KDM5 Lysine Demethylases Are Involved in Maintenance of 3'UTR Length [RNA-Seq]

We discovered that the Saccharomyces cerevisiae lysine demethylase, Jhd2 (also known as KDM5), recruits 3'UTR processing machinery and promotes alteration of 3'UTR length in a demethylase-dependent manner. Interaction of Jhd2 with both chromatin and RNA suggests that Jhd2 affects selection of polyadenylation sites through a transcription-coupled mechanism. For 3'READs analysis, wild-type yeast or yeast with JHD2 deleted were grown to mid-log phase and RNA was extracted as mentioned above. cDNA libraries enriched for 3'UTRs were prepared as previously published and as noted in Figure 4a [28]. Samples were then subjected to RNA sequencing on the Illumina Hiseq 2000 using 50bp single end reads. Data was analyzed as previously [Hoque M] with the following modifications: The adapter sequences were trimmed off the single-ended reads. The remaining reads longer than 15 bp were mapped to yeast genome SacCer3 using bowtie2 [Langmead B 2012] with the setting '-5 4 --local', trimming off 4bp from 5' and allowing soft clipping (S) at both ends. The alignments were then filtered by 1) MAPQ>=10, 2) mismatches <= 5%, 3) unaligned 5' Ts >=2 to get the PASS reads. The last aligned positions of the PASS reads were grouped to pA clusters with a clustering size of 24 bp. Each pA cluster was assigned to one of the genes defined by SGD [Cherry JM]. The 3' end of a gene was extended 1000bp from the end of CDS until it overlaps with another gene in the same direction. The pA Clusters were then filtered by contains 1) >= 3 PASS reads 2) >= 5% of all PASS reads mapped to the gene.

我们发现，酿酒酵母（Saccharomyces cerevisiae）的赖氨酸去甲基化酶Jhd2（亦称KDM5）能够招募3'非翻译区（3' untranslated region, 3'UTR）加工复合物，并以去甲基化酶依赖的方式促进3'UTR长度的改变。Jhd2与染色质及RNA的相互作用提示，其可通过转录偶联机制影响多腺苷酸化位点的选择。
针对3'端测序reads（3'READS）的分析实验中，我们将野生型酵母或敲除JHD2的酵母培养至对数生长中期，随后按照前述方法提取RNA。参照已发表方法并结合图4a[28]中的流程，我们制备了富集3'UTR的cDNA文库。随后使用Illumina Hiseq 2000平台对样本进行RNA测序，采用50bp单端reads测序策略。
数据分析流程参照已发表方法（Hoque M等）并做如下修改：首先切除单端reads的接头序列；保留长度大于15bp的reads，使用bowtie2[Langmead B 2012]将其比对至酵母参考基因组SacCer3，比对参数设置为'-5 4 --local'，即从5'端切除4bp，并允许两端发生软剪切（S）。随后通过以下标准过滤得到合格（PASS）reads：1）比对质量值MAPQ≥10；2）错配率≤5%；3）5'端未比对的T碱基数目≥2。将合格reads的最后一个比对位点以24bp为聚类窗口，分组为多腺苷酸化簇（polyadenylation cluster, pA簇）。每个pA簇被分配至酵母基因组数据库（Saccharomyces Genome Database, SGD, [Cherry JM]）所定义的一个基因。将基因的3'端从编码区（Coding Sequence, CDS）末端向外延伸1000bp，直至与同方向的其他基因发生重叠。随后再通过以下标准对pA簇进行过滤：1）包含≥3个合格reads；2）该簇的合格reads数目占对应基因所有合格reads的比例≥5%。