = 150 bp
Introns = 3500bp (up to 500kb!)

34
Q

What are exonic splicing enhancers?

A

Sequences within the exon that promote exon joining furing splicing

ex: SR proteins

35
Q

What are SR proteins? Their role?

A

SR = Serine-Arginine proteins (rich in SR)
- RNA binding proteins with RRM domains that decorate the exons to help U2AF identify where to sit down/where AG will be
- protein:protein interaction domains (faciliates interactions with other splicing factors of spliceosome)
- Bind exonic splicing enhancer sequences (doesn’t bind the intron directly)
- Facilitate U1snRNP binding (at 5’ splicing site of intron around GU) and U2snRNP binding (at branch point)

36
Q

What is the cross-exon recognition complex?

A

SR protein:protein/snRNA interactions form cross-exon recognition complex

37
Q

How can the human express so many proteins and be so complex but have only about 20 000 genes?

A

By alternative splicing
Regulated in a temporal and tissue specific manner

38
Q

Explain the alternative splicing of the fibronectin gene.

A

Fibronectin gene DNA has EIIIB and EIIIA exon sequences that encode for sticky domains on proteins

Fibroblast fibronectin mRNA contains EIIIB and EIIIA exons → protein will have sticky domains → interact with extracellular matrix so cells stick to it strongly

Hepatocyte fibronectin mRNA doesn’t have EIIIB and EIIIA exons → liver cells that circulate in the blood, important in clotting, don’t want it to stick to your veins

39
Q

What is responsible for sex determination in Drosophila?

A

Alternative splicing
Expression of Sex-lethal causes this alternative splicing

40
Q

Where and How is Sex-lethal expressed?

A
  • Protein expressed in female drosophila in early embrogenesis
  • Later in dev, female-specific promoter repressed, but different “late” Sxl promoter activated in male and female
  • “Late” Sxl pre-mRNA is alternatively spliced, functional Sxl requires presence of “early Sxl protein to specifiy appropriate splicing (only found in females)

Sxl protein binds at 3’-end of intron between exon 2 and 3 to block U2AF from interacting with RNA at that point
In female: Sxl protein = exon 2-4 (3 is spliced out)
In male: RNA pol II encouters in frame stop in exon 3, pol falls off, no Sxl protein synthesized

41
Q

How is Tra (transformer) expressed in drosophila?

A

In females: Sxl protein binds to at 3’-end of intron between exon 1-2 so U2AF can’t bind, so no recognition of exon 2 in splicing → Tra protein with exons 1-3…

In males: Sxl protein not there to permit splicing so RNA pol will go through exon 2 and see an inframe stop

42
Q

What is the role of Tra protein (expressed only in female drosophila)

A

Tra form complex with TRA2 and RBP1 and together bind to exonic splicing enhancers in exon 4 of Dsx gene so it is spliced in the mRNA of femal double sex protein (exons 3-4-5)

In male Dsx: exons 3-5-6…

RBP1 and TRA2 = SR proteins (bind RNA)

43
Q

What is double sex protein?

A

Dsx = transcription factor that will activate genes that are important for sex expression (specific characteristics)

44
Q

What is RNA editing?

A

After RNA editing, the sequence of the mature mRNA differs from the sequence of the coding region of the genomic DNA
(Not same as splicing, bc in splicing, we can find all sequences in initial DNA gene)

  • RNA editing is widespread in the mitochrondia and plasmids
  • Observed (uncommonly) in the nuclear genoms of higher eukaryotes
  • Regulated process
  • ONLY A → I or C → U (deamination reactions)
45
Q

How is RNA editing important in different forms of mammalian Apolipoprotein B?

A

Apo-B100 found in Liver x2 length of Ap-B48 found in intestine
RNA editing changes CAA codon → UAA which is a stop codon in the middle of exon 26

*Not affected by NMD

46
Q

Are all mRNA transcripts polyadenylated?

A

All mRNAs are polyadenylated except histone mRNAs
Histones have unique secondary structure in their 3’UTRs (untranscripted regulatory regions), so are not degraded even if they don’t have a poly(A) tail

47
Q

How is the poly(A) tail added to the mRNA? (part of mRNA processing)

A
  1. Transcription + 5’ capping (transcription continues/termination sites are past poly(A) site)
  2. Cleavage at Poly(A) site by endonuclease
  3. Polyadenylation by Poly(A) polymerase (PAP) + ATP
48
Q

What is the polyadenylation signal? and its importance?

A

AAUUAAA
Recruits complex that binds to pre-mRNA and catalyses cleavage

49
Q

What steps lead to polyadenylation from RNA synthesis?

A
  1. RNA pol II released from the template at terminator sites (past poly(A) site)
  2. A 3’ AAUUAAA xxxxxx G/U sequence recongnized by cleavage and polyadenylation factors (cleavage proteins bind in all the region between poly signal and G/U)
    *contain polyadenylation signal = AAUAAA
  3. 3’ end of pre-mRNA cleaved
  4. Poly A Polymerase (PAP) attaches direclty when cleavage and catalyses the formation of a poly A tail
50
Q

What are the 2 phases of actual polyadenylation

A
  1. Slow phase: (bc PAP is not very efficient)
    Poly A Polymerase (PAP) adds 12 A residues to cleaved 3’ end directly when cleaved to protect from degradation
  2. Structure recognized by Poly A Binding Protein (PABPN1) → forms complex with PAP (enhances ability of PAP) → rapid addition of ~200 A residues

*ATP dependent

51
Q

How many X and Y chromosomes are present in female and male Drosophilas?

A

Females: 2X
Males: 1X, 1Y

52
Q

What is dosage compensation?

A

Mechanisms that help maintain a proper amount of gene product.
Alternative splicing plays a role in dosage compensation as gives rise to different mRNA isoforms
ex: inactivation of one of the 2 X-chromosomes in females
ex: presence of embryonic Sxl protein in female drosophilas

53
Q

What is the role of CPSF?

A

Cleavage and Polyadenylation Specificity Factor

  • CSPF recognizes and binds to AAUAAA (polyadenylation signal on pre-mRNA)
  • Facilitates cleavage at appropriate site (downstream of AAUAAA)
  • Stimulates addition of poly (A) tail at 3’ end of cleaved pre-mRNA
54
Q

Which proteins are involved in cleavage pre-polyadenylation of pre-mRNA 3’-end?

A

5’ —pre-mRNA–AAUAAA-xxxxx-G/U-3’

CPSF = Cleavage and Polyadenylation Specific factor → binds to AAUAAA
CStF = Cleavage Stimulation Factor → binds to G/U
CF1 = Cleavage factor 1
CFII = Cleavage factor II

*After cleavage, PAP recognizes the free 3’-OH and adds 12 A residues (not very efficient but comes in fast to protect)
When slow polyadenylation occurs (ATP dependent) → CStF, CFI, CFII CPSF stays on

BIOL 200 (11 decks)

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