poly a tail length measurement of genes from drosophila brain

Kvantificering af poly(A)-længden af gener fra Drosophila-nervesystemet

Most eukaryotic mRNAs are posttranscriptionally polyadenylated at their 3&#8242; 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&#39; 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&#160;of the 3&#39; 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&#39;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.

Forfatter Spotlight: Udforskning af grænsen for mRNA-forskning med Poly A Tail Analysis Techniques

Måling af Poly A halelængde fra Drosophila larve hjerne og cellelinje

Polyadenylation is a post-transcription modification. This add poly-A tails to the three-prime end of mRNA molecules. Dysregulation of polyadenylation has been associated with abnormal gene expression and various diseases, including neurological disorders.Our method provides a cost-effective, high-resolution analysis for measuring poly-A lengths. High-throughput methods for poly-A tail analysis, like next generation sequencing, and a high-throughput sequencing provides high-content information. However, analyzing rare transcripts can be extremely challenging with these methods.PCR-based methods are usually low-throughput. However, they can be easily used to analyze a small number of transcripts. The GI-tailing method is one of them.With this method, we discovered that Dscam mRNAs contain short poly-A tails. Dscam is a neuronal cell adhesion molecule. It is involved in many important neurodevelopmental processes.Because Dscam mRNA contains short poly-A tails unlike most mRNAs, Dscam mRNA translation is under unique gene regulation, which is one of the questions we are trying to answer.

Poly A halelængde måling af gener fra Drosophila hjerne

For at begynde G / I-tailing skal du forberede en 20-mikroliters blanding på is med total RNA, halebufferblanding og haleenzymblanding. Inkuber ved 37 grader Celsius i 60 minutter i en termocykler. Derefter tilsættes halestopopløsningen og opbevares på is i to minutter.Udfør omvendt transkription og PCR-forstærkning. Efter gelelektroforese med høj opløsning af PCR-produkter skal du få adgang til data ved hjælp af softwaren. Åbn XAD-filen, og vælg eksempelnavne eller stige i panelet Trævisning.Zoom ind og ud på elektroferogrammer og gellignende billeder for detaljeret visning. For at opnå topstørrelserne skal du åbne elektroferogrammet for en valgt prøve. Højreklik på elektroferogrammet og vælg manuel integration for manuelt at vælge toppe ved at trække den vandrette linje.Overhold topværdierne i toptabellen, og identificer toppen med den største tophøjde. Aktivér ikonet Vis/skjul sætpunkter i topmenuen. Et nyt panel vises på højre side.Vælg Avanceret, rul ned til Udfør udtværingsanalyse, og marker afkrydsningsfeltet. Dette tilføjer tabellen Region til fanen Elektroferogrammet. Vælg derefter et elektroferogramme, og gå til tabellen Region.I menuen Fra basepar og til basepar skal du indstille start- og slutbaseparret ved at højreklikke på elektroferogrammet og vælge et område, der skal tilføjes. Højreklik på en hvilken som helst celle i tabellen Region, og vælg Rediger regioner for at lave en lille ny vinduespopup for at indstille brugerdefinerede regioner. Brug denne ligning til at beregne poly(A)halelængden på det interessante mRNA.Næste på fanen Elektroferogrammer skal du kontrollere Vis størrelser for automatisk at konvertere regionstabellen fra basispar til runtime. Åbn CSV-filen, og vælg de værdier, der er opnået for runtime for at generere en graf. Ved hjælp af denne protokol blev poly(A)halelængder af Dscam1- og GAPDH-gener fra Drosophila-larvehjerner analyseret, mens PCR-produkter fra genspecifikke primerpar viste et enkelt bånd.PCR-produkter fra de genspecifikke universelle primerpar viste forskellige udstrygningsmønstre, hvilket indikerer differentiel poly(A)længde af mRNA'er. De gennemsnitlige poly(A)halelængder blev opnået ved hjælp af udtværingsanalyse. Og rækkevidden af halelængderne var repræsenteret af elektroferogrammer.Tilsvarende viste poly(A)halelængdeanalyse på S2-celler differentialpoly(A)halelængder for SV43 prime UTR- og GAPDH-generne.

Research

JoVE Journal

Neuroscience

Poly A Tail Length Measurement of Genes from Drosophila Brain

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

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 ...

Isolation of Brain from Drosophila Larvae and Harvest of S2 Schneider Cells

Isolering af hjernen fra Drosophila larver og høst af S2 Schneider celler

Total RNA Extraction from Drosophila Larvae Brain and S2 Schneider Cells

Total RNA-ekstraktion fra Drosophila-larver , hjerne- og S2-Schneider-celler