poly a tail length measurement of genes from drosophila brain

Cuantificación de la longitud de Poly(A) de los genes del sistema nervioso de Drosophila

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.

Autor destacado: Explorando la frontera de la investigación de ARNm con técnicas de análisis de cola de Poly A

Medición de la longitud de la cola de Poly A a partir del cerebro y la línea celular de la larva de Drosophila

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.

Medición de la longitud de la cola de Poly A de genes del cerebro de Drosophila

Para comenzar el relé G/I, prepare una mezcla de 20 microlitros en hielo con ARN total, mezcla de tampón de cola y mezcla de enzimas de cola. Incubar a 37 grados centígrados durante 60 minutos en un termociclador. Luego, agregue la solución de tope de cola y manténgala en hielo durante dos minutos.Realizar transcripción inversa y amplificación por PCR. Después de la electroforesis en gel de alta resolución de los productos de PCR, acceda a los datos utilizando el software. Abra el archivo XAD y seleccione los nombres de ejemplo o la escalera en el panel Vista de árbol.Acérquese y aléjese de los electroferogramas y las imágenes en forma de gel para una visualización detallada. Para obtener los tamaños de pico, abra el electroferograma de una muestra seleccionada. Haga clic con el botón derecho en el electroferograma y seleccione integración manual para seleccionar manualmente los picos arrastrando la línea horizontal.Observe los valores máximos de la tabla de picos e identifique el pico con la mayor altura de pico. Habilite el icono Mostrar/Ocultar puntos de ajuste en el menú superior. Aparecerá un nuevo panel en el lado derecho.Seleccione Avanzado, desplácese hacia abajo hasta Realizar análisis de frotis y seleccione la casilla de verificación. Esto agregará la tabla Región a la pestaña Electroferograma. A continuación, seleccione un electroferograma y vaya a la tabla Región.En el menú From Base Pair y To Base Pair, establezca el par de bases inicial y final haciendo clic con el botón derecho en el electroferograma y seleccionando una región para agregar. Haga clic con el botón derecho en cualquier celda de la tabla Región y seleccione Modificar regiones para crear una pequeña ventana emergente nueva para establecer regiones personalizadas. Utilice esta ecuación para calcular la longitud de la cola de poli(A) en el ARNm de interés.A continuación, en la pestaña Electroferograma, active Mostrar tamaños para convertir automáticamente la tabla de regiones de par base a tiempo de ejecución. Abra el archivo CSV y seleccione los valores obtenidos para el tiempo de ejecución para generar un gráfico. Utilizando este protocolo, se analizaron las longitudes de la poli(A)cola de los genes Dscam1 y GAPDH de cerebros de larvas de Drosophila, mientras que los productos de PCR de pares de cebadores específicos de genes mostraron una sola banda.Los productos de PCR de los pares de cebadores universales específicos del gen mostraron patrones de frotis distintos, lo que indica una poli(A)longitud diferencial de los ARNm. Las longitudes medias de la cola de poli(A) se obtuvieron mediante el análisis de frotis. Y el rango de las longitudes de la cola estaba representado por electroferogramas.De manera similar, el análisis de la longitud de la cola de poli(A) en células S2 mostró longitudes diferenciales de la cola de poli(A) para los genes SV43 prime UTR y GAPDH.

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 ...

Investigación

Neurociencias

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

Aislamiento del cerebro de las larvas de Drosophila y recolección de células S2 de Schneider

Total RNA Extraction from Drosophila Larvae Brain and S2 Schneider Cells

Extracción de ARN total del cerebro de larvas de Drosophila y células S2 de Schneider