In all major functions of eukaryotic mRNA, there is an involvement of a poly(A)-tail. The addition of a poly(A)-tail to the 3′-termini of RNA molecules influences stability, nuclear export, and efficiency of translation. The translational silence and activation, in the cytoplasm, is regulated by the dynamic changes in the length of the poly(A)-tail. Thus, measurement of the poly(A)-tail associated with any given mRNA at steady-state can serve as a surrogate readout of its translation-state. This addition of a poly(A) tail is identified as the final quality control step. After the final step, every nascent mRNA undergoes prior to nuclear export.
The purpose of the poly(A)-tail is to promote circularization of the RNA molecule in a closed-loop configuration which promotes initiation of translation. During the synthesis process, the length of the poly(A)-tail is generally uniform for any given system and it is species-dependent. The length of yeast and mammalian this is approximately 60-250 adenosine residues. The steady-state length distribution of poly(A)-tails, in the cytoplasm, can vary dramatically for transcripts of different functional classes.
Targeted deadenylation is achieved by the binding of microRNA or RNA binding proteins. Activating cytoplasmic adenylation can modulate the polyadenylation state of the transcriptomes of many eukaryotes. The function of RNA adenylation to destabilize mitochondrial, structural, and noncoding RNA is employed by eukaryotic cells. Other gene expression parameters, such as ribosome occupancy and protein abundance is highly correlated with the adenylation state of the transcriptome. This serves the purpose that the measurement of mRNA poly(A)-tail length. This serves as a surrogate for translation-state measurements.
Different methods are developed measuring poly(A)-tail length. RNase H digest combination with high-resolution Northern blot is the most direct method. Ligation-Mediated Poly(A) Test (LM-PAT) assay was also used measuring poly(A) tail. T4 RNA ligase has been used to either circularize mRNA. This can be used to ligate adaptors to the 3′-end of mRNA. These assays serve measure poly(A)-tail length. Ever assay has its own limitation. These high-resolution Northern blots have the limitations are labor-intensive and require a lot of RNA. The LM-PAT assay has the following properties such as low-resolution and a tendency toward exaggerated apparent short-tailed mRNA, and adaptor ligation approaches are relatively inefficient.
A simple assay is illustrated in this article. It has the characteristic of high-resolution. The assay is very efficient for measuring the poly(A)-tail of mRNA. The below method assigns 3′ UTR unambiguously of the specific transcripts. The use of Klenow polymerase to extend the 3′-termini of specific RNA molecules with dNTPs. The intrinsic activity can be harnessed to tag the 3′-end of RNA molecules as an extension-mediated poly(A)- tail length measurement as well as for other applications that require 3′-end labeling. The intrinsic property of Escherichia coli DNA polymerase I to extend an RNA primer using a DNA template is used in this method to 3′-tag adenylated RNA in total RNA samples. This tag is used as an anchor for cDNA synthesis. This tag is used in subsequent gene-specific PCR to assess poly (A)-tail length. In the new method defined in this article, Escherichia coli DNA polymerase I is used to extend an RNA primer using a DNA template. This polymerase tags a 3′-adenylated RNA in total RNA. The tag serves the purpose of anchor for cDNA synthesis, which in turn is used to assess poly(A)-tail length in gene-specific PCR. This test is called as extension Poly(A)- Test(ePAT). The efficiency of this method is on par with traditional Ligation- Mediated Poly(A) Test (LM-PAT) assays. This new method has efficiently avoided problems like traditional Ligation- Mediated Poly(A) Test (LM-PAT) assays. The accuracy of this methods has found the application of these methods in traditional Ligation- Mediated Poly(A) Test (LM-PAT) assays.
Materials & Methods
- The By4741 yeast strain was grown to exponential phase (OD600 ~0.6) in rich medium (2% peptone and 1% yeast extract) with 2% raffinose as the sole carbon source.
- Transcription of GAL genes was transiently induced by addition of 2% galactose and then repressed after 10 min by the addition of 2% glucose.
- At each indicated time point, 5 mL of culture was removed into 15 mL tubes containing 50 mL 10% sodium azide prechilled in an ice bath.
- Once all samples were collected, the cells were harvested by centrifugation (4000g for 2 min), washed once in ice-cold water containing sodium azide (0.1%), snap frozen, and stored at -80°C.
- elegans wild-type Bristol N2 and gld-2(q497) strains were maintained at 20°C using standard methods.
- Total RNA from yeast cells was prepared according to the hot phenol method.
- Total C. elegans RNA was prepared by suspending between 50 and 100 snap frozen worms in 1 mL of Trizol.
- After addition of ~200 mL ziconia beads, the sample was homogenized for 30 sec using a Mini-Beadbeater.
- Trizol extraction was performed according to the manufacturer’s instructions, except that 2 mL glycogen was added prior to precipitation with isopropanol.
- To improve the generally poor Nano-drop QC A260/A230 ratios that result from Trizol purifications, resuspended the resulting pellet in 100 mL dH2O and reprecipitated the RNA with 1/10 volume of 3 M NaOac [pH 5.2] and 2.5 volumes of ethanol.
- RNA quantification was performed with a Nano-Drop 1000.
The ePAT method and product detection
- The ePAT approach uses the same PAT-anchor primer (PAT anchor 5′- GCGAGCTCCGCGGCCGCGTTTTTTTTTTTT), was stored as small 100-mM aliquots at -20°C. Of note, an alternative anchor sequences were equally efficient in our laboratory.
- The incubation steps of the assay were most conveniently performed in a thermocycler with an accessible lid programmed with a series of temperature hold/pause settings, where the pause maintains the temperature while allowing access to the tubes.
- To assemble the ePAT reaction, 1 mg of total RNA (or less) and 1 mL of PAT-anchor primer were combined and brought to a volume of 8 mL with dH2O in a 200-mL PCR tube.
- The mixture was incubated at 80°C for 5 min and then cooled to room temperature.
- Once cooled, the sample was flash-centrifuged, and 12 mL of a master mix was added that contained 4 mL dH2O, 4 mL 5X Superscript III buffer, 1 mL 100 mM DTT,
- 1 mL 10 mM dNTPs, 1 mL RNaseOUT, and 1 mL (5 U) Klenow polymerase per reaction.
- The sample was mixed thoroughly by inversion, flash-centrifuged, and then incubated at 25°C for 1 h.
- The polymerase was then inactivated by increasing the temperature to 80°C for 10 min prior to cooling the reaction to 55°C for 1 min.
- While maintaining the tubes at that temperature in the block, 1 mL (200 U) of Superscript III was added to the tubes.
- The tubes were closed and mixed rapidly by flick-inversion.
- Incubation was then resumed at 55°C for 1 h, followed by inactivation of the reverse transcriptase by increasing the temperature to 80°C for 10 min. It is critical to maintain the temperature during the reverse transcription step because internal priming can occur if the temperature drops.
- At 55°C, only priming from an end-extended RNA molecule is possible. It can also be useful to include spiked-in RNA from an unrelated organism as ballast for dilute RNA reactions and to control for equal assay efficiency across samples. Spike total HeLa RNA in yeast samples and RNA from a deadenylase deficient yeast strain in metazoan RNA samples is recommended.
- For the PCR reactions, cDNA was diluted 1:6 by the addition of 120 mL dH2O. The PCR reactions were typically conducted in a total volume of 20 mL using 5 mL of diluted cDNA input and a fast-start polymerase, such as Fast-Start or Amplitaq Gold 360 master. It can also be useful to include a TVN-PAT reaction as a size control for the size of the amplicon with a fixed A12 poly(A)-tail.
- Both the LM-PAT and the TVN-PAT reactions were performed.
- The cycle number was dependent on the abundance of the transcript of interest in the sample but normally ranges between 23–33 cycles.
- To detect the PCR amplicons from TVN-PAT, ePAT, and LMPAT PCR reactions, 50% of a 20-mL PCR reaction was loaded per lane into a 2% high-resolution agarose gel that was prestained with SYBR safe.
- The primers used are supplied.
- To estimate the PCR product sizes and to quantify the mass of PCR amplicons from such gels, the band intensity and migration was determined relative to a 100-bp ladder using an LAS 3000 imager and multigauge software.
- To track deadenylation kinetics, the migration of the highest peak intensity of each band was determined for each lane and expressed relative to the migration of the TVN-PAT peak.
- The length of poly(A) at time zero was then normalized to 100%, and the peaks of subsequent time points were expressed relative to the normalized control.
- The graphs and statistical analyses were prepared using GraphPad Prism software.
- Efficiency calculations used to estimate the number of cDNA input molecules were based on a calculated mass of 110 ng and an amplicon length of 120-bp at an estimated efficiency of 98% as user inputs for oma-2 in the PCR calculator.
The extension Poly(A) Test (ePAT) is a two-step assay. Both the steps are conducted in one reaction tube. The main principle of this method is the intrinsic property of the Klenow polymerase to extend RNA molecules with dNTPs from an annealed oligonucleotide. This oligonucleotide is prepared by annealing DNA template in standard reverse transcription buffers. This reaction is controlled by increasing the reaction temperature to 55°C prior to addition of reverse transcriptase.
This temperature increase ensures that only the DNA oligos that have annealed to an extended 3′ terminus, which have a melt-temperature sufficiently high, to prime reverse transcription. This technique is used to control or eliminate unwanted priming from internal poly(A)-tracts. Further amplification of the oligonucleotide is done by using two primers: gene-specific forward primer and a universal reverse primer. The resultant amplicons of the PCR reaction, reflects the distribution of lengths of the poly(A)-tail on endogenous mRNA.
The length of the poly(A)-tail is calculated using a separate ‘‘TVN-PAT’’ reaction. The reaction reports the size of the amplicon with an invariant 12-(A) poly(A)-tail, irrespective of the actual poly(A)-lengths in the sample. In this TVN-PAT reaction, the cDNA is prepared using an identical primer sequence is similar to that of ePAT. The variation comes in the addition of two 3′ variable bases V and N (where V is A, G, or C, and N is any base). The purpose of these variable bases is to lock the primer to the polyadenylation site during reverse transcription.
Characteristics of ePAT test
Applicability of ePAT over a range of RNA concentration
ePAT has the capability of reporting poly(A)-tail length over a range of RNA concentrations. This property of ePAT is validated using a test using a sample of yeast DNA with Human (HeLa) total RNA. The sample is prepared by serial (1⁄2) dilutions of total yeast RNA to spike into a fixed concentration of Human (HeLa) total RNA. The yeast RNA contains two genes are taken into consideration GAL10 and APQ12. After the RNA isolation from yeast, a 10-min incubation with galactose is carried out. After galactose induction GAL10 shows a uniform length of ~40 adenosine residues. But, APQ12, is not a galactose-responsive gene. This results in a smear of amplicons representing both aged and new transcripts. The poly(A)- tail reported in this test in invariant of the across the range of concentration. Densitometry studies validate this method for quantification studies. The limation arises from the quantitative limitation of the PCR and the fluorescence detection. APQ12 and human GAPDH signals are linear do not vary with concentrations of total RNA. GAL10 response over a certain range of the input sample.
Comparison of different Poly(A) Tail tests
The efficacy of ePAT is compared to the standard LM-PAT method. The standard method relies on the parameters such as serial ligation of p(dT)12–18 and an anchor primer. The role of the primer is to generate the cDNA that encompasses the full poly(A)-tail. For the purpose of comparison invitro synthesized and adenylated TOM5 transcripts are used. The transcripts are then spiked with HeLa RNA to assess the accuracy of the ePAT in length calling. The estimation of the average tail length, of both PCR products and invitro transcripts, is calculated based on the relative migration RNA to DNA ladders. Both the assays are effective and reflect the length of the invitro synthesized poly(A)-tail. LM-PAT assays had a distinctive feature of a slight bias over specific sizes. This assay reported mRNA having longer poly(A)-tails in these sizes.
Activation and repression of gene expression are also studied by this test. For this purpose adenylation state of specific endogenous transcripts are tested in response to various states of gene expression. The response of Saccharomyces cerevisiae to a transcriptional pulse-chase regimen involving activation of gene expression by galactose followed by repression by the addition of glucose is used as the test condition. Yeast cells were harvested 10 min after galactose induction and at the indicated time points in the pulse period after glucose repression. During repression process, the assays showed an increase in the length of poly(A) tail. Both the assays were equally efficient at generating cDNA, which represented the state of adenylation in the transcripts. LM-PAT assay is limited on the resolution it is contributed to the fact that the process depends on the serial annealing of fixed length oligo-(pdT) oligonucleotides.
The kinetics of deadenylation is studied using densitometry and peak-identification. Using ePAT data linear deadenylation rates were calculated. Formerly this required high-resolution northern blots. Thus ePAT is considered to be more accurate and more detailed in the estimation of poly (A)-tail length distribution and deadenylation kinetics.
Alternate poly(A) site usage is revealed by ePAT
The presence of alternate Poly(A) site is widespread and dynamic in case of eukaryotes. It is recommended to use shorter 3′ UTRs(Untranslated regions) to eradicate the influence of post-transcriptional regulatory modules within themselves. It is done while analyzing microRNA and regulatory protein binding sites. The priming of cDNA in ePAT assay requires the extension of the 3′ end of the RNA molecule. This avoids priming of the internal poly(A) stretches. Certains genes which have the tendency to change their subnuclear position in response to activation by galactose in a 3′ UTR-dependent manner. This implies the function of 3′ processing in gene activation and/or repression. Alternative polyadenylation is studied by the ePAT method and the evidence for such polyadenylation is found using the pulse-chase met