1. INTRODUCTION TO MOLECULAR BIOLOGY
Sebastian Kadener

2. What I am going to be teaching?
mRNA processing (Today)
Co/post-transcriptional regulation (16/12)
miRNAs (6/1)

3. My leitmotif
"In science, self-satisfaction is death. Personal self-satisfaction is the
death of the scientist. Collective self-satisfaction is the death of the research. It is restlessness, anxiety, dissatisfaction, agony of mind that nourish science"
Jacques Monod.
Basically, don’t be afraid to ask any question everything even if it sounds (and/or is) stupid.

4. Lecture 8
pre-mRNA processing

5. The central dogma: from DNA to protein
RNA
PROTEIN

6. The central dogma in Prokaryotes
Figure 6-21b Molecular Biology of the Cell (© Garland Science 2008)

7. The central dogma is evident in bacteria, also spatially

8. An idealized eukaryotic transcriptional unit

9. However, it does not look like this!

10. Some sequences in the DNA are not present in the mRNA
These sequences are called INTRONS!

11. Genes have introns! Introns are removed!

12. Most eukaryotic pre-mRNAs have exons and introns
Introns are removed by the process of RNA splicing, which occurs only in cis on an individual RNA molecule.
Only mutations in exons can affect protein sequence;
however, mutations in introns can affect processing of the RNA, preventing production of protein.

13. Continuous open reading frames are created when introns are removed from RNA.

14. The order of exons does not change between DNA and RNA.

15. Typical structure of an eukaryotic mRNA

16. The central dogma in Eukaryotes
DNA
RNA
RNA PROCESSING
PROTEIN

17. RNA molecules are very unstable mainly because of the existence of 3’ and 5’ exo-ribo-nucleases
How can the cell protect the mRNAs from them?

18. Protections are added to the 3’ and 5’ ends of mRNAs
PolyA tail
CAP
They are in the mRNA but not in the DNA!

19. Overview of pre-mRNA processing and export

20. Electrophoresis
To separate DNA or RNA according to size, shape and topological properties
The nucleic acid is subjected to an electric field.
charged negatively (why?)=>
migrate to the positive pole
Gel matrix is an inserted, jello-like porous material that support and allows macromolecules to move through.

22. DNA separation by gel electrophoresis
large
moderate
small

23. 2. Hybridization
DNA (or RNA) Hybridization can be used to identify specific DNA
(or RNA) molecules
Using labeled nucleic acids, the detection (and quantification is possible)

24. Probe: a labeled, defined sequence used to search mixtures of nucleic acids for molecules containing a complementary sequence

25. Dot Blott
Tell us:
If a given RNA is expressed
Not really quantitative

27. Northern blotting

28. Southern blot hybridization

29. Pre-mRNA processing
Capping
PolyAdenilation
Splicing

30. The 5’ end of eukaryotic mRNA is capped
A 5’ cap is formed by adding a G to the terminal base of the transcript via a 5’–5’ link.
1-3 methyl groups are added to the base or ribose of the new terminal guanosine.
Figure 7.18

31. 5’-capping reaction Occurs after ~25 b 1. Phosphatase
2. Guanylyl transferase
3. Methyl transferase
CBC
Figure Molecular Biology of the Cell (© Garland Science 2008)

32. Pre-mRNA processing
Capping
PolyAdenilation
Splicing

33. The 3’ terminus of eukaryotic mRNAs is polyadenilated
A length of poly(A) ∼200 nucleotides long is added to a nuclear transcript after transcription.
The poly(A) is bound by a specific protein (PABP).
The poly(A) stabilizes the mRNA against degradation.

34. A 3’ end can be generated by cleavage.

35. The 3’ Ends of mRNAs Are Generated by Cleavage and Polyadenylation
The sequence AAUAAA is a signal for cleavage to generate a 3’ end of mRNA that is polyadenylated.
The reaction requires a protein complex that contains:
a specificity factor
an endonuclease
poly(A) polymerase
Figure 26.34

36. There is a single 3’ end-processing complex.
Once PBP binds, increase
processivity of PAP

37. Pre-mRNA processing
Capping
PolyAdenilation
Splicing

38. Most eukaryotic pre-mRNAs have exons and introns
Introns are removed by the process of RNA splicing, which occurs only in cis on an individual RNA molecule.
Only mutations in exons can affect protein sequence;
however, mutations in introns can affect processing of the RNA, preventing production of protein.

39. Pre-mRNA splicing proceeds through a lariat.
Splicing requires the 5’ and 3’splice sites and a branch site just upstream of the 3’splice site.
The branch sequence is conserved in yeast but less well conserved in higher eukaryotes.

40. Pre-mRNA splicing proceeds through a lariat.
A lariat is formed when:
the intron is cleaved at the 5’ splice site
the 5’end is joined to a 2’ position at an A at the branch site in the intron
The intron is released as a lariat when:
it is cleaved at the 3’ splice site
the left and right exons are then ligated together
Figure 26.6

41. The reactions occur by transesterifications: a bond is transferred from one location to another.
Figure 26.7

42. Two trans-esterification reactions release the lariat
1st reaction
Exon 1 A
Exon 2
Exon 1
Exon 2 A
2nd reaction
Exon 1
Exon 2 A

43. Splicing is mediated by the SpliceosomeU1 U2 U4 U5 U6
snRNAs (snRNPs)
+ ~ 200 proteins

44. 1. U1 snRNP Initiates Splicing
The pre-mRNA splicing mechanism
1. U1 snRNP Initiates Splicing
Figure 6-29 (part 1 of 2) Molecular Biology of the Cell (© Garland Science 2008)

45. The pre-mRNA splicing mechanism

46. The pre-mRNA splicing mechanism
2. Recognition of the branching point site by BBP and U2AF
Figure 6-29 (part 1 of 2) Molecular Biology of the Cell (© Garland Science 2008)

47. The pre-mRNA splicing mechanism
3. U2 binds to the branch point displacing BBP and U2AF

48. The pre-mRNA splicing mechanism
4. U4/U6-U5 snRNP bind

49. The pre-mRNA splicing mechanism
5. Lariat formation, exit of U1,U4, RNA-RNA rearrangements

50. Rearrangement 1 The 5’ splice site is passed from U1 to U6
Figure 6-30a Molecular Biology of the Cell (© Garland Science 2008)

51. Rearrangement 2
U4 dissociates allowing U2 to attack the 5’ splice site and form
the lariat.
Figure 6-30b Molecular Biology of the Cell (© Garland Science 2008)

52. Rearrangement 3 Lariat formation! Splicing!
Figure 6-30c Molecular Biology of the Cell (© Garland Science 2008)

53. snRNA pairing is important in splicing.

54. U6 snRNA can pair with either U4 or U2.

55. The splicing cycle

56. Intron definition model
Intron ends can be recognized by either of two pathways
Intron definition model
5`ss
U1 snRNP
U1 70K
pppG7m
SC35
ASF/SF2
U2AF65
U2AF35
SF1/BBP A
ESE
ESE 3`
(Py)n
3`ss
5`ss
Exon definition model
SC35
ASF/SF2 A
(Py)n
3`ss
5`ss
U2AF65
U2AF35
U1 snRNP
U1 70K
SF1/BBP
ESE 3` 5`

57. Self-splicing
Figure Molecular Biology of the Cell (© Garland Science 2008)

58. Two types of splicing errors
Figure Molecular Biology of the Cell (© Garland Science 2008)

59. Abnormal processing of β-globin in β thalassemia
Figure Molecular Biology of the Cell (© Garland Science 2008)

61. Alternative processing
And alternative splicing….

62. Alternative splicing involves differential use of splice junctions
Specific exons may be excluded or included in the RNA product by using or failing to use a pair of splicing junctions.
Exons may be extended by changing one of the splice junctions to use an alternative junction.
Figure 26.21

63. Structural and functional consequences of alternative splicing
Ion channel signaling behaviors altered
-Glutamate receptor: different receptors display different activities
2) Protein-protein binding surface altered
-Neurexins: AS changes ligand binding
3) Enzyme active sites altered
-Phosphotyrosine phosphatase: different isoforms have different substrate specificities
4) Enzyme allosteric site altered
-Pyruvate kinase: AS alteres allosteric regulation
5) DNA binding modules shuffled
-Lola: different isozymes have different zinc finger combinations leading to different target specifities

64. Alternative Pre-mRNA Splicing Can Create Enormous Diversity

65. Alternative Pre-mRNA Splicing Can Create Enormous Diversity

66. How is alternative splicing achieved?
Alternative exons often have suboptimal splice sites and/or length
Splicing of regulated exons is modulated:
Proteins – SR proteins and hnRNPs
cis elements in introns and exons – splicing enhancers and silencers
Differences in the activities and/or amounts of general splicing
factors and/or gene-specific splicing regulators during
development or in differnt tissues can cause alternative splicing

67. How is alternative splicing achieved?
What are these intronic or exonic elements?
Splicing enhancers or splicing silencers

68. SR proteins - nuclear phosphoproteins, localized in speckles
RRM RRM SR
RRM SR
RRM Zn SR
- nuclear phosphoproteins, localized in speckles
- phosphorylation status regulates their
subcellular localisation and protein-protein
interactions
- shuttling proteins (h9G8, hSRp20, hSF2/ASF)
- constitutive splicing
- alternative 5` splice site selection
- alternative 3` splice site selection
exon-(in)dependent
- found in all eukaryotes except in S. cerevisiae

69. 5`and 3`splice site selection – role for SR proteins
Specific sequence independent – over both intron and exon
5`ss
U1 snRNP
U1 70K
pppG7m
SC35
ASF/SF2
U2AF65
U2AF35
SF1/BBP A
ESE
ESE 3`
(Py)n
3`ss
5`ss
Specific sequence dependent - over both intron and exon
SC35
ASF/SF2 A
(Py)n
3`ss
5`ss
U2AF65
U2AF35
U1 snRNP
U1 70K
SF1/BBP
ESE 3` 5`

70. Negative and Positive Control of Alternative
Pre-mRNA Splicing

71. Specific sequence required
U2AF recruitment model
Specific sequence required
SR protein binds to ESE and promote binding of U2AF to Py tract, which results in activation of adjacent 3‘ss
This is mediated by interaction of RS domain of SR protein with the small subunit (U2AF35) of U2AF

72. Alternative splicing can be regulated

73. Sex determination in Drosophila involves a series of alternative splicing events in genes coding for successive products of a pathway.

74. The idea of exon definition by SR proteins
Figure Molecular Biology of the Cell (© Garland Science 2008)

75. Major Vs minor (U12 type) introns
An alternative pathway uses another set of snRNPs that comprise the U12 spliceosome.
The target introns are defined by longer consensus sequences at the splice junctions. They usually include the same GU-AG junctions.

76. Major Vs minor (U12 type) introns
Figure 6-34b Molecular Biology of the Cell (© Garland Science 2008)

77. The E complex forms by interactions involving both splice sites.

78. The splicing cycle Release of U1 snRNP:
allows U6 snRNA to interact with the 5′ splice site
converts the B1 spliceosome to the B2 spliceosome
When U4 dissociates from U6 snRNP, U6 snRNA can pair with U2 snRNA to form the catalytic active site.
Figure 26.13

79. Intron ends can be recognized by either of two pathways
U1 snRNP to bind at the 5’ splice site
U2AF to bind at a pyrimidine tract between the branch site and the 3’ splice site
U2AF at the pyrimidine tract
U1 snRNP at a downstream 5’ splice site