15 – Coordination of Processing PDF

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03-07-2017 -

Polyadenylation of other examined eukaryotes o mRNAs of plants and yeast are also polyadenylated, but the signals are different o Yeast usually lack AAUAAA and its often hard to detect a signal other than an AU rich motif upstream of polyA site o Plants may have AAUAAA but the variations in the sequence that are permitted are different from animals  Animal signals do not function when placed at the end of plant genes

Cleavage and Polyadenylation of Pre-mRNA -

Pre-mRNA cleavage required several proteins: 1. Cleavage and polyadenylation specificity factor (CPSF) which binds recognizes and binds to AAUAAA signal (mRNA) 2. Cleavage stimulation factor (CstF) binds to G/U region after the cleavage site a. 1 and 2 bind to flank the sides of the cleavage site and bind cooperatively 3. Cleavage factors I and II (CFI and II) a. These actually make the cut 4. Poly A polymerase (PAP) strongly associated because no cleaved unpolyadenylated RNAs at the 3’ end are found a. Immediately after the cut, the upstream piece is going to get polyadenylated 5. RNA polymerase II: RNAs made by Pol II are capped, spliced and polyadenylated whereas RNAs made by Pol II lacking CTD of the largest subunit are not efficiently spliced and polyadenylated a. Important for bringing in the cleavage and polyadenylation machinery b. The CTD of pol II when it gets phosphorylated binds to the CPSF, and is already bound to the polymerase as it is transcribing the AAUAAA sequence c. This is why you never find any unpolyadenylated pieces of RNA (as soon as it runs over the sequence, the other factors for polyadenylation are recruited)

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Testing the importance of CTD in cleavage: o Expressed the Pol II CTD as a fusion protein with the glutathione S-transferase and purified the protein by glutathione affinity chromatography o Phosphorylated a portion of the fusion protein prep on its CTD and tested phosphorylated (CTD P) vs. unphosphorylated (CTD) proteins o The CTD fusions were incubated with the other protein factors and 32P-labeled adenovirus L3 pre-mRNA o Electrophoresed products and autoradiographed gel to see if pre-mRNA was cleaved at the right place o Results:  Both forms of CTD stimulated cleavage and gave more product with increasing concentrations, but the phosphorylated form was 5x better  Cleavage reaction is therefore promoted by pol II  The CTD is phosphorylated in Pol IIO, which is the form that carries out transcription elongation

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Model for the precleavage complex (right) o Polymerase is bound to all of the proteins shown (CPSF, PAP, CFI/II, and CstF) o The cut occurs through CFs in between the AAUAAA and GU sequences and the PAP is right beside the cut site o When it is cut, there is a new 3’ end, and PAP is ready to go when the cut is made, and adds As onto it o After the cut, polymerase keeps going  Now there is a free 5’ end with no cap, and this acts as a substrate and this piece of transcribed RNA gets degraded

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Polyadenylation occurs in two phases: o 1. Initiation: depends on AAUAAA signal and involves slow addition of at least 10 As to the pre-mRNA  Recognizes the AAUAAA signal and adds As there (wont do this on a random 3’ free end; needs this signal to work) o 2. Elongation: independent of AAUAAA but dependent on oligoA, involves rapid of 200+ As (addition  Doesn’t rely on the AAUAAA signal before, just depends on the previous 10 As that were put on  Addition happens much faster

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AAUAAA and G-U/U rich sequence is actually a cleavage signal – these cannot be the polyadenylation signal since the G-U/U rich region has been removed after cleavage PolyA signal is actually the AAUAAA sequence plus approximately 8 nucleotides Evidence that the initiation and elongation phases of polyadenylation are distinct: o (Left) When using a AAUAAA polyadenylation signal, polyA can be added to either just the signal or the signal with 40 A’s added, which corresponds to after the initiation phase (large smear at the top is the larger sequence due to the addition of As)  Poly cannot be added to the AAUAAA followed by non A nucleotides (still had the AAUAAA sequence, but not the As after)  This then shows that you need the AAUAAA and initiation sequence (more As) in order to get polyadenylation elongation occurring o (Right) When an AAGAAA polyadenylation signal is used (mutated polyA signal), polyA can no longer be added to just this signal (elongation cannot occur because initiation cant occur)  Polyadenylation still occurs when this mutated signal is followed by 40 A’s (have bypassed initiation)  When both the AAUAAA sequence is mutated and bases other than A are added are added after, you don’t get elongation  Therefore the recognition of the initiation and elongation polyadenylation is distinct  Initiation recognizes AAUAAA and elongation recognizes the As that have been added after (in the initiation phase)  The elongation phase therefore relies on the initiation phase

Initiation of PolyA - The following proteins are needed for the initiation phase: o 1. PolyA polymerase o 2. CPSF, binds directly to AAUAAA - In vitro experiments show that PAP alone can add As to the 3’ end of RNA as long as substrate concentrations are high, but at low concentrations, PAP needs CPSF o In vivo, its likely that CPSF is helping recruit PAP - The general hypothesis: o After pre-mRNA cleavage, polyadenylation depends on AAUAAA signal and CPSF until PolyA is at least 10 As long o Then polyadenylation depends on the PolyA (As you just put on) at the 3’ end  It no longer requires CPSF but is stimulated by it (works better with it) Elongation of PolyA - PolyA polyermase binds to an elongates the PolyA very poorly by itself o Requires PolyA binding protein II, which covers the polyA tail - PAB II: 49kD, binds to polyA tail o PABII is nuclear, which binds the polyA tail during polyadenylation in the nucleus (PABI works in the cytoplasmic when the mRNA is exported out of the nucleus)  When PABII falls off in the cytoplasm, it will be replaced by PABI o Stimulates polyadenylation of RNAs that already have oligoA tails o Can direct efficient polyadenylation of RNA with a mutant AAGAAA signal, as long as oligoA is present

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Model for polyadenylation: (the beige line is the RNA that is coming out of the pol)

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1. RNA Pol II transcribes through the cleavage signal

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2. CPSF, CStF, and CFI and II assemble on pre-mRNA at their respective sites  CPSF at the AAUAAA, CstF at the GU/U element (which then recruits CFs)

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3. Cleavage occurs, stimulated by CTD of Pol II

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4. PolyA polymerase, helped being recruited by CPSF, initiates polyA synthesis, adding at least 10 As

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5. PAB II allows rapid extension of oligoA (with around 10 nucleotides) to full length PolyA, enhanced by CPSF (no longer requires AAUAAA)  PAB II recognizes the 10 As that were initially put on

Turnover of PolyA - PolyA tails of mRNAs in cytoplasm eventually get shorter - They are shortened by mRNAses and extended by cytoplasmic PolyA polymerase - Shown by: o Incubated HeLa cells with 3H for 48 hrs, then isolated nuclear and cytoplasmic PolyA+ RNAs o 32P 5S rRNA was a size marker o Left side of graph is top of gel = larger things o Nuclear polyA is 210 ± 20nt and was homogeneous (very narrow peak because most of them are the same size in the nucleus: ~200nt) o Cytoplasmic PolyA is 190 ± 20nt (shorter) and is a much broader peak  The cytoplasmic peak tails off to mRNAs that were much smaller  PolyA tail in the cytoplasm is not a homogeneous length (is a heterogrneous length), and is gradually getting degraded  The tail can be added back (which is why there is so much fluctuation) but the general trend is towards shortening -

PolyA turns over in the cytoplasm o RNases tear it down from the 3’ end o Cytoplasmic polyA polyermase builds it back up o The general trend for an mRNA in the cytoplasm is toward shortening o When the polyA is gone (too short to bind PAP) the mRNA is slated for destruction

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UUUUAAU is needed for cytoplasmic polyadenylation o There are certain times when cytoplasmic polyadenylation is done in a regulated way o In xenopus oocytes, there are mRNAs that are synthesized in the cytoplasm of the egg but are not translated, and have little to no polyA tail o When the egg is stimulated with progesterone (hormonal signal to turn on the egg) those mRNAs in the cytoplasm of the egg re-acquire polyA tails through cytoplasmic PAP o Shown by:  Cytoplasmic polyadenylation not only relies on the AAUAAA sequence, it also needs UUUUUAU (cytoplasmic polyadenylation element/CPE), which is upstream of AAUAAA sequence (the cytoplasmic PAP looks for both of these sequences)  Injected radiolabelled SV40 3’-mRNA with/without cytoplasmic polyA signal sequences (P = progesterone) into oocyte cytoplasm  Incubated 12 h, then retrieved RNA and separated on gel  In the presence of the P (P+) the polyA tail was added only when UUUUAU was there too (not without it) AAUAAA is needed for both cytoplasmic and nuclear polyadenlyation o Both motifs (AAUAAA and UUUUUAU) are required for cytoplasmic polyadenylation 03-07-2017

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15 – Coordination of Processing -

In reality, 5’ cap formation, poly A addition, and splicing is thought to occur co-transcriptionally, and defects in one process can affect the efficiency of other types of mRNA processing o Likewise, deficiencies in mRNA processing can result in defects in mRNA export from the nucleus to the cytoplasm o First, we will discuss the importance of 5’ cap formation on splicing of the first intron

Dependence of Splicing on the Cap -

It was found that optimal splicing of the first intron in a pre-mRNA requires the presence of the 5’ cap. How this was shown: o Created promoters and terminators so that the first intron is now different (exons = boxes; lines = introns) o Transcribed a section of gene in vitro to create labeled, synthetic message with different caps at 5’ end of messages o In vitro transcripts were incubated in HeLa cell extracts for various times, to allow splicing to occur o Mixtures were electrophoresed to separate products from precursors (below)

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Green line: product with the first intron spliced out Blue line: product with the second intron spliced out Red line: product with both introns spliced out GpppG appears to work as well as m7GpppG  End up getting the splicing of both introns Absence of cap selectively inhibits splicing of the first intron  Don’t get any of the final product (both introns spliced)  Get accumulation of the product with the second intron spliced out  Therefore having no cap, the splicing of the first intron there is a problem

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Does the order of exons matter, or does capping simply stimulate splicing of first intron because it is closest to cap? o They did the same experiment but reversed the order of the introns (i.e. instead of 13-14-15, change to 15-14-15), and the closest intron was still spliced (intron between 15 and 14 was spliced in this scenario) o So therefore it is not due to particular sequence in an intron or borders of exons (just the position) o Shows that the splicing of the first intron is dependent on the cap only (not due to a recognition sequence)

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The nuclear cap-binding complex is important for cap-dependent stimulation of splicing o Consists of 2 cap-binding proteins, 80 kDa (CPB80) and 20 kDa (CPB20) o An antibody against the CBP80 immunoprecipitates the complex of both proteins (because they are associated with one another) o Immunoprecipitation (removal) of CBC with anti-CBP80 from HeLa cell extracts greatly diminishes splicing o Splicing activity could be restored by adding back CBC o CBC is involved in spliceosome complex formation/assembly at the first intron

Effect of PolyA on Splicing

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3 labeled substrates containing an intron, either with w/t polyA signal AAUAAA, mutant AAGAAA, or unrelated RNA at 3’ end with no polyA signal Added to splicing extracts for various times, then electrophoresed to detect any splicing o Without the polyA tail, there is no splicing of the last intron o Therefore the polyA tail promotes the splicing of the last intron Does polyA tail affect splicing of only the closest intron? (similar to previous 5’ cap experiment) o 2 labeled pre-mRNAs (with 2 introns each), one with wild-type polyA signal, the other with mutated AAGAAA signal o Added to splicing extracts for various times, then electrophoresed to detect any splicing o Results  First intron of non-polyA substrate is spliced well but second intron is not  Both introns are spliced well in polyA substrate (wild type)  PolyA is required for removal of nearest intron/last intron  Splicing of all other introns occurs at normal rate, even without polyA

How are Capping, Polyadenlyation, and Splicing Coupled to RNA Polymerase Co-transcriptionally? -

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Evidence for binding of mRNA processing proteins to CTD of Rpb1 (large subunit of pol II): o Guanylyl transferase (enzyme in the capping reaction that takes a GTP and ligates to the 5’ end of the mRNA) activity was shown bound to CTD, but only to its phosphorylated form o Used a GST pulldown (things that are fused to GST will fuse to the beads) o L: material loaded on glutathione column (has guanylyl transferase activity in the cell) o FT: column flowthrough (anything that flowed through the column; still had some guanylyl transferase activity) o Several different GST fusions were made so that they stick to the beads and in each, lysates (with active guanylyl transferase) were poured into them  GST: activity of guanylyl transferase bound to GST alone (nothing bound)  mut CTD: the CTD of the pol II subunit was mutated such that it cant be phosphorylated anymore (nothing bound)  wt CTD: wildtype CTD that is not phosphorylated (nothing bound)  wt CTD-PO: wt CTD that has been phosphorylated  Guanylyl transferase activity was affinity purified only when column was loaded with wild type Rpb1 CTD that was phosphorylated  This means that the guanylyl transfera se bound to the wt CTD that was phosphorylated  Capping machinery therefore directly contacts pol II o Similar evidence links the yeast cleavage/polyadenylation factor CF1A to the phosphorylated tail of RNA pol II This is evidence for the coordination of transcription and these processes (transcription events, like phosphorylation of the CTD, recruits all of these factors)

Evidence that the yeast splicing factor, Prp40 (component of U1 snRNP), is bound to phosphorylated CTD o Far-Western blot:  Proteins of interest (Prp40 and other splicing factors, as well as some other related factors) are separated by SDS-PAGE and transferred to a membrane, as per a western blot  Radiolabelled CTD of RNA polymerase II is used to probe the blot, instead of an antibody

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Prp40 stuck to the CTD of pol II the same way an antigen would stick to a protein This tells you that Prp40’s nature structure is not required to bind to pol II (this is an SDS PAGE so it was denatured)  Perhaps the recognition sequence is not that long or doesn’t need to be folded in a certain way This is also evidence that the splicing machinery makes direct contact to the CTD of pol II What does this analysis reveal about the nature of the interaction between the CTD of RNA pol II and these splicing factors? Does it require native protein structure in the splicing factors?

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Guanylyl transferase activity is found at the 5’ end of an mRNA, while polyadenlyation is associated with the 3’ end. Can these activities differentially associate with the RNA pol II CTD? How can the polyA, 5’ cap, and splicing machinery all bind to RNA pol at the same time? o CTD is large, but they cant all bind at once o ChIP crosslinks proteins with DNA, shears DNA, and then you immunoprecipitate the protein which will be associated with the piece of DNA it was bound do o For each immunoprecipitation, there were primers that annealed to the promoter of the gene of interest, and within the gene (in the ORF; denoted by CDS) o ChIP using an antibody against the yeast guanylyl transferase (Ceg1) preferentially immunoprecipitates DNA at or near the promoters of 3 genes (ADH1, PMA1, and PDR5)  Get a large band for the promoter but not within the gene  Capping is therefore physically linked to the DNA (and therefore the CTD at pol II) at the promoter o An antibody against a polyadenylation factor (Hrp1) precipitates more DNA downstream from the promoter  Polyadenylation factors are therefore more associated with DNA and pol II that is further from the promoter o Control (alpha-HA-Rbb3): pull down a subunit of RNA polymerase, and shows that you get DNA at all lanes  This shows that all of the gene fragments are there, and that the proteins of interest just aren’t binding to them  However, you don’t get a band for intergenic DNA (DNA between genes) – because this is not expected to be transcribed o All of the different enzymes are therefore coming on and off of RNA pol II depending on where it is in the gene o What is the mechanism for this?

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Recall that the CTD of Rpb1 is differentially phosphorylated at and downstream of the promoter: serine 5 of CTD is phosphorylated when polymerase is near promoters but serine 2 is phosphorylated far from promoters o ChIP using an antibody specific for serine 5 phosphorylation of the CTD preferentially immunoprecipitates DNA near the promoter o However, ChIP using an antibody specific for serine 2 phosphorylation of the CTD preferentially immunoprecipitates DNA downstream of the promoter  There is also a gradual increasing intensity of the bands as you go down the gene  This is because during initiation, TFIIH phosphorylates the CTD at position 5  But as the pol II clears the promoter, it sometimes stalls because the phosphate is removed

When the phosphate is removed, PTEFB puts it back on in the serine 2 position instead of the serine 5 position A general antibody against the CTD immunoprecipitates DNA from the entire transcribed region It is hypothesized that these different marks allow for the differential association of processing factors (there are many other different modifications as well) 

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Model for RNA processing organized by CTD o The capping complex is first associated with the CTD  As the RNA comes out, it gets capped right away o As the polymerase moves down the DNA, phosphorylation marks change and splicing complexes are recruited o As the polymerase gets near the end of the DNA, (more serine 2 marks), the cleavage and polyadenylation machinery is recruited, so that when the AAUAAA sequence comes out, these enzymes are ready to add the polyA tail

Termination of Transcription -

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Cleavage and polyadenylation mean that the mature 3’ end of mRNA is upstream of actual termination site o Termination is difficult to study because of unstable 3’ cleaved piece When the cell sees free 3’ and 5’ ends (after the polyadenylation cut) those strands get degraded

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Basic mechanism of transcription termination by pol II: the torpedo model o a) As the RNA pol II nears the end of the gene, it transcribes a cleavage and polyadenylation site (AAUAAA in the mRNA) o b) mRNA cleavage and polyadenylation factors (already associated with the RNA pol II CTD) cut the mRNA at this site  RNA pol II continues transcribing o c) The mRNA, newly cut at the 3’ end is polyadenylated  At the new, free 5’ end of the RNA, a 5’ to 3’ exonuclease (Rat1 in yeast) binds and cleaves the still elongating transcript, chasing after RNA pol II (the torpedo)  This exonuclease doesn’t normally degrade mRNA because it has a cap o d) The torpedo reaches RNA pol II (because it mo...