poly a tail length measurement of genes from drosophila brain

量化 果蝇 神经系统基因的 Poly（A） 长度

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.

作者聚焦：使用 Poly A Tail 分析技术探索 mRNA 研究的前沿

果 蝇 幼虫脑和细胞系的Poly A尾长测量

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 果 蝇 脑基因的尾长测量

要开始 G/I 拖尾，在冰上制备含有总 RNA、尾部缓冲液混合物和尾部酶混合物的 20 微升混合物。在 37 摄氏度下在热循环仪中孵育 60 分钟。然后，加入止尾溶液，在冰上保持两分钟。进行逆转录和PCR扩增。对PCR产物进行高分辨率凝胶电泳后，使用软件访问数据。打开 XAD 文件，然后在“树视图”面板中选择样品名称或梯形图。放大和缩小电泳图和凝胶状图像，以进行详细显示。要获得峰尺寸，请打开所选样品的电泳图。右键单击电泳图，然后选择手动积分，通过拖动水平线手动选择峰。观察峰表中的峰值，并确定峰高最大的峰。启用顶部菜单上的“显示/隐藏设定点”图标。右侧将弹出一个新面板。选择“高级”，向下滚动到“执行涂片分析”，然后选中该复选框。这会将 Region 表添加到 Electropherogram 选项卡。然后选择一个电泳图并转到“区域”表。在“从碱基对”和“到碱基对”菜单中，通过右键单击电泳图并选择要添加的区域来设置开始和结束碱基对。右键单击“区域”表中的任何单元格，然后选择“修改区域”以弹出一个小的新窗口以设置自定义区域。使用此公式计算目标 mRNA 上的 poly（A） 尾长度。接下来，在 Electropherogram 选项卡上，选中 Show Sizes 以自动将 Region 表从基对转换为运行时。打开 CSV 文件，选择为运行时获取的值以生成图形。使用该方案，分析了来自果蝇幼虫大脑的Dscam1和GAPDH基因的poly（A）尾长，而来自基因特异性引物对的PCR产物显示单条带。来自基因特异性通用引物对的PCR产物显示出不同的涂片模式，表明mRNA的poly（A）长度不同。使用拖尾分析获得平均poly（A）尾长。尾部长度的范围由电泳图表示。同样，对 S2 细胞的 poly（A） 尾长分析显示 SV43 主要 UTR 和 GAPDH 基因的 poly（A） 尾长不同。

poly-a-tail-length-measurement-of-genes-from-drosophila-brain

Research

JoVE Journal

Neuroscience

Poly A Tail Length Measurement of Genes from Drosophila Brain

52 次观看.  University of Nevada, Reno. 要开始 G/I 拖尾，在冰上制备含有总 RNA、尾部缓冲液混合物和尾部酶混合物的 20 微升混合物。在 37 摄氏度下在热循环仪中孵育 60 分钟。然后，加入止尾溶液，在冰上保持两分钟。进行逆转录和PCR扩增。对PCR产物进行高分辨率凝胶电泳后，使用软件访问数据。打开 XAD 文件，然后在“树视图”面板中选择样品名称或梯形图。放大和缩小电泳图和凝胶状图像，以进行?...

视频: Poly A 果 蝇 脑基因的尾长测量

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 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.

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.

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 ...

Poly A 果 蝇 脑基因的尾长测量

Poly A 果蝇脑基因的尾长测量