Post‐Transcriptional Gene Regulation RNA Processing: and editing pre mRNA processing Polyadenylation

• Most eukaryotic mRNAs contain 20‐250 residues at their 3' ends that are added in the nucleus • Polyadenylation occurs on nascent RNA (growing or pre‐ mRNA being synthesized) • Transcription proceeds downstream of the 3’end of mRNAs and the 3’ end is generated by endonucleolytic cleavage • Nuclear vs. cytoplasmic polyadenylation in – Primary poly(A) tail added in nucleus = 200‐250 – Subsequent modulation of mature poly(A) tail lenth occurs in the cytoplasm of the cell (Length can be modified in cytoplasm = 10‐250) Pre - mRNA 3’ End Processing - Polyadenylation cis Elements and the 2-Step Reaction Terminal 10-30 nt

3’ SS AAUAAA CA U or GU-rich

Hexanucleotide 10 - 50 nt Highly Conserved ~50-90%? Step 1 - Cleavage

AAUAAA CA

Step 2 - Poly A addition

AAUAAA CA AAAAAAAAA(200-300) Reactions can be uncoupled in vitro Importance of the Hexanucleotide sequence

A new A‐rich class of PolyA site

Eukaryotic Polyadenyation AAUAAA and G/U cis elements define poly(A) site

CPSF (Cleavage‐and‐polyadenylation specificity factor)

CstF (Cleavage stimulation factor) Assembly of the polyadenylation complex

Cleavage and Cleavage Stimulation Factor Polyadenylation Specific Factor CPSF

Curr Opin Cell Biol. 2004 Jun;16(3):272-8

Molecular Cell Biology Figure 11-12. Model for cleavage and poly-adenylation of pre-mRNAs in mammalian cells Mechanism of polyadenylation

a) Transcription Cleavage/Polyadenylation (CF) Specificity Factor (CPSF) b) Cleavage

PolyA-Polymerase (PAP) c) Polyadenylation phase I (AAUAAA/CPSF-dependent)

PolyA-Binding protein II d) Polyadenylation phase II (PAB II) Oligo-A/PABII-dependent

II. mRNA deadenylation Function of Polyadenylation

• Stability of mRNAs in eukaryotes (protection from nucleases) • Transcription termination • Translational efficiency • Enhancement of Splicing • Generation of diversity (alternative polyA)

• stability and translation efficiency can be affected by alternative polyadenylation (this will be discussed later in miRNAs function) • Globin RNA injected into oocytes

• Rate of translation measured

• All changes attributed to mRNA stability Function of Polyadenylation Stability of mRNAs in Eukaryotes

polyA tail protects 3' end (PolyAdenilatedBindingProtein bound to An) from 3' exonucleolytic degradation – Degree of stability related to length of tail – Reduction in tail to a minimal length often signals for degradation of mRNA (<10‐15 in yeast is target for degrading RNA) Function of Polyadenylation Tx Termination

• Pol I and Pol III – Promoters often have discrete termination elements that signal the RNA polymerase to stop • Pol II transcription termination – Discrete elements involved in signaling the RNA polymerase II to stop are not as well known – Evidence suggests that termination is coupled to polyadenylation for some genes – Coupling of 3' processing and Tx termination which may prevent premature Tx termination The allosteric and torpedo models for PolII transcription termination

Function of Polyadenylation Translational Efficiency • Tail length = mechanism of translation control – Short tailed = poor translation substrate – RNAs with longer tails are better for translation – Poly A tails bind proteins (ex. PABP), which interacts via protein‐protein interaction with cap of the mRNA effecting translation and stability

Figure 2. Model for the 48S initiation complex. The interactions among eIF1, eIF2, eIF3, eIF4A, eIF4E, eIF4G, eIF5, Mnk, PABP, mRNA, and the 40S ribosomal subunit are shown. The thin line represents mRNA, with the wavy line indicating mRNA secondary structure. Meti is the initiator tRNA. The sizes of protein depictions are roughly proportion to their molecular masses. Function of Polyadenylation Translational Efficiency

• Effect of poly(A) and cap tested in rabbit reticulocyte lysate

• Poly(A) artificially encoded in gene • Here, no difference was observed in stability (no nucleases present reticulocyte lysate) Function of Polyadenylation Splicing

• Element associated with 3' terminal removal (Return to this after splicing lectures on exon definition to understand better) – Removal of last intron in pre‐mRNA appears to depend on a 3'‐processing signal downstream Regulation of Polyadenylation the autoregulatory feedback of U1A

• very few examples available • the autoregulatory feedback of U1A upstrem regulatory elements • U1A is an RNA binding protein that is part of hnRNP particle which is fundamental in splicing • The amount of U1A protein regulates its expression inhibiting polydenylation of its nascent transcript Histone mRNA is not Polyadenylated

• The 3’ end of histone mRNA is protected from degradation by a double‐stranded stem loop structure • Fold and unfolding of this stem loop is critical in regulating histone mRNA stability – Unstable during most of cell cycle when high histone synthesis not necessary. Instability elements exposed – Highly stable during S phase (DNA synthesis so many histones required). Protein binds and “hides” instability sequences • 3' end is formed by specific cleavage event carried out by other RNAs and proteins that are distinct from machinery involved in polyadenylation Alternative polyadenilation

• about 50% of human genes are alternatively polyadenylated • frequently tissue specific (for example brain mRNAs has longer 3’UTR) • What is the function of alternative polyadenylation? • Mechanism still unknown Polyadenylation Generation of diversity

Generation of diversity

• Most mammalian genes include alternative poly A signals

• Alternative poly A signals can be in the same or different • Using different poly A sites generates multiple mRNA products from a single gene Millevoi and Vagner, Nuc. Acids Res. (2009): 1‐18 Types of alternative polyadenylation.

Coding Region APA skipped‐exon associated 3′ UTRs

composite exons associated 3′ UTRs

UTR APA single terminal exons with two or more polyA sites Tandem polyA

Experimental Cell Research 316, 1 May 2010, 1357‐1364

Genome wide analysis of APA

Alternative Polyadenylation (APA) regulation • Largely unknown • Role of cis sequences. Consensus sequence elements and strength of upstream and downstream elements determine efficient of reaction. In APA one third of proximal polya sites are non canonical or weak but have more U or UG rich sequences. Combinatorial control • Regulation by core or accessory elements. Relative concentration of polyadenylation complexs and its proteins can affect APA • Regulation by splicing factors that bind to 3’UTRs (PTB, U1 hnRNP and NOVA) • Chromatin‐mediated regulation ? Genome wide map of the Neuronal splicing factor NOVA shows its role in APA

How to identify the end of a transcript

• 3’ RACE (Rapid Amplification Complementary Ends) • RNAse protection

• Northern Blotting From Southern to Northern and Western an historical view Restriction enzymes RFLPs the invention of Southern Analysis of Restriction Enzyme Sites:

1. Restriction sites can be mapped by cutting DNA with restriction enzymes, electrophoresing the DNA on an agarose gel, and visualizing the DNA banding pattern with ethidium bromide.

2. Positions of restrictions sites can be used to create a linkage map or compare presence/absence of positions among different individuals (forensics, systematics, population genetics).

3. DNA is cut with different enzymes, and each DNA‐enzyme mixture is loaded on a separate lane in the gel.

4. Negatively charged DNAs separate by size in the electric field (smaller fragments move faster than larger fragments).

5. Fragment pattern produced by gel stained with ethidium bromide is photographed or imaged and processed by software.

6. Distance each band migrates is calibrated with known size standards of pre‐cut DNA (i.e., ladder).

7. Resulting pattern of different numbers and sizes of fragments cut by different enzymes is interpreted to make a restriction site map. Applications of Restriction Enzyme Site Analysis:

1. Analyze intron organization, gene structure, mapping.

2. Restriction Fragment Length Polymorphism (RFLPs) can be used for forensic and phylogenetic analyses.

3. Cut DNAs can be probed without cloning using the Southern Blot (also can be used to determine if a gene has been duplicated). Southern Blot:

• Invented by Edward Southern.

• After restriction enzyme cutting and electrophoresis, genomic DNA appears as a continuous smear on agarose gel.

• Denature DNA in the gel with an alkaline solution.

• Neutralize gel and place on blotting paper that spans a glass plate and reaches into buffer solution.

• Place a Hybridization membrane over the gel, and stack paper towels and a weight on top of the membrane.

• Buffer solution is wicked up by blotting paper to the paper towels, passing through the gel and transferring DNA to membrane.

• Fragments on the filter are arranged the same as on the gel.

• Saturate the membrane with a labeled probe (radioactive or nonradioactive) and expose to x‐ray film. Fig. 7.18, Southern Blot Northern Blot:

• Northern blot was not invented by a person named Northern.

• Components are not arranged upside down or in reverse order, but the same as the Southern Blot.

• Used for RNA.

• Can be used to determine mRNA size, e.g., detect differences in the promoter and terminator sites, etc.

• Can be used to determine if a particular gene is expressed, and if so, how much, what tissue type, and when in the life cycle? ma chick y tur e chick olk sac en liv

Northern er Blot Analysis

alpha‐fetoprotein RNA probe

Fig. 1.6 3’ - RACE Rapid Amplification Complementary Ends

mRNA 7-mG AAAAAAAA-3’ Trascrittasi inversa Sintesi primo filamento cDNA + P(dT) 7-mG AAAAAAAA-3’ TTTTT P Sintesi secondo filamento cDNA GSP1 complementarietà al primer P(dT) GSP1

3’ TTTTT P 5’

complementarietà al primer GSP1 I° gruppo di amplificazioni

5’ GSP1 AAAAA P’ 3’

3’ GSP1’ TTTTT P 5’

II° gruppo di amplificazioni

3’ GSP1’ TTTTT P 5’ GSP2 5’ GSP2 AAAAA P’ 3’

P TTTTT 3’ GSP2’ TTTTT P 5’ 5’ GSP1 AAAAA P’ 3’ Prodotto PCR finale 3’RACE PCR and APA polyA

3 1 polyA polyA 2 3

polyA polyA 3 3 X X

polyA polyA 4 X 3

4 1 2 3

PolyA of globin – 430bp

cryptic PolyA – 356bp RNA Editing

Definition: any process, other than splicing, that results in a change in the sequence of a RNA transcript such that it differs from the sequence of the DNA template

• Discovered in trypanosome mitochondria • Also common in plant mitochondria • Also occurs in few nuclear genes in mammals Mammalian genes with RNA Editing

– (C  U)

– Glutamate receptor B (A  I () – Serotonin Receptor 5HT (A  I (inosine) – ADAR2 (A  I (inosine) TRANSCRIPT AND PROTEIN DIVERSITY THROUGH RNA EDITING

MODIFICATIONS of NUCLEOTIDES

A to I base modification MOST WIDESPREAD in HIGHER EUKARYOTES C to U base modification

– Apolipoprotein B (C  U)

– Glutamate receptor B (A  I (inosine) – Serotonin Receptor 5HT (A  I ) – ADAR2 (A  I ) Identification of editing sites

• RNA • cDNA • cloning • sequencing • comparison with DNA

• Efficiency from few (5%) to 100% Editing of Apolipoprotein B in Mammals

1. Large nuclear gene 2. Editing is C6666  U6666 in exon 26 of the 14 Kb mRNA 3. This creates a Stop codon, producing a truncated form of the protein - both forms circulate in blood but have different functions - the long form is endocytosed via the LDL receptor; the short form is not APOB100 pre‐mRNA EDITING C to U editing

1987‐ Apobec 1 – Zn‐dependent citydine deaminase only few other cases known Editing of Apolipoprotein B – The Editosome 1. A deaminase activity is involved – apobec (apoB mRNA editing enzyme catalytic subunit) 2. Another protein, ACF (apobec complementation factor) is also required 3. Both recognize sequences flanking the C to be edited specificity provided by a mooring sequence UGAUCAGCAUA A to I base modification

Any A to I change in a protein coding sequence is equivalent to making an A to G mutation. Mechanism of A to I Editing

• dsRNA-dependent adenosine deaminase (ADAR) 1. converts A  I in 2 Glut Receptor B exons (changes the amino acids, I read as guanosine during translation) 2. recognizes secondary structure around site to be edited 3. requires intron and exon sequences - acts on unspliced receptor pre-mRNA 4. has dsRNA binding domains as well as a catalytic center similar to the cytosine deaminase Three major types of A to I RNA editing targets and their fates glutamate receptor subunit B (GluR‐B) Pre‐mRNA editing by Adenosine deamination edited at two exonic sites termed the Q/R and R/G, lower Ca2þ permeability of the channe CAG_>CGG Arg Glu

Glutamate receptor Human genes serotonin ADAR2 receptor

Drosophila genes

From Reenan Trends in Genetics (2001), v17 p53 Editing By Adenosine Deamination (ADAR Enzymes)

NH2

• 3 ADARs in humans

• A to I changes Q codon to R codon

• One protein affected is a Reenan, Trends in Genetics, 17 (2001), 53‐56 brain Ca2+ channel, GluR

• Recent work using deep sequencing identified more than 200 new sites Li et al, Science 324 (2009), 1210‐1213 Adenosine deaminases acting on RNA (ADARs)

zinc RNA double BS strand Three major types of A to I RNA editing targets and their fates Alu repeats

• ar e SINE (short interspersed nuclear elements) primate specific about 300bp • c ontains an internal polymerase III promoter sequence (not always functional) •mos t abundant sequence in the human genome, copy number of 1,000,000 • ha ve high GC content •es timates that 98% are A to I edited in brain •fr equently located in and in the 3’UTR •c an become exons during evolution through acquisition of point mutations (Exonization) Ways of exon expansion: RNA‐editing‐mediated exon evolution

Alu retroelements are specific to primates and abundant in the human genome. Through mutations that create functional splice sites within intronic Alus, these elements can become new exons in a process denoted exonization. It was recently shown that Alu elements are also heavily changed by RNA editing in the human genome. Here we show that the human nuclear prelamin A recognition factor contains a primate‐specific Alu‐ exon that exclusively depends on RNA editing for its exonization. We demonstrate that RNA editing regulates the exonization in a tissue‐dependent manner, through both the creation of a functional AG 3' splice site, and alteration of functional exonic splicing enhancers within the exon. Furthermore, a premature stop codon within the Alu‐exon is eliminated by an exceptionally efficient RNA editing event. The sequence surrounding this editing site is important not only for editing of that site but also for editing in other neighboring sites as well. Our results show that the abundant RNA editing of Alu sequences can be recruited as a mechanism supporting the birth of new exons in the human

Genome Biology 2007, 8:R29(doi:10.1186/gb‐2007‐8‐2‐r29) Levels of Alu‐exon inclusion and RNA editing in the endogenous human NARF gene. The antisense Alu is essential for exonization Three major types of A to I RNA editing targets and their fates miRNA editing estimates 16% of miRNAs are edited possible functions

• prevent miRNA expression –inhibit cleavage by DROSHA or Dicer on miRNA precursors

• modify the specificity (change the seed sequence ‐ mir 376)

• change in 3’ UTR binding sites

(we will discuss later in miRNA lesson) Winter et al. Nat. Cell Biol. 2009 Readings polyA and RNA editing

• HITS‐CLIP yields genome‐wide insights into brain alternative RNA processing. Licatalosi DD, et al (The CLIP identify NOVA as regulator of Alterantive PolyAdenilation) • Mechanisms and consequences of alternative polyadenylation.Di Giammartino DC, Nishida K, Manley JL.Mol Cell. 2011 Sep 16;43(6):853‐66. • Molecular diversity through RNA editing: a balancing act.Farajollahi S, Maas S.Trends Genet. 2010 May;26(5):221‐30. Epub 2010 Apr 13. Review.

In red those papers that are part of the exam (i.e. one question will be to comment a figure of these papers)