INTRODUCTION TO MOLECULAR BIOLOGY Sebastian Kadener skadener@cc.huji.ac.il

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

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.

Lecture 8 pre-mRNA processing

The central dogma: from DNA to protein RNA PROTEIN

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

The central dogma is evident in bacteria, also spatially

An idealized eukaryotic transcriptional unit

However, it does not look like this!

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

Genes have introns! Introns are removed!

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.

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

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

Typical structure of an eukaryotic mRNA

The central dogma in Eukaryotes DNA RNA RNA PROCESSING PROTEIN

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?

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

Overview of pre-mRNA processing and export

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.

DNA separation by gel electrophoresis large moderate small

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)

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

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

Northern blotting

Southern blot hybridization

Pre-mRNA processing Capping PolyAdenilation Splicing

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

5’-capping reaction Occurs after ~25 b 1. Phosphatase 2. Guanylyl transferase 3. Methyl transferase CBC Figure 6-24 Molecular Biology of the Cell (© Garland Science 2008)

Pre-mRNA processing Capping PolyAdenilation Splicing

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.

A 3’ end can be generated by cleavage.

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

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

Pre-mRNA processing Capping PolyAdenilation Splicing

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.

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.

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

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

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

Splicing is mediated by the Spliceosome U1 U2 U4 U5 U6 snRNAs (snRNPs) + ~ 200 proteins

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)

The pre-mRNA splicing mechanism

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)

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

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

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

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

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)

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

snRNA pairing is important in splicing.

U6 snRNA can pair with either U4 or U2.

The splicing cycle

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`

Self-splicing Figure 6-36 Molecular Biology of the Cell (© Garland Science 2008)

Two types of splicing errors Figure 6-31 Molecular Biology of the Cell (© Garland Science 2008)

Abnormal processing of β-globin in β thalassemia Figure 6-35 Molecular Biology of the Cell (© Garland Science 2008)

Alternative processing And alternative splicing….

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

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

Alternative Pre-mRNA Splicing Can Create Enormous Diversity

Alternative Pre-mRNA Splicing Can Create Enormous Diversity

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

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

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

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`

Negative and Positive Control of Alternative Pre-mRNA Splicing

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

Alternative splicing can be regulated

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

The idea of exon definition by SR proteins Figure 6-33 Molecular Biology of the Cell (© Garland Science 2008)

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.

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

The E complex forms by interactions involving both splice sites.

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

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