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Second Lesson Polyade Editing .Pptx Post‐Transcriptional Gene Regulation RNA Processing: Polyadenylation and editing pre mRNA processing Polyadenylation • Most eukaryotic mRNAs contain 20‐250 adenosine 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 eukaryotes – 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 Exon 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 Factor 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 RNAs = 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 intron 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 exons • 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
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