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MCB 2 > I. DNA & RNA | 16. Processing of the eukaryotic mRNA > Flashcards

I. DNA & RNA | 16. Processing of the eukaryotic mRNA Flashcards

1
Q

Eukaryotic cells convert the initial primary transcript, synthesized by RNA polymerase II, into a ____

A

matured + functional mRNA (pre-mRNA -> mature mRNA)

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2
Q

What are the 3 major events of maturation of mRNA?

A
  • The sequence of events takes place in a co-transcriptional manner, as the 5’ capping starts shortly after the RNA polymerase has initiated transcription.
  • Export towards the cell cytosol starts shortly after processing and is assisted by specific RNA-transport proteins.
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3
Q

I. 5’ capping
1. General description of 5’ capping

A
  • As soon as the RNA strand reaches a length of about 25 nucleotides, a protective cap, composed of 7-methylguanosine (and methylation of the first 2 riboses), is added to the first nucleotide at its 5’-end by 5’-5’ triphosphate bridge.
  • The cap is added by the enzyme, guanylyl transferase, which may first interact with the RNA polymerase. Binding to RNA polymerase and activation of the capping enzyme by the 5— phosphorylated CTD (C-terminal domain), results in a characteristic sequence of events:
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4
Q

I. 5’ capping
2. Describe 3-step mechanism of 5’capping?

A
  1. RNA triphosphatase (1 of 3 capping enzymes) removes the γ-phosphate from the 5’- end, while the remaining α- and β-phosphates remain associated with the cap
  2. Guanylyl transferase transfers the GMP moiety from GTP to the 5’-diphosphate of the growing transcript, creating the guanosine-5’-5’-triphosphate bridge
  3. In the final steps, different types of the methyltransferase transfer methyl groups (CH3) from S-adenosylmethionine to the: (1) N7 position of guanine and (2) to the 2’ oxygens of the first one/two nucleotides
    - the process allows RNA polymerase II to enter into a faster mode of elongation
    - The net effect of this mechanism (besides protection on the developing strand) is that the polymerase waits for the growing RNA to be capped before elongating at a rapid rate
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5
Q

I. 5’ capping
3. What is the role of RNA triphosphatase (1 of 3 capping enzymes)?

A

RNA triphosphatase (1 of 3 capping enzymes) removes the γ-phosphate from the 5’- end, while the remaining α- and β-phosphates remain associated with the cap

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6
Q

I. 5’ capping
4. What is the role of Guanylyl transferase (1 of 3 capping enzymes)?

A

Guanylyl transferase transfers the GMP moiety from GTP to the 5’-diphosphate of the growing transcript, creating the guanosine-5’-5’-triphosphate bridge

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7
Q

I. 5’ capping
5. What is the role of Guanylyl transferase (1 of 3 capping enzymes)?

A

In the final steps, different types of the methyltransferase transfer methyl groups (CH3) from S-adenosylmethionine to the: (1) N7 position of guanine and (2) to the 2’ oxygens of the first one/two nucleotides

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8
Q

II. 3’ cleavage and polyadenylation
1. What are definition and features of Polyadenylation?

A
  • Polyadenylation is the addition of the poly-A-tail which contains around 250 adenosine residues and is considered to have an important role in the protection of the RNA strand from degrading enzymes.
  • During this process, multiprotein complexes assemble through a network of specific protein- nucleic acid and protein-protein interactions.
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9
Q

II. 3’ cleavage and polyadenylation
2. What is the 9-step mechanism?

A
  1. Specific sequence signals in the pre-mRNA direct the binding of protein factors to the pre-mRNA. A conserved AAUAAA sequence is necessary and G/U-rich sequence downstream (DSE) is also important
  2. Cleavage + polyadenylation specificity factor (CPSF) binds to the upstream AAUAAA polyadenylation signal
  3. Cleavage stimulation factor (CStF) interacts with a downstream GU- or U-rich sequence and with bound CPSF, will form a loop in the RNA
  4. Binding of cleavage factor (CFI + CFII) helps stabilize the complex
  5. Binding of polyadenylate polymerase (PAP) stimulates cleavage at a poly-(A)-cleavage-site (which is usually 15- 30 nucleotides 3’ of the upstream polyadenylation signal)
  6. The cleavage factors are released, as well as the downstream RNA cleavage product, which is rapidly degraded
  7. Bound PAP then adds about 12 A residues at a slow rate to the 3’-OH group produced by the cleavage reaction
  8. Binding polyadenylate binding protein (PAB) to the initial short poly-(A)-tail will speed up the adenylation
  9. After 200-250 A residues have been added, PAB signals PAP to stop
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10
Q

II. 3’ cleavage and polyadenylation
3. What is the role of Cleavage + polyadenylation specificity factor (CPSF)?

A

Cleavage + polyadenylation specificity factor (CPSF) binds to the upstream AAUAAA polyadenylation signal

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11
Q

II. 3’ cleavage and polyadenylation
4. What is the role of Cleavage stimulation factor (CStF)?

A

Cleavage stimulation factor (CStF) interacts with a downstream GU- or U-rich sequence and with bound CPSF, will form a loop in the RNA

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12
Q

II. 3’ cleavage and polyadenylation
5. What is the role of cleavage factor (CFI + CFII)?

A

Binding of cleavage factor (CFI + CFII) helps stabilize the complex

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13
Q

II. 3’ cleavage and polyadenylation
6. What is the role of polyadenylate polymerase (PAP)?

A
  • Binding of polyadenylate polymerase (PAP) stimulates cleavage at a poly-(A)-cleavage-site (which is usually 15- 30 nucleotides 3’ of the upstream polyadenylation signal)
  • Bound PAP then adds about 12 A residues at a slow rate to the 3’-OH group produced by the cleavage reaction
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14
Q

II. 3’ cleavage and polyadenylation
7. What is the role of polyadenylate binding protein (PAB)?

A
  • Binding polyadenylate binding protein (PAB) to the initial short poly-(A)-tail will speed up the adenylation
  • After 200-250 A residues have been added, PAB signals PAP to stop
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15
Q

III. RNA splicing
1. What is RNA splicing?

A
  • During formation of a mature + functional mRNA, introns are removed and exons are spliced together.
  • The process of splicing involves 2 trans-esterification mechanisms, in which one ester bond is cleaved and replaced by a new one, connecting two adjacent exons.
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16
Q

III. RNA splicing
2. What does RNA splicing require?

A

Splicing requires the presence of small nuclear RNAs (snRNAs), which base-pair with the pre-mRNA to form a spliceosome

17
Q

III. RNA splicing
3. Describe the structure of spliceosome

A

Specific snRNPs sequences are defined:
- U1: 5’ site binding
- U2: branch point binding
- U5: 3’ site binidng
- U4 U6: complex assembly and catalytic activity
=> Create an assembly of large ribonucleoprotein complex called SPLICEOSOME

18
Q

III. RNA splicing
4. What is the 6-step mechanism of RNA splicing?

A
  1. After U1 base-pairs with the 5’-splice site, SF1 (splicing factor 1) binds the branch-point-A. U2AF (U2 associated factor) associates with the polypyrimidine tract and 3’-splice-site
  2. The U2 snRNP associates with branch-point-A via base-pairing interactions displacing SF1
  3. Spliceosome is formed when a snRNP complex of U4, U5 and U6 joins the initial complex
  4. Rearrangements of base-pairing interactions convert the spliceosome into an active conformation, and release the U1 and U4 snRNPs
  5. The 1st trans-esterification reaction is catalyzed by the catalytic core (U6 + U2), forming an intermediate containing a 2’,5’- phosphodiester bond
  6. The 2nd trans-esterification reaction joins the two exons by a 3’,5’-phosphodiester bond and releases the intron as a lariat (circular molecule with a short tail) structure, together with remaining snRNAs
    => The cut out lariat intron is converted into a linear RNA by debranching enzymes = may be degraded or used for synthesis of microRNA
19
Q

III. RNA splicing
5. Describe The 1st trans-esterification reaction

A

The 1st trans-esterification reaction is catalyzed by the catalytic core (U6 + U2), forming an intermediate containing a 2’,5’- phosphodiester bond

20
Q

III. RNA splicing
6. Describe The 2nd trans-esterification reaction

A

The 2nd trans-esterification reaction joins the two exons by a 3’,5’-phosphodiester bond and releases the intron as a lariat (circular molecule with a short tail) structure, together with remaining snRNAs
=> The cut out lariat intron is converted into a linear RNA by debranching enzymes = may be degraded or used for synthesis of microRNA

21
Q

III. RNA splicing
6. What is the fate of lariat intron

A

The cut out lariat intron is converted into a linear RNA by debranching enzymes = may be degraded or used for synthesis of microRNA

22
Q

III. RNA splicing
7. What is the role of U2AF (U2 associated factor)

A
  • U2AF (U2 associated factor) associates with the polypyrimidine tract and 3’-splice-site
  • The U2 snRNP associates with branch-point-A via base-pairing interactions displacing SF1

MCB 2 (59 decks)

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