Processes of Polyadenylation

Processes of Polyadenylation DNA once transcribed into mRNA it is transported to the cytoplasm. All mRNA’s including specific unspliced mRNA precursors contain the poly A tail with histon mRNA as an exception. But once they are transported to the cytoplasm there exist a poly (A) tail shift that is brought about by the degradation by RNases and rebuilding by cytoplasmic poly (A) polymerase. Discovery: James Darnell and his coworkers carried out various experiments to study and understand the process of polyadenylation. To begin with, they concentrated on the isolation of the poly (A) tail from the newly synthesized mRNA of the HeLa cell line using two subtypes of the enzyme RNase. The enzymes were; 1. RNase A which function as nucleases that cut after the pyrimidine nucleotides C and G and 2. RNase T1 which cuts after G nucleotides. Both these enzymes together helped in selection of pure runs of A’s. They then carried out centrifugation to separate the nucleus and cytoplasm to separate them according to their sizes and exposed them to the scintillation counter. The results obtained showed that both peaks of the nucleus and cytoplasm electrophoresed even slower than the 4S-tRNA and 5S-rRNA markers used (size markers). It also confirms the little difference in size that exist between the nuclear and cytoplasmic mRNA poly (A)’s. Position confirmation: To confirm the 3-prime position of the poly (A) tail they subjected mRNA to an enzyme RNase. On complete digestion it yielded one molecule of adenosine and about 200 molecules of AMP. This result also aided in concluding the size of the poly (A) tail to be about 200 nucleotides long but recent advances and studies have confirmed the size of the poly (A) tail to be about 250nt long. Activity of poly (A) polymerase: Furthermore it had to confirmed that the poly A tail hadn’t come from DNA transcription as the DNA doesn’t contain long runs of T’s. Therefore being a post transcription modification it stresses on the activity of the poly (A) polymerase that adds AMP residues one at a time to the mRNA synthesized during the transcription process. This can be confirmed with the use of actinomycin D that inhibits DNA-directed transcription but doesn’t inhibit polyadenylation. Role of the poly (A) tail: 1. Protects mRNA from degradation – Michel Revel and his colleagues studied the same by injecting globin mRNA with and without poly A tail into Xenopus oocyes and measured the rate of its synthesis at various intervals. They found a little difference at first but after 6 hours only the mRNA without the poly (A) tail couldn’t support translation. The simplest explanation they gave regarding the same was that the mRNA with the poly (A) tail had a longer shelf life therefore its protective in nature. 2. Stimulates translation of the attached mRNA- Poly (A)-binding protein (PAB 1) in eukaryotes boost the efficiency of the mRNA translation. This is confirmed by the invitro experiment that contained a capped and poladenylated mRNA excess poly (A) tails. When comparing with the control that lacked the excess poly (A) tails lower rates of translation was observed. This suggested that the free poly (A) tails competed with the poly (A) tails on the mRNA. Another control confirmed that in the absence of the mRNA the transciption rates were very low as it can’t bind to PAB1 efficiently. David Munroe and Allan Jacobson studied the effect of both capping and polyadenylation on the transcription of two synthetic mRNA’s(rabbit ÃŽ ² globin gene-RBG and vesicular stomatitis virus N gene –VSN.N under the influence of phage SP6 promoter) in rabbits reticulocytes. a) Polysome profi les. Munroe and Jacobson mixed  32P-labeled poly(A)1 (blue) and 3H-labeled poly(A)2 (red) mRNA with a  rabbit reticulocyte extract, then separated polysomes from monosomes by sucrose gradient ultracentrifugation. The arrow  denotes the monosome peak; fractions to the left of this peak are  polysomes, and one can see the disome, trisome, and even higher  polysome peaks. The poly(A)1 mRNA is clearly better at associating  mRNA stability and translatability. The Basic Mechanism: Polyadenylation is assumed to occur either at the 3à ¡Ã‚ ¿Ã‚ ½-end of the primary transcript synthesized or at the polyadenylation site upsteam to the last coding site of the transcript. But polyadenylation begins even before the transcripts is synthesized as it involves a pre-transcriptional step of clipping of mRNA and then adding poly(A) tail to the newly exposed 3à ¡Ã‚ ¿Ã‚ ½-end. Thus the RNA polymerase can still be functioning as somewhere upstream the polyadenylation apparatus has already located a signal which can cut the mRNA upstream and polyadenylate it. Nevins and Darnell eliminated the first hypothesis by creating hybrids of radioactive RNA made in cells late in infection to DNA fragments of the major late region. If transcription halted at the first few genes after the first polyadenylation sites then much more transcripts would bind to the 5à ¡Ã‚ ¿Ã‚ ½-end rather than the 3à ¡Ã‚ ¿Ã‚ ½-end of the major late region. But it was seen that the RNA hybridized equally to both the ends confirming that once the transcription of the late gene has begun it runs all the way as there is only one transcription terminator at the end of the gene. Thus this region can be called as a transcription unit due to its ability to be transcribed as a whole though it contains multiple genes. They also went on to confirm the clipping of the mRNA pre translation. Erhard Hofer and James Darnell isolated labeled globin encoding RNA that was induced by dimethyl sulphoxide-DMSO and hybridized it to the ÃŽ ²-globin gene and regions downstream to the gene. They observed hybridization to fragments within the ÃŽ ²-globin gene and upto 500 bp downstream to the polyadenylation site. Thus confirming that transcription terminated about 500 bp beyond the polyadenylation site in both cellular and viral transcripts. Hofer and Darnell isolated nuclei from DMSO-stimulated Friend  erythroleukemia cells and incubated them with [32P]UTP to label  run-on RNA—mostly globin pre-mRNA. Then they hybridized this  labeled RNA to DNA fragments A–F, whose locations and sizes are  given in the diagram at top. The molarities of RNA hybridization to  each fragment are given beneath each, with their standard deviations  (s.d.). In the physical map at top, the exons are in red and the introns  are in yellow. Polyadenylation signals: The polyadenylation signals depends on the kind of cell that is being transcribed. The signaling process of plants and animals also differ. At the DNA level in mammalian cells the 20 bp- ‘AATAAA’ sequence was discovered as the polyadenylation sequence by various molecular biologist in 1981. And at the RNA level, in mammalian and plants cells the ‘AAUAAA’ sequence about 20-nt upstream of their poly (A) is considered as the polyadenylation sequence. Another common variant ‘AUUAAA’ is also 80% efficient as ‘AAUAAA’. The other variants are less efficient and less common. Molly Fitzgerald and Thomas Shenk studied the importance of the RNA polyadenylation site. They created a recombinant SV40 virus with duplicate polyadenylation sequence 240 bp apart and carried out an S1 assay. They then carried out an S1 assay of the 3à ¡Ã‚ ¿Ã‚ ½-end which showed two signals 240 bp apart confirming the activity of both the sites. They then deleted one of the two polyadenylation sites one at a time and carried out the S1 assay again. The inserted polyadenylation site beyond the pre-mRNA couldn’t function if the site within the pre-mRNA was absent. Several other scientist studied this phenomenon and discovered another sequence present immediately downstream to the polyadenylation recognition site that affects polyadenylation. But the difficulty in further discovery of details regarding the same was difficult as this wasn’t a conserved sequence among invertebrates. This region was usually found to be a GU- or U- rich region 20 bp downstream to the polyadenylation recognition site. Anna Gil and Nicholas Proudfoot studied this hypothesis in detail and observed the following results: Added an extra copy of the whole polyadenylation signal upstream and carried out an S1 assay. This cloned DNA showed 90% efficiency. Deleted the 35-bp fragment containing the GU- and U- rich region. Polyadenylation process was hampered which explains its importance. Reconstructed clones containing either a GU- rich or a U- rich region. These clones showed however only 30% efficiency. Clones GU- and U- rich regions by an excess of 5-bp sequence between them. These clones showed only 30% efficiency confiming the importance of the spacing between them. Based on all these manipulations they concluded that for an efficient polyadenylation signal; (a)A polyadenylation recognition motif ‘AAUAAA’ followed by (b)A 23-25-bp GU-rich motif downstream immediately followed by (c) A U- rich motif. POLY (A) POLYMERASE: The poly (A) polymerase (PAP) was discovered by James Manley in 1991. He cloned their genome and discovered two different cDNA’s that had variable 3à ¡Ã‚ ¿Ã‚ ½ends due to two alternative splicing methods giving rise to two different PAP’s (PAP-à Ã¢â‚¬  , PAP-à Ã‚  [the most important], four additional PAP). They differ in the amino acids sequences present at their carboxy termini but the PAB-à Ã‚  consists of consensus sequences that overlap with the known functional sequences of proteins. The genome consists of :1. RNA-binding domain (RBD), 2. Polymerase module (PM), 3. Two nuclear localization signals (NLS-1 and NLS-2), 4. Serine/Threonine-rich regions S/T. Polyadenylation at the amino terminal. The mRNA is polyadenylated before leaving the cytoplasm as well as after entering the cytoplasm. However these two adenylations could be distinguished by Sheiness and Darnell due to their slight difference in size. They confirmed the same by carrying out various assays against the isolated mRNA that was grown in labeled RNA for 48 hours. The nuclear RNA, cytoplasmic RNA, and 5sRNA marker showed peaks as seen in the figure alongside. The major peaks thus obtained are 210 ±20nt and 190 ±20nt for the nuclear and cytoplasmic poly (A) tail respectively. About 50nt RNA’s are present in this broad peak. Maurice Sussman, in 1970 gave the ‘ticketing’ hypothesis which encompasses the theory of each RNA having a ticket to gain entry to the ribosome and further ticket punching everytime it got translated. Thus after a particular limit, it can’t longer undergo protein synthesis which another reason for the shortening of the poly (A) tail. Thus the 3à ¡Ã‚ ¿Ã‚ ½-end shortening of the poly(A) tail clearly depends on the some other factor other than translation or the ticket like some post-transcriptional modification. It has been observed that the poly(A) tail has not only been shortened in the cytoplasm but it also turns over. This inverted poly(A) tail is susceptible to RNase degradation and elongation by the cytoplasmic poly(A) polymerase simultaneously. This continues till the mRNA looses all or almost all of the nuclear poly(A) tail. This happens when its almost time for the demise of the mRNA. Cytoplasmic polyadenylation This process is best studied in Xenopus oocytes. Administration of progesterone to their oocytes cause stimulation of the deadenylation of maternal mRNA’or maternal message. Polyadenylation the actual process: The process involves the recognition of that conserved polyadenylation motif, RNA cleavage and polyadenylation. Pre mRNA cleavage: The proteins responsible for this cleavage are: Shrenk and his colleagues carried out various experiments confirming the importance of these cleavage factors. Cleavage polyadenylation specificity factor (CPSF)- Its one of the most important factors. Its subunit CPSF-73is related to ELAC that cleaves pre-tRNA’s to generate their 3à ¡Ã‚ ¿Ã‚ ½-end. They are known as ÃŽ ²-lactamase superfamily of Zn (as they contain 2 Zn ions at their active site necessary for RNase activity) dependent hydrolases. Cleavage stimulating factor (CSF) – Its one of the most important ones. It bindings to the GU- rich region, together and stably. Cleavage factors (CF à Ã¢â‚¬   and CF à Ã‚ )- The poly (A) polymerase- This immediate coupling is so strong that no cleaved unpolyadenylated RNA’s can be found. The RNA polymerase à Ã‚ (containing the carboxy terminal domain-CTD and its phosphorylation status). Yukata Hirose and James Manley expressed CTD as a fusion protein with glutathionine-s transferase. They then purified the protein by glutathionine affinity chromatography and the phosphorylated and non-phosphorylated forms were exposed to the cleavage assay with adenovirus L3 pre-mRNA. The results obtained confirmed that (a) the activity of CTD is independent of transcription and (b) After incubating the phosphorylated and non-phosphorylated forms of the enzyme along with all the other cleavage factors showed that the phosphorylated forms five times batter cleavage. This can be explained as the phosphorylated form of CTD is present in the polymerase à Ã‚  that carries out transcription. Polyadenylation initiation: Once the pre-mRNA is cleaved using the factors described above its polyadenylation process takes place in two phases. The first initiations phase consists of the slow addition of the first 10 A’s. This phase depends on the ‘AAUAAA’ signal. The second phase is independent of the initial ‘AAUAAA’ signal but it depends on the existing 10 A’s added to the pre-mRNA. This phase involves rapid addition of about 200 or more A’s along the length, thus called elongation. The initiation signal that carries out polyadenylation is none other than the cleavage signal which attracts the cleavage enzyme that specifically recognizes the AAUAAA motif and cuts the RNA 20 nucleotide downstream. This thaught was discarded because as the cleavage enzyme prior to polyadenylation has already cut the downstream GU-rich and U-rich sequence. Thus it’s this 8 nucleotide GU/U-rich sequence post the AAUAAA motif that brings about this adenylation. Marvin Wickens and his colleagues used two parameters (a) a poly (A) polymerase and (b) a specificity factor CPSF that binds to the pre mRNA. Both these factors work well when substrates are in high concentration but the assay carried out was using low substrate concentrations. The figure alongside explains their experiments, Lane 1- shows no polyadenylation by poly (A) polymerase by itself in low concentrations of substrates, Lane 2- shows no polyadenylation as the CPSF alone can’t detect the AAUAAA motif, Lane 3- shows polyadenylation with both factors together and Lane 4- shows that both factors can’t polyadenylate a substrate with an aberrant signal like AAUAAA. But however this dependency is temporary i.e. after the addition of the first 10 nucleotides it enters the elongation phase that’s independent of these two factors. ELONGATION OF POLY (A) TAIL: While studying the fact that the initiation is independent of the CPSF factor, another interesting fact came to be known that a purified poly (A) polymerase could carry out elongation very poorly on its own. Whale further explored this by designing experiments which consisted of purification of the poly (A) polymerase and its polyadenylation capability comparison in various conditions. Purification of the poly (A) polymerase using PAGE gave two fractions – a major 49-kD polypeptide (PAB-à Ã¢â‚¬  ) as well as a minor 70kD polypeptide (PAB-à Ã‚ ). This latter band however was found to have a variable nature and was even absent in some preparations. Whale’s experiments further showed high activity of this 49-kD polypeptide coinciding with high activity of the poly (A) polymerase using a nitrocellulose filter binding assay. He also tested this fraction’s capability of polyadenylation in the presence of the CPSF and poly (A) polymerase and found the same results. He therefore named this fraction as poly (A) binding proteinà Ã‚  (PAB- à Ã‚ ). Thus he confirmed that PAB-à Ã‚  acts like CPSF but binds to poly (A) polymerase instead of the AAUAAA motif on the RNA. Its activity is high only in the elongation phase but is found absent in the initiation phase. He carried out another experiment to find the interdependence of these two factors with their interdependence on the polyadenylation process using the poly (A) polymerase. When either CPSF or PAB-à Ã‚  was added to a solution that contained mRNA and poly (A) polymerase, the polyadenylation process was found to be active. But it showed higher polyadenylation capabilities in presence of both the factors. Thus this whole process can be summarized by the proposed figure below:-