Measurement of Poly A Tail Length from Drosophila Larva Brain and Cell Line

Published: January 12, 2024

doi:

¹Department of Biology,University of Nevada, Reno

Summary

The protocol describes an efficient and reliable method for quantifying the poly(A) length of the gene of interest from the Drosophila nervous system, which can be easily adapted to tissues or cell types from other species.

Abstract

Polyadenylation is a crucial posttranscriptional modification that adds poly(A) tails to the 3′ end of mRNA molecules. The length of the poly(A) tail is tightly regulated by cellular processes. Dysregulation of mRNA polyadenylation has been associated with abnormal gene expression and various diseases, including cancer, neurological disorders, and developmental abnormalities. Therefore, comprehending the dynamics of polyadenylation is vital for unraveling the complexities of mRNA processing and posttranscriptional gene regulation.

This paper presents a method for measuring poly(A) tail lengths in RNA samples isolated from Drosophila larval brains and Drosophila Schneider S2 cells. We employed the guanosine/inosine (G/I) tailing approach, which involves the enzymatic addition of G/I residues at the 3′ end of mRNA using yeast poly(A) polymerase. This modification protects the RNA’s 3′ end from enzymatic degradation. The protected full-length poly(A) tails are then reverse-transcribed using a universal antisense primer. Subsequently, PCR amplification is performed using a gene-specific oligo that targets the gene of interest, along with a universal sequence oligo used for reverse transcription.

This generates PCR products encompassing the poly(A) tails of the gene of interest. Since polyadenylation is not a uniform modification and results in tails of varying lengths, the PCR products display a range of sizes, leading to a smear pattern on agarose gel. Finally, the PCR products are subjected to high-resolution capillary gel electrophoresis, followed by quantification using the sizes of the poly(A) PCR products and the gene-specific PCR product. This technique offers a straightforward and reliable tool for analyzing poly(A) tail lengths, enabling us to gain deeper insights into the intricate mechanisms governing mRNA regulation.

Introduction

Most eukaryotic mRNAs are posttranscriptionally polyadenylated at their 3′ terminus in the nucleus by the addition of non-templated adenosines by canonical poly(A) polymerases. An intact poly(A) tail is pivotal throughout the lifecycle of mRNA, as it is essential for mRNA nuclear export¹, facilitates interaction with poly(A)-binding proteins to enhance translational efficiency², and imparts resistance against degradation³. In certain cases, the poly(A) tail can also undergo extension in the cytoplasm, facilitated by noncanonical poly(A) polymerases⁴. In the cytoplasm, poly (A) tail length dynamically changes and influences the life span of the mRNA molecule. Numerous polymerases and deadenylases are known for modulating tail length⁵^,⁶^,⁷. For example, the shortening of poly(A) tails correlates with translational repression, whereas the lengthening of poly(A) tails enhances translation⁸^,⁹.

Accumulating genomic studies have demonstrated the fundamental significance of the poly(A) tail length across various facets of eukaryotic biology. This includes roles in germ-cell development, early embryonic development, neuronal synaptic plasticity for learning and memory, and the inflammatory response¹⁰. There have been numerous methods and assays developed for measuring poly(A) tail lengths. For example, the RNase H/oligo(dT) assay takes advantage of RNase H in the presence or absence of oligo(dT) to study poly(A) tail length¹¹^,¹². Other methods to study poly(A) tail include the PCR amplification of 3' ends such as rapid amplification of cDNA ends poly(A) test (RACE-PAT)¹²^,¹³ and the ligase-mediated poly(A) test (LM-PAT)¹⁴. Further modifications of the PAT assay include ePAT¹⁵ and sPAT¹⁶. Enzymatic G-tailing¹⁷^,¹⁸ or G/I-tailing of the 3' end are other variations of the PAT assay. Further modification of these techniques includes the use of fluorescently labeled primers along with capillary gel electrophoresis for high-resolution analysis, referred to as the high-resolution poly(A) test (Hire-PAT)¹⁹. These PCR-driven assays allow fast and high-sensitivity poly(A) length quantitation.

With the development of next-generation sequencing, a high-throughput sequencing method, such as PAL-seq²⁰ and TAIL-seq²¹, allows polyadenylation analyses at a transcriptome-wide scale. However, these methods provide only short sequencing reads of 36-51 nucleotides. Therefore, FLAM-Seq²² was developed for global tail length profiling of full-length mRNA and provides long reads. Nanopore technology²³ provides PCR-independent, direct RNA, or direct cDNA sequencing for poly(A) tail length estimations. However, these high-throughput methods are not without limitations. They require large amounts of starting materials, are expensive, and time-consuming. Moreover, analyzing rare transcripts can be extremely challenging with high-throughput methods, and low-throughput PCR-based methods still provide an advantage when a small number of transcripts need to be analyzed, for pilot experiments, and validation of other methods.

We have recently demonstrated that Dscam1 mRNAs contain short poly(A) tails in Drosophila, which necessitates a non-canonical binding of the cytoplasmic poly(A)-binding protein on Dscam1 3'UTR using the G/I tailing method²⁴. Here we provide a streamlined procedure for tissue preparation and quantifying poly(A) length of mRNAs from the Drosophila nervous system and Drosophila S2 cells.

Protocol

1. Rearing and selecting Drosophila larvae Maintain/culture the fly strain (w1118, wildtype) on standard fly food medium at 25 ˚C in a humidified incubator. Select 10 wandering 3rd instar larvae 72 h after egg laying. Place the larvae in a 35 mm empty Petri dish and gently wash them by transferring the larvae to the new dish containing tap water using forceps. Do this 2x to remove any remaining …

Representative Results

Here, we analyzed the poly(A) tail length of Dscam1 and GAPDH from Drosophila larval brains (Figure 4). Isolated RNAs were visualized on an agarose gel for quality control. A single RNA band at around 600 nucleotide size indicates intact RNA preparation (Figure 2A). RNAs were subjected to the G/I tailing and high-resolution capillary electrophoresis using an Agilent 2100 bioanalyzer. The gel images were exported using the Agilent 2100 …

Discussion

In this protocol, we describe the technique to dissect the Drosophila larval brain from wandering 3^rd instar stage as well as the sample preparation from Drosophila S2 cells. Due to the labile nature of mRNAs, sample collection requires extra caution. For larval brain dissection, brains should not be damaged during isolation and should not be kept in solution for a prolonged duration. Keeping dissection time to 8-10 min for a round of dissection is essential. It may also be beneficial to supp…

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

This study was supported by the National Institute of Neurological Disorders and Stroke Grant R01NS116463 to J.K., and the Cellular and Molecular Imaging Core facility at the University of Nevada, Reno, which was supported by National Institutes of Health Grant P20GM103650 and used for research reported in this study.

Materials

3-(N-morpholino) propanesulfonic acid (MOPS)	Research Product Internation (RPI)	M92020
Agilent High Sensitivity DNA Kit	Agilent Technologies	5067-4626
Agilent software 2100 expert free download demo	Agilent Technologies		https://www.agilent.com/en/product/automated-electrophoresis/bioanalyzer-systems/bioanalyzer-software/2100-expert-software-228259
Apex 100 bp-Low DNA Ladder	Genesee Scientific	19-109
Bioanalyzer	Agilent 2100 Bioanalyzer G2938C
Diethyl pyrocarbonate (DEPC)	Research Product Internation (RPI)	D43060
DNA dye (Gel Loading Dye, Purple (6x)	New England biolabs	B7024S
Drosophila S2 cell line	Drosophila Genomics Resource Center stock #181
Drosophila Schneider’s Medium	Thermo Fisher Scientific	21720024
Ehidium bromide	Genesee scientific	20-276
Fetal bovine serum (FBS)	Sigma-Aldrich	F4135
Forceps Dumont 5	Fine Science tools	11254-20
Nuclease free water	Thermo Fisher Scientific	AM9932
PBS 10x	Research Product Internation (RPI)	P32200
Poly(A) Tail-Length Assay Kit	Thermo Fisher Scientific	764551KT
RiboRuler Low Range RNA Ladder	Thermo Fisher Scientific	SM1833
RNA Gel Loading Dye (2x)	Thermo Fisher Scientific	R0641
RNA microprep kit	Zymoresearch	R1050
RNA miniprep kit	Zymoresearch	R1055
Scissors-Vannas Spring Scissors – 2.5 mm Cutting Edge	Fine Science tools	15000-08
TopVision Agarose Tablets	Thermo Fisher Scientific	R2802
Tris-Acetate-EDTA (TAE)	Thermo Fisher Scientific	B49

Referenzen

Stewart, M. Polyadenylation and nuclear export of mRNAs. Journal of Biological Chemistry. 294 (9), 2977-2987 (2019).
Machida, K., et al. Dynamic interaction of poly(A)-binding protein with the ribosome. Scientific Reports. 8 (1), 17435 (2018).
Eisen, T. J., et al. The dynamics of cytoplasmic mRNA metabolism. Molecular Cell. 77 (4), 786-799 (2020).
Liudkovska, V., Dziembowski, A. Functions and mechanisms of RNA tailing by metazoan terminal nucleotidyltransferases. Wiley Interdisciplinary Reviews RNA. 12 (2), e1622 (2021).
Goldstrohm, A. C., Wickens, M. Multifunctional deadenylase complexes diversify mRNA control. Nature Reviews Molecular Cell Biology. 9 (4), 337-344 (2008).
Schmidt, M. J., Norbury, C. J. Polyadenylation and beyond: emerging roles for noncanonical poly(A) polymerases. Wiley interdisciplinary reviews RNA. 1 (1), 142-151 (2010).
Laishram, R. S. Poly(A) polymerase (PAP) diversity in gene expression – Star-PAP vs canonical PAP. FEBS Letters. 588 (14), 2185-2197 (2014).
Salles, F. J., Lieberfarb, M. E., Wreden, C., Gergen, J. P., Strickland, S. Coordinate initiation of Drosophila development by regulated polyadenylation of maternal messenger RNAs. Science. 266 (5193), 1996-1999 (1994).
Wreden, C., Verrotti, A. C., Schisa, J. A., Lieberfarb, M. E., Strickland, S. Nanos and pumilio establish embryonic polarity in Drosophila by promoting posterior deadenylation of hunchback mRNA. Development. 124 (15), 3015-3023 (1997).
Passmore, L. A., Coller, J. Roles of mRNA poly(A) tails in regulation of eukaryotic gene expression. Nature Reviews Molecular Cell Biology. 23 (2), 93-106 (2021).
Murray, E. L., Schoenberg, D. R. Assays for determining poly(a) tail length and the polarity of mRNA decay in mammalian cells. Methods in Enzymology. 448, 483-504 (2008).
Salles, F. J., Strickland, S. Analysis of poly(a) tail lengths by PCR: The PAT assay. Methods in Molecular Biology. 118, 441-448 (1999).
Salles, F. J., Darrow, A. L., O’Connell, M. L., Strickland, S. Isolation of novel murine maternal mRNAs regulated by cytoplasmic polyadenylation. Genes and Development. 6 (7), 1202-1212 (1992).
Salles, F. J., Strickland, S. Rapid and sensitive analysis of mRNA polyadenylation states by PCR. Genome Research. 4 (6), 317-321 (1995).
Janicke, A., Vancuylenberg, J., Boag, P. R., Traven, A., Beilharz, T. H. ePAT: A simple method to tag adenylated RNA to measure poly(a)-tail length and other 3′ RACE applications. RNA. 18 (6), 1289-1295 (2012).
Minasaki, R., Rudel, D., Eckmann, C. R. Increased sensitivity and accuracy of a single-stranded DNA splint-mediated ligation assay (sPAT) reveals poly(a) tail length dynamics of developmentally regulated mRNAs. RNA Biology. 11 (2), 111-123 (2014).
Martin, G., Keller, W. Tailing and 3′-end labeling of RNA with yeast poly(A) polymerase and various nucleotides. RNA. 4 (2), 226-230 (1998).
Kusov, Y. Y., Shatirishvili, G., Dzagurov, G., Verena, G. M. A new G-tailing method for the determination of the poly(a) tail length applied to hepatitis a virus RNA. Nucleic Acids Research. 29 (12), 57 (2001).
Bazzini, A. A., Lee, M. T., Giraldez, A. J. Ribosome profiling shows that miR-430 reduces translation before causing mRNA decay in zebrafish. Science. 336 (6078), 233-237 (2012).
Subtelny, A. O., Eichhorn, S. W., Chen, G. R., Sive, H., Bartel, D. P. Poly(a)-tail profiling reveals an embryonic switch in translational control. Nature. 508 (1), 66-71 (2014).
Chang, H., Lim, J., Ha, M., Kim, V. N. TAIL-seq: Genome-wide determination of poly(a) tail length and 3′ end modifications. Molecular Cell. 53 (6), 1044-1052 (2014).
Legnini, I., Alles, J., Karaiskos, N., Ayoub, S., Rajewsky, N. FLAM-seq: Full-length mRNA sequencing reveals principles of poly(A) tail length control. Nature Methods. 16 (9), 879-886 (2019).
Garalde, D. R., et al. Highly parallel direct RNA sequencing on an array of nanopores. Nature Methods. 15 (3), 201-206 (2018).
Singh, M., Ye, B., Kim, J. H. Dual leucine zipper kinase regulates Dscam expression through a noncanonical function of the cytoplasmic poly(A)-binding protein. Journal of Neuroscience. 42 (31), 6007-6019 (2022).
Macharia, R. W., Ombura, F. L., Aroko, E. O. Insects’ RNA profiling reveals absence of "hidden break" in 28S ribosomal RNA molecule of onion thrips, Thrips tabaci. Journal of Nucleic Acids. 2015, 965294 (2015).
Miura, P., Sanfilippo, P., Shenker, S., Lai, E. C. Alternative polyadenylation in the nervous system: to what lengths will 3′ UTR extensions take us. Bioessays. 36 (8), 766-777 (2014).
Sement, F. M., et al. et al Uridylation prevents 3′ trimming of oligoadenylated mRNAs. Nucleic Acids Research. 41 (14), 7115-7127 (2013).

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Singh, M., Kim, J. H. Measurement of Poly A Tail Length from Drosophila Larva Brain and Cell Line. J. Vis. Exp. (203), e66116, doi:10.3791/66116 (2024).