Next generation sequencing identifies polyadenylated species of hTR. Homo sapiens

The paired-end next-generation sequencing of all hTR versions of less than 200 nucleotides in length was used to analyze the 3’ end distribution of hTR associated to Flag-tagged versions of PABPN1, hTERT and Dyskerin. In total, we obtained 2,716,342, 3,152,013, and 3,077,395 hTR-specific reads for the PABPN1, hTERT, and Dyskerin purifications, respectively. We found that the majority of polyadenylated telomerase RNA recovered in the PABPN1, hTERT, and Dyskerin purifications had poly(A) tails starting immediately after the annotated hTR 3’ end. However, a greater proportion of poly(A) tails were found on genome-encoded 3’-extentions in the PABPN1-bound fraction compared to the population of hTR that was copurified with hTERT and DKC1: 15.5% for PABPN1, 6.9% for hTERT, and 6.5% for Dyskerin. Notably, the population of polyadenylated telomerase RNA recovered with PABPN1 was significantly different in terms of poly(A) tail length compared to polyadenylated hTR retrieved in the hTERT- and Dyskerin-bound fractions (P-value=5.551e-16, Kolmogorov-Smirnov test), showing a tendency toward longer poly(A) tails in the PABPN1-bound fraction. The enrichment of telomerase RNA with long poly(A) tails in the PABPN1-bound fraction is consistent with a role of PABPN1 in a maturation pathway that depends on hTR 3’ end polyadenylation. Moreover, our results indicate that a fraction of hTERT and Dyskerin are associated with polyadenylated versions of hTR. Overall design: Total RNA was immunoprecipitated with FLAG-PABPN1, FLAG-hTERT, and FLAG-DKC1 by the RNA-Immunoprecipitate (RIP) method as previously described. The RNA Ligase-Mediated Rapid Amplification of cDNA End (RLM-RACE) approach was applied to prepare RNA libraries. Briefly, Co-IPed RNAs were ligated with a linker-3 oligo (IDT) followed by a reverse transcription reaction. Three consecutive PCR reactions were performed with specific primers containing unique barcodes for each RNA sample to amplify RNA libraries, which were analyzed by Illumina MiSeq technology.

本研究通过对所有长度小于200 nt的hTR（人端粒酶RNA，human telomerase RNA）变体开展双端下一代测序（paired-end next-generation sequencing），分析与FLAG标签（FLAG-tag）融合的PABPN1、hTERT及Dyskerin蛋白结合的hTR的3'端分布特征。其中，针对PABPN1、hTERT及Dyskerin的纯化组，我们分别获得了2716342、3152013和3077395条hTR特异性读段（reads）。
我们发现，在PABPN1、hTERT及Dyskerin纯化组分中回收的多聚腺苷酸化端粒酶RNA，其poly(A)尾（poly(A) tail）大多直接起始于已注释的hTR 3'端。然而，与hTERT和DKC1共纯化得到的hTR群体相比，PABPN1结合组分中带有基因组编码3'端延伸序列的poly(A)尾占比更高：PABPN1组为15.5%，hTERT组为6.9%，Dyskerin组为6.5%。
值得注意的是，与hTERT和Dyskerin结合组分中回收的多聚腺苷酸化形式的hTR相比，PABPN1结合组分中回收的多聚腺苷酸化端粒酶RNA群体，其poly(A)尾长度分布存在显著差异（P值=5.551e-16，Kolmogorov-Smirnov检验（Kolmogorov-Smirnov test）），且PABPN1结合组分中的poly(A)尾整体更长。
PABPN1结合组分中长poly(A)尾端粒酶RNA的富集现象，与PABPN1在依赖于hTR 3'端多聚腺苷酸化的成熟通路中发挥的作用相一致。此外，我们的研究结果表明，有一部分hTERT和Dyskerin蛋白与多聚腺苷酸化形式的hTR相结合。

实验整体设计：按照此前报道的方法，采用RNA免疫沉淀（RNA Immunoprecipitation, RIP）技术，分别利用FLAG-PABPN1、FLAG-hTERT和FLAG-DKC1对总RNA进行免疫沉淀富集。采用RNA连接介导的cDNA末端快速扩增（RNA Ligase-Mediated Rapid Amplification of cDNA Ends, RLM-RACE）技术构建RNA测序文库。简要流程如下：将免疫共沉淀得到的RNA与IDT公司合成的linker-3寡核苷酸进行连接，随后开展反转录反应。使用带有针对每个RNA样本的独特条形码（barcode）的特异性引物，连续开展三轮PCR反应以扩增RNA文库，最终通过Illumina MiSeq测序平台完成文库测序分析。