Transcriptome-wide profiling of Yeast Phosphofructokinase 1 and 2 RNA targets. Transcriptome-wide profiling of Yeast Phosphofructokinase 1 and 2 RNA targets

The RNA targets of the yeast Pfk1 and Pfk2 were mapped on a transcriptome scale using formaldehyde-assisted crosslinking and RNA-protein immunoprecipitation followed by high throuput sequencing (fRIP-seq). Tandem affinity purification (TAP) tagged Pfk1 and Pfk2 were usesd to capture the transcritps bound by Pfk1 and Pfk2. We also included a mock IP control i.e. wild type treated exactly the same way as the fRIP-seq samples to account for non-specific RNA interactions with the beads. Moreover, we ran total RNA-seq on wild type control for data normalization. We found that Pfk1 and Pfk2 bind mainly to mRNAs that code for proteins invovled in distinct functions . Our data suggests that, besides their well-characterized functions as glycolytic enzymes, they perfom additional functions as RNA-binding involved in posttranscriptional regulation of gene expression. Overall design: Our aim was to map transcripts bound by the yeast Pfk1 and Pfk2 to explore their RNA-bining functions and to obtain and insight into their potential secondary function as posttranscriptional regulators of gene expression. We performed formaldehyde assisted crosslinking followed by RNA-protein immuniprecipitation (fRIP-seq) to identify RNAs bound by Pfk1 and Pfk2 under normal physiological conditon. Diploid yeast cells expressing either Pfk1 or Pfk2 with Tandem Affinity purification (TAP) tags as well as dipliod wild type cells for mock IP control were grown in rich YPD media to mid-log phase (OD600 = 0.6) then crosslinked with low concentration of formaldehyde to a final concentration of 0.3% followed by quenching of crosslinking using glycine (final concentration 125 mM). Cells were pelleted then washed three times with 1X PBS then flash frozen in liquid nitroen. Frozen cell pellets were ground under cryogenic conditions using mortar and pestel to fine powders then powders were resuspended in fRIP lysis buffer. Pfk1:TAP-RNA and Pfk2:TAP-RNA were captured using Pan mouse IgG beads. Crosslinking was reveresed followd by proteinase K digestion of proteins then RNA was extracted using standard acidic phenol method. RNA was then converted to cDNA followed by second strand synthesis then double-stranded cDNA was sheared using sonication. Sequencing libraries were constructed using Bioline JetSeq libraray preparation kit as described in product manual. We also perfomed total RNA-seq from wild type diploid cells for data normalization. Samples were sequenced on Illumina HiSeq2500 with minimum of 5 million reads per library. Our data showed that both Pfk1 and Pfk2 bind to hunderds on mRNAs coding for proteins involved in various cellular functions. Interestingly, our data suggests that Pfk1 and Pfk2 bind to mRNAs coding for proteins involved in distinct functions indicating that the two proteins are involved in different cellular functions as posttranscriptional regulators.

本研究通过甲醛辅助交联-RNA蛋白免疫沉淀结合高通量测序（formaldehyde-assisted crosslinking and RNA-protein immunoprecipitation followed by high-throughput sequencing, fRIP-seq），在转录组水平上定位了酿酒酵母Pfk1与Pfk2的RNA靶标。我们使用串联亲和纯化（Tandem Affinity Purification, TAP）标签标记的Pfk1与Pfk2，捕获二者结合的转录本。同时设置了Mock免疫沉淀对照：即与fRIP-seq样本处理流程完全一致的野生型样本，以排除与磁珠发生非特异性RNA结合的干扰。此外，我们对野生型对照样本进行了总RNA测序，用于后续数据标准化。

研究发现，Pfk1与Pfk2主要结合编码不同功能蛋白的mRNA。数据表明，除了作为糖酵解酶的经典功能外，二者还可作为RNA结合蛋白参与基因表达的转录后调控。

总体实验设计：本研究旨在定位酿酒酵母Pfk1与Pfk2结合的转录本，以探究二者的RNA结合功能，并深入解析其作为基因表达转录后调控因子的潜在次要功能。我们采用甲醛辅助交联结合RNA-蛋白免疫沉淀测序（fRIP-seq），在正常生理条件下鉴定Pfk1与Pfk2结合的RNA。

将分别携带TAP标签的Pfk1或Pfk2的二倍体酵母细胞，以及用于Mock对照的野生型二倍体酵母细胞，在丰富YPD培养基中培养至对数中期（OD₆₀₀=0.6）；随后用终浓度0.3%的低浓度甲醛进行交联，再用终浓度125 mM的甘氨酸终止交联反应。收集细胞并以1×PBS洗涤三次，之后置于液氮中快速冷冻。将冷冻的细胞沉淀置于液氮环境下，使用研钵与研杵研磨成细粉，随后将粉末重悬于fRIP裂解缓冲液中。使用Pan小鼠IgG磁珠捕获Pfk1:TAP-RNA与Pfk2:TAP-RNA复合物。之后逆转交联反应，经蛋白酶K消化去除蛋白后，采用标准酸性苯酚法提取RNA。将提取的RNA反转录为cDNA并合成第二链，随后通过超声破碎获得双链cDNA片段。使用Bioline JetSeq文库制备试剂盒，按照产品说明书构建测序文库。同时，我们对野生型二倍体酵母细胞进行总RNA测序，用于数据标准化。所有样本均在Illumina HiSeq2500平台上进行测序，每个文库的测序读长不低于500万条。

数据分析显示，Pfk1与Pfk2均可结合数百个编码不同细胞功能蛋白的mRNA。值得注意的是，二者结合的mRNA编码的蛋白功能存在显著差异，表明这两种蛋白作为转录后调控因子参与了不同的细胞生理过程。