poly a tail length measurement of genes from drosophila brain

Kvantifisering av poly(A)-lengden til 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: Utforske grensen for mRNA-forskning med Poly A Tail Analysis Techniques

Måling av poly A hale lengde 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 hale lengde måling av gener fra Drosophila Brain

For å begynne G/I-haling, lag en 20-mikroliters blanding på is med total RNA, halebufferblanding og haleenzymblanding. Inkuber ved 37 grader Celsius i 60 minutter i en termosyklist. Deretter legger du til halestoppløsning og holder på is i to minutter.Utfør revers transkripsjon og PCR-amplifikasjon. Etter høyoppløselig gelelektroforese av PCR-produkter, få tilgang til data ved hjelp av programvaren. Åpne XAD-filen og velg eksempelnavn eller stige i Trevisning-panelet.Zoom inn og ut av elektroferogrammer og gellignende bilder for detaljert visning. For å oppnå toppstørrelsene, åpne elektroferogrammet til en valgt prøve. Høyreklikk på elektrofherogrammet og velg manuell integrasjon for å manuelt velge topper ved å dra den horisontale linjen.Observer toppverdiene i topptabellen og identifiser toppen med den største topphøyden. Aktiver Vis / skjul settpunkter ikonet på toppmenyen. Et nytt panel vil dukke opp på høyre side.Velg Avansert, bla ned til Utfør smøreanalyse og merk av i avmerkingsboksen. Dette vil legge til Region-tabellen i kategorien Elektroferogramme. Velg deretter et elektroferogram og gå til regiontabellen.I menyen From Base Pair og To Base Pair angir du start- og sluttbaseparet ved å høyreklikke på elektroferogrammet og velge en region du vil legge til. Høyreklikk på en hvilken som helst celle i regiontabellen og velg Endre regioner for å lage et lite nytt vindu popup for å angi egendefinerte regioner. Bruk denne ligningen til å beregne poly (A) halelengden på mRNA av interesse.Neste på Electropherogram-fanen, sjekk Vis størrelser for automatisk å konvertere regiontabellen fra basepar til kjøretid. Åpne CSV-filen og velg verdiene som er oppnådd for kjøretid for å generere en graf. Ved hjelp av denne protokollen ble poly (A) halelengder av Dscam1 og GAPDH gener fra Drosophila larvehjerner analysert mens PCR-produkter fra genspesifikke primerpar viste et enkelt bånd.PCR-produkter fra genspesifikke universelle primerpar viste distinkte smøremønstre, noe som indikerer differensialpoly (A) lengde av mRNA. De gjennomsnittlige poly(A)halelengdene ble oppnådd ved hjelp av smøreanalyse. Og rekkevidden av halelengdene ble representert av elektroferogrammer.Tilsvarende viste poly (A) halelengdeanalyse på S2-celler differensialpoly (A) halelengder for SV43 prime UTR og GAPDH gener.

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 av hjernen fra Drosophila larver og høsting av S2 Schneider-celler

Total RNA Extraction from Drosophila Larvae Brain and S2 Schneider Cells

Total RNA-ekstraksjon fra Drosophila larver hjerne og S2 Schneider celler